Co-reporter:Caroline Schuabb, Salome Pataraia, Melanie Berghaus, Roland Winter
Biophysical Chemistry 2017 Volume 231(Volume 231) pp:
Publication Date(Web):1 December 2017
DOI:10.1016/j.bpc.2016.10.006
•The sRNAh structure is not fully unfolded even at 90 °C.•Pressure up to 400 MPa induces small conformational perturbations, only.•Cy3/Cy5 labeling of the sRNAh structure changes its stability.RNAs perform multiple vital roles within cells, including catalyzing biological reactions and expression of proteins. Small RNA hairpins (sRNAh) are the smallest functional entities of nucleic acids and are involved in various important biological functions such as ligand binding and tertiary folding initiation of proteins. We investigated the conformational and free energy landscape of the sRNAh gcUUCGgc over a wide range of temperatures and pressures using fluorescence resonance energy transfer, Fourier-transform infrared and UV/Vis spectroscopy as well as small-angle X-ray scattering on the unlabeled and/or fluorescently labeled sRNAh. The sRNAh shows a broad melting profile with continuous increase of unpaired conformations up to about 60 °C. However, the sRNAh structure might not be fully unfolded at temperatures as high as 90 °C and still comprise various partially unfolded compact conformations. Pressure up to 400 MPa has a small effect on the base pairing and base stacking interactions of the sRNAh, indicating small conformational perturbations, only, which might originate from minor changes in packing and hydration of the RNA molecule upon compression. Pressurization at 70 °C, i.e. at a temperature above the melting transition, has no significant effect on the conformational ensemble of the sRNAh, i.e., it does not promote formation of new native stem connections after thermal denaturation. Finally, we noticed that Cy3/Cy5 labeling of the sRNAh changes, probably via stacking interactions between the fluorescent dyes and the nucleotide rings, the stability of the sRNAh, thereby rendering FRET analysis of the conformational dynamics of such small RNA structure inappropriate.Download high-res image (178KB)Download full-size image
Co-reporter:S. R. Al-Ayoubi;P. H. Schummel;M. Golub;J. Peters;R. Winter
Physical Chemistry Chemical Physics 2017 vol. 19(Issue 22) pp:14230-14237
Publication Date(Web):2017/06/07
DOI:10.1039/C7CP00705A
We studied the effects of temperature and hydrostatic pressure on the dynamical properties and folding stability of highly concentrated lysozyme solutions in the absence and presence of the osmolytes trimethylamine-N-oxide (TMAO) and urea. Elastic incoherent neutron scattering (EINS) was applied to determine the mean-squared displacement (MSD) of the protein's hydrogen atoms to yield insights into the effects of these cosolvents on the averaged sub-nanosecond dynamics in the pressure range from ambient up to 4000 bar. To evaluate the additional effect of self-crowding, two protein concentrations (80 and 160 mg mL−1) were used. We observed a distinct effect of TMAO on the internal hydrogen dynamics, namely a reduced mobility. Urea, on the other hand, revealed no marked effect and consequently, no counteracting effect in an urea–TMAO mixture was observed. Different from the less concentrated protein solution, no significant effect of pressure on the MSD was observed for 160 mg mL−1 lysozyme. The EINS experiments were complemented by Fourier-transform infrared (FTIR) spectroscopy measurements, which led to additional insights into the folding stability of lysozyme under the various environmental conditions. We observed a stabilization of the protein in the presence of the compatible osmolyte TMAO and a destabilization in the presence of urea against temperature and pressure for both protein concentrations. Additionally, we noticed a slight destabilizing effect upon self-crowding at very high protein concentration (160 mg mL−1), which is attributable to transient destabilizing intermolecular interactions. Furthermore, a pressure–temperature diagram could be obtained for lysozyme at these high protein concentrations that mimics densely packed intracellular conditions.
Co-reporter:Nikolai Smolin;Vladimir P. Voloshin;Alexey V. Anikeenko;Alfons Geiger;Nikolai N. Medvedev
Physical Chemistry Chemical Physics 2017 vol. 19(Issue 9) pp:6345-6357
Publication Date(Web):2017/03/01
DOI:10.1039/C6CP07903B
We performed all-atom MD simulations of the protein SNase in aqueous solution and in the presence of two major osmolytes, trimethylamine-N-oxide (TMAO) and urea, as cosolvents at various concentrations and compositions and at different pressures and temperatures. The distributions of the cosolvent molecules and their orientation in the surroundings of the protein were analyzed in great detail. The distribution of urea is largely conserved near the protein. It varies little with pressure and temperature, and does practically not depend on the addition of TMAO. The slight decrease with temperature of the number of urea molecules that are in contact with the SNase molecule is consistent with the view that the interaction of the protein with urea is mainly of enthalpic nature. Most of the TMAO molecules tend to be oriented to the protein by its methyl groups, a small amount of these molecules contact the protein by its oxygen, forming hydrogen bonds with the protein, only. Unlike urea, the fraction of TMAO in the hydration shell of SNase slightly increases with temperature (a signature of a prevailing hydrophobic interaction between TMAO and SNase), and decreases significantly upon the addition of urea. This behavior reflects the diverse nature of the interaction of the two osmolytes with the protein. Using the Voronoi volume of the atoms of the solvent molecules (water, urea, TMAO), we compared the fraction of the volume occupied by a given type of solvent molecule in the hydration shell and in the bulk solvent. The volume fraction of urea in the hydration shell is more than two times larger than in the bulk, whereas the volume fraction of TMAO in the hydration shell is only slightly larger in the binary solvent (TMAO + water) and becomes even less than in the bulk in the ternary solvent (TMAO + water + urea). Thus, TMAO tends to be excluded from the hydration shell of the protein. The behavior of the two cosolvents in the vicinity of the protein does not change much with pressure (from 1 to 5000 bar) and temperature (from 280 to 330 K). This is also in line with the conception of the “osmophobic effect” of TMAO to protect proteins from denaturation also at harsh environmental conditions. We also calculated the volumetric parameters of SNase and found that the cosolvents have a small but significant effect on the apparent volume and its contributions, i.e. the intrinsic, molecular and thermal volumes.
Co-reporter:Julian Schulze, Johannes Möller, Jonathan Weine, Karin Julius, Nico König, Julia Nase, Michael Paulus, Metin Tolan and Roland Winter
Physical Chemistry Chemical Physics 2016 vol. 18(Issue 21) pp:14252-14256
Publication Date(Web):05 May 2016
DOI:10.1039/C6CP01791F
We present results from small-angle X-ray scattering and turbidity measurements on the effect of high hydrostatic pressure on the phase behavior of dense lysozyme solutions in the liquid–liquid phase separation region, and characterize the underlying intermolecular protein–protein interactions as a function of temperature and pressure under charge-screening conditions (0.5 M NaCl). A reentrant liquid–liquid phase separation region is observed at elevated pressures, which may originate in the pressure dependence of the solvent-mediated protein–protein interaction. A temperature-pressure-concentration phase diagram was constructed for highly concentrated lysozyme solutions over a wide range of temperatures, pressures and protein concentrations including the critical region of the liquid–liquid miscibility gap.
Co-reporter:Nelli Erwin, Benjamin Sperlich, Guillaume Garivet, Herbert Waldmann, Katrin Weise and Roland Winter
Physical Chemistry Chemical Physics 2016 vol. 18(Issue 13) pp:8954-8962
Publication Date(Web):26 Feb 2016
DOI:10.1039/C6CP00563B
In a combined chemical-biological and biophysical approach we explored the membrane partitioning of the lipidated signaling proteins N-Ras and K-Ras4B into membrane systems of different complexity, ranging from one-component lipid bilayers and anionic binary and ternary heterogeneous membrane systems even up to partitioning studies on protein-free and protein-containing giant plasma membrane vesicles (GPMVs). To yield a pictorial view of the localization process, imaging using confocal laser scanning and atomic force microscopy was performed. The results reveal pronounced isoform-specific differences regarding the lateral distribution and formation of protein-rich membrane domains. Line tension is one of the key parameters controlling not only the size and dynamic properties of segregated lipid domains but also the partitioning process of N-Ras that acts as a lineactant. The formation of N-Ras protein clusters is even recorded for almost vanishing hydrophobic mismatch. Conversely, for K-Ras4B, selective localization and clustering are electrostatically mediated by its polybasic farnesylated C-terminus. The formation of K-Ras4B clusters is also observed for the multi-component GPMV membrane, i.e., it seems to be a general phenomenon, largely independent of the details of the membrane composition, including the anionic charge density of lipid headgroups. Our data indicate that unspecific and entropy-driven membrane-mediated interactions play a major role in the partitioning behavior, thus relaxing the need for a multitude of fine-tuned interactions. Such a scenario seems also to be reasonable recalling the high dynamic nature of cellular membranes. Finally, we note that even relatively simple models of heterogeneous membranes are able to reproduce many of the properties of much more complex biological membranes.
Co-reporter:Paul Hendrik Schummel, Andreas Haag, Werner Kremer, Hans Robert Kalbitzer, and Roland Winter
The Journal of Physical Chemistry B 2016 Volume 120(Issue 27) pp:6575-6586
Publication Date(Web):June 17, 2016
DOI:10.1021/acs.jpcb.6b04738
Actin can be found in nearly all eukaryotic cells and is responsible for many different cellular functions. The polymerization process of actin has been found to be among the most pressure sensitive processes in vivo. In this study, we explored the effects of chaotropic and kosmotropic cosolvents, such as urea and the compatible osmolyte trimethylamine-N-oxide (TMAO), and, to mimic a more cell-like environment, crowding agents on the pressure and temperature stability of globular actin (G-actin). The temperature and pressure of unfolding as well as thermodynamic parameters upon unfolding, such as enthalpy and volume changes, have been determined by fluorescence spectroscopy over a wide range of temperatures and pressures, ranging from 10 to 80 °C and from 1 to 3000 bar, respectively. Complementary high-pressure NMR studies revealed additional information on the existence of native-like conformational substates of G-actin as well as a molten-globule-like state preceding the complete pressure denaturation. Different from the chaotropic agent urea, TMAO increases both the temperature and pressure stability for the protein most effectively. The Gibbs free energy differences of most of the native substates detected are not influenced significantly by TMAO. In mixtures of these osmolytes, urea counteracts the stabilizing effect of TMAO to some extent. Addition of the crowding agent Ficoll increases the temperature and pressure stability even further, thereby allowing sufficient stability of the protein at temperature and pressure conditions encountered under extreme environmental conditions on Earth.
Co-reporter:Melanie Berghaus, Michael Paulus, Paul Salmen, Samy Al-Ayoubi, Metin Tolan, and Roland Winter
The Journal of Physical Chemistry B 2016 Volume 120(Issue 29) pp:7148-7153
Publication Date(Web):July 7, 2016
DOI:10.1021/acs.jpcb.6b05639
The effect of hydrostatic pressure on the structure of a bicontinuous microemulsion in the presence of a solid interface has been studied by X-ray reflectometry and compared to the bulk behavior determined by small-angle X-ray scattering. Surface-induced lamellar ordering is observed close to the hydrophilic interface, which persists upon compression. The lamellar domains are compressed, but the correlation length of lamellar order does not change with pressure. SAXS measurements on the bulk microemulsion revealed an increased order upon pressurization. Although pressure can cause the formation of highly ordered lamellar phases from ordered bicontinuous cubic phases, such a scenario is not observed for the disordered analogue studied here. High pressure increases the stiffness of the interfacial surfactant layer, but this is not sufficient to overcome the loss in conformational entropy that would result from a transition to an ordered lamellar phase. Possible technological and biological implications of our results are briefly discussed.
Co-reporter:Dr. Satyajit Patra;Nelli Erwin ; Rol Winter
ChemPhysChem 2016 Volume 17( Issue 14) pp:2164-2169
Publication Date(Web):
DOI:10.1002/cphc.201600179
Abstract
Ras proteins are small GTPases and are involved in transmitting signals that control cell growth, differentiation, and proliferation. Since the cell cytoplasm is crowded with different macromolecules, understanding the translational dynamics of Ras proteins in crowded environments is crucial to yielding deeper insight into their reactivity and function. Herein, the translational dynamics of lipidated N-Ras and K-Ras4B is studied in the bulk and in the presence of a macromolecular crowder (Ficoll) and the compatible osmolyte and microcrowder sucrose by fluorescence correlation spectroscopy. The results reveal that N-Ras forms dimers due to the presence of its lipid moiety in the hypervariable region, whereas K-Ras4B remains in its monomeric form in the bulk. Addition of a macromolecular crowding agent gradually favors clustering of the Ras proteins. In 20 wt % Ficoll N-Ras forms trimers and K-Ras4B dimers. Concentrations of sucrose up to 10 wt % foster formation of N-Ras trimers and K-Ras dimers as well. The results can be rationalized in terms of the excluded-volume effect, which enhances the association of the proteins, and, for the higher concentrations, by limited-hydration conditions. The results of this study shed new light on the association state of these proteins in a crowded environment. This is of particular interest for the Ras proteins, because their solution state—monomeric or clustered—influences their membrane-partitioning behavior and their interplay with cytosolic interaction partners.
Co-reporter:Saba Suladze; Suleyman Cinar; Benjamin Sperlich
Journal of the American Chemical Society 2015 Volume 137(Issue 39) pp:12588-12596
Publication Date(Web):September 14, 2015
DOI:10.1021/jacs.5b07009
Phospholipases A2 (PLA2) catalyze the hydrolysis reaction of sn-2 fatty acids of membrane phospholipids and are also involved in receptor signaling and transcriptional pathways. Here, we used pressure modulation of the PLA2 activity and of the membrane’s physical–chemical properties to reveal new mechanistic information about the membrane association and subsequent enzymatic reaction of PLA2. Although the effect of high hydrostatic pressure (HHP) on aqueous soluble and integral membrane proteins has been investigated to some extent, its effect on enzymatic reactions operating at the water/lipid interface has not been explored, yet. This study focuses on the effect of HHP on the structure, membrane binding and enzymatic activity of membrane-associated bee venom PLA2, covering a pressure range up to 2 kbar. To this end, high-pressure Fourier-transform infrared and high-pressure stopped-flow fluorescence spectroscopies were applied. The results show that PLA2 binding to model biomembranes is not significantly affected by pressure and occurs in at least two kinetically distinct steps. Followed by fast initial membrane association, structural reorganization of α-helical segments of PLA2 takes place at the lipid water interface. FRET-based activity measurements reveal that pressure has a marked inhibitory effect on the lipid hydrolysis rate, which decreases by 75% upon compression up to 2 kbar. Lipid hydrolysis under extreme environmental conditions, such as those encountered in the deep sea where pressures up to the kbar-level are encountered, is hence markedly affected by HHP, rendering PLA2, next to being a primary osmosensor, a good candidate for a sensitive pressure sensor in vivo.
Co-reporter:Trung Quan Luong and Roland Winter
Physical Chemistry Chemical Physics 2015 vol. 17(Issue 35) pp:23273-23278
Publication Date(Web):12 Aug 2015
DOI:10.1039/C5CP03529E
We investigated the combined effects of cosolvents and pressure on the hydrolysis of a model peptide catalysed by α-chymotrypsin. The enzymatic activity was measured in the pressure range from 0.1 to 200 MPa using a high-pressure stopped-flow systems with 10 ms time resolution. A kosmotropic (trimethalymine-N-oxide, TMAO) and chaotropic (urea) cosolvent and mixtures thereof were used as cosolvents. High pressure enhances the hydrolysis rate as a consequence of a negative activation volume, ΔV#, which, depending on the cosolvent system, amounts to −2 to −4 mL mol−1. A more negative activation volume can be explained by a smaller compression of the ES complex relative to the transition state. Kinetic constants, such as kcat and the Michaelis constant KM, were determined for all solution conditions as a function of pressure. With increasing pressure, kcat increases by about 35% and its pressure dependence by a factor of 1.9 upon addition of 2 M urea, whereas 1 M TMAO has no significant effect on kcat and its pressure dependence. Similarly, KM increases upon addition of urea 6-fold. Addition of TMAO compensates the urea-effect on kcat and KM to some extent. The maximum rate of the enzymatic reaction increases with increasing pressure in all solutions except in the TMAO:urea 1:2 mixture, where, remarkably, pressure is found to have no effect on the rate of the enzymatic reaction anymore. Our data clearly show that compatible solutes can easily override deleterious effects of harsh environmental conditions, such as high hydrostatic pressures in the 100 MPa range, which is the maximum pressure encountered in the deep biosphere on Earth.
Co-reporter:Christopher Rosin, Paul Hendrik Schummel and Roland Winter
Physical Chemistry Chemical Physics 2015 vol. 17(Issue 13) pp:8330-8337
Publication Date(Web):07 Nov 2014
DOI:10.1039/C4CP04431B
We studied the effects of kosmotropic and chaotropic cosolvents, trimethylamine-N-oxide (TMAO) and urea, as well as crowding agents (dextran) on the polymerization reaction of actin. Time-lapse fluorescence intensity and anisotropy experiments were carried out to yield information about the kinetics of the polymerization process. To also quantitatively describe the effects, cosolvents and crowding impose on the underlying rate constants of the G-to-F-transformation, an integrative stochastic simulation model was applied. Drastic and diverse changes in the lag phase and association rates as well as the critical actin concentration were observed under different solvent conditions. The association rate constant is drastically increased by TMAO but decreased by urea. In mixtures of these osmolytes, TMAO counteracts not only the deleterious effect of urea on protein structure and stability, but also on the protein–protein interactions in the course of actin polymerization. Owing to the excluded volume effect, cell-like macromolecular crowding conditions increase the nucleation and association rates by one order of magnitude. Our results clearly reveal the pronounced sensitivity of the actin polymerization reaction to changes in cosolvent conditions and the presence of macromolecular crowding, and suggest that such effects should be taken into account in any discussion of the actin polymerization reaction in vivo.
Co-reporter:Janine Seeliger, Nelli Erwin, Christopher Rosin, Marie Kahse, Katrin Weise and Roland Winter
Physical Chemistry Chemical Physics 2015 vol. 17(Issue 11) pp:7507-7513
Publication Date(Web):12 Feb 2015
DOI:10.1039/C4CP05845C
Not only drastic temperature- but also pressure-induced perturbations of membrane organization pose a serious challenge to the biological cell. Although high hydrostatic pressure significantly influences the structural properties and thus functional characteristics of cells, this has not prevented life from invading the high pressure habitats of marine depths where pressures up to the 100 MPa level are encountered. Here, the temperature- and pressure-dependent structure and phase behavior of giant plasma membrane vesicles have been explored in the absence and presence of membrane proteins using a combined spectroscopic and microscopic approach. Demixing into extended liquid-ordered and liquid-disordered domains is observed over a wide range of temperatures and pressures. Only at pressures beyond 200 MPa a physiologically unfavorable all gel-like ordered lipid phase is reached at ambient temperature. This is in fact the pressure range where the membrane–protein function has generally been observed to cease, thereby shedding new light on the possible origin of this observation.
Co-reporter:Vladimir P. Voloshin, Nikolai N. Medvedev, Nikolai Smolin, Alfons Geiger and Roland Winter
Physical Chemistry Chemical Physics 2015 vol. 17(Issue 13) pp:8499-8508
Publication Date(Web):09 Feb 2015
DOI:10.1039/C5CP00251F
Understanding the physical basis of the structure, stability and function of proteins in solution, including extreme environmental conditions, requires knowledge of their temperature and pressure dependent volumetric properties. One physical–chemical property of proteins that is still little understood is their partial molar volume and its dependence on temperature and pressure. We used molecular dynamics simulations of aqueous solutions of a typical monomeric folded protein, staphylococcal nuclease (SNase), to study and analyze the pressure dependence of the apparent volume, Vapp, and its components by the Voronoi–Delaunay method. We show that the strong decrease of Vapp with pressure (βT = 0.95 × 10−5 bar−1, in very good agreement with the experimental value) is essentially due to the compression of the molecular volume, VM, ultimately, of its internal voids, VemptyM. Changes of the intrinsic volume (defined as the Voronoi volume of the molecule), the contribution of the solvent to the apparent volume, and of the contribution of the boundary voids between the protein and the solvent have also been studied and quantified in detail. The pressure dependences of the volumetric characteristics obtained are compared with the temperature dependent behavior of these quantities and with corresponding results for a natively unfolded polypeptide.
Co-reporter:Caroline Schuabb;Melanie Berghaus;Christopher Rosin ; Dr. Rol Winter
ChemPhysChem 2015 Volume 16( Issue 1) pp:138-146
Publication Date(Web):
DOI:10.1002/cphc.201402676
Abstract
A combined temperature- and pressure-dependent study was employed to reveal the conformational and free-energy landscape of phenylalanine transfer RNA (tRNAPhe), a known model for RNA function, to elucidate the features that are essential in determining its stability. These studies also help explore its structural properties under extreme environmental conditions, such as low/high temperatures and high pressures. To this end, fluorescence and FTIR spectroscopies, calorimetric and small-angle scattering measurements were carried out at different ion concentrations over a wide range of temperatures and pressures up to several hundred MPa. Compared with the pronounced temperature effect, the pressure-dependent structural changes of tRNAPhe are small. A maximum of only 15 % unpaired bases is observed upon pressurization up to 1 GPa. RNA unfolding differs not only from protein unfolding, but also from DNA melting. Its pressure stability seems to be similar to that of noncanonical DNA structures.
Co-reporter:Caroline Schuabb;Melanie Berghaus;Christopher Rosin ; Dr. Rol Winter
ChemPhysChem 2015 Volume 16( Issue 1) pp:
Publication Date(Web):
DOI:10.1002/cphc.201590002
Co-reporter:Christopher Rosin;Kathrin Estel;Jessica Hälker ; Rol Winter
ChemPhysChem 2015 Volume 16( Issue 7) pp:1379-1385
Publication Date(Web):
DOI:10.1002/cphc.201500083
Abstract
In vivo studies have shown that the cytoskeleton of cells is very sensitive to changes in temperature and pressure. In particular, actin filaments get depolymerized when pressure is increased up to several hundred bars, conditions that are easily encountered in the deep sea. We quantitatively evaluate the effects of temperature, pressure, and osmolytes on the kinetics of the polymerization reaction of actin by high-pressure stopped-flow experiments in combination with fluorescence detection and an integrative stochastic simulation of the polymerization process. We show that the compatible osmolyte trimethylamine-N-oxide is not only able to compensate for the strongly retarding effect of chaotropic agents, such as urea, on actin polymerization, it is also able to largely offset the deteriorating effect of pressure on actin polymerization, thereby allowing biological cells to better cope with extreme environmental conditions.
Co-reporter:Mimi Gao ;Dr. Rol Winter
ChemPhysChem 2015 Volume 16( Issue 17) pp:3681-3686
Publication Date(Web):
DOI:10.1002/cphc.201500633
Abstract
Actin polymerization is an essential process in eukaryotic cells that provides a driving force for motility and mechanical resistance for cell shape. By using preformed gelsolin–actin nuclei and applying stopped-flow methodology, we quantitatively studied the elongation kinetics of actin filaments as a function of temperature and pressure in the presence of synthetic and protein crowding agents. We show that the association of actin monomers to the pointed end of double-stranded helical actin filaments (F-actin) proceeds via a transition state that requires an activation energy of 56 kJ mol−1 for conformational and hydration rearrangements, but exhibits a negligible activation volume, pointing to a compact transition state that is devoid of packing defects. Macromolecular crowding causes acceleration of the F-actin elongation rate and counteracts the deteriorating effect of pressure. The results shed new light on the combined effect of these parameters on the polymerization process of actin, and help us understand the temperature and pressure sensitivity of actin polymerization under extreme conditions.
Co-reporter:Dr. Trung Quan Luong;Dr. Shobhna Kapoor ; Rol Winter
ChemPhysChem 2015 Volume 16( Issue 17) pp:3555-3571
Publication Date(Web):
DOI:10.1002/cphc.201500669
Abstract
Now that the centennial anniversary of the first report on pressure denaturation of proteins by Nobel Laureate P. W. Bridgman can be celebrated, this Review on the application of high pressure as a key variable for studying the energetics and interactions of proteins appears. We demonstrate that combined temperature–pressure-dependent studies help delineate the free-energy landscape of proteins and elucidate which features are essential in determining their stability. Pressure perturbation also serves as an important tool to explore fluctuations in proteins and reveal their conformational substates. From shaping the free-energy landscape of proteins themselves to that of their interactions, conformational fluctuations not only dictate a plethora of biological processes, but are also implicated in a number of debilitating diseases. Finally, the advantages of using pressure to explore biomolecular assemblies and modulate enzymatic reactions are discussed.
Co-reporter:Dr. Trung Quan Luong;Dr. Shobhna Kapoor ; Rol Winter
ChemPhysChem 2015 Volume 16( Issue 17) pp:
Publication Date(Web):
DOI:10.1002/cphc.201501003
Abstract
The front cover artwork is provided by the group of Prof. Roland Winter at TU Dortmund University. The image shows the effect of pressure on protein′s energy landscape, which is controlled by the hydration sphere. Read the full text of the article at 10.1002/cphc.201500669
Co-reporter:Dr. Trung Quan Luong;Dr. Shobhna Kapoor ; Rol Winter
ChemPhysChem 2015 Volume 16( Issue 17) pp:
Publication Date(Web):
DOI:10.1002/cphc.201501004
Co-reporter:Vladimir P. Voloshin, Nikolai N. Medvedev, Nikolai Smolin, Alfons Geiger, and Roland Winter
The Journal of Physical Chemistry B 2015 Volume 119(Issue 5) pp:1881-1890
Publication Date(Web):January 15, 2015
DOI:10.1021/jp510891b
We used molecular dynamics simulations of a typical monomeric protein, SNase, in combination with Voronoi–Delaunay tessellation to study and analyze the temperature dependence of the apparent volume, Vapp, of the solute. We show that the void volume, VB, created in the boundary region between solute and solvent, determines the temperature dependence of Vapp to a major extent. The less pronounced but still significant temperature dependence of the molecular volume of the solute, VM, is essentially the result of the expansivity of its internal voids, as the van der Waals contribution to VM is practically independent of temperature. Results for polypeptides of different chemical nature feature a similar temperature behavior, suggesting that the boundary/hydration contribution seems to be a universal part of the temperature dependence of Vapp. The results presented here shine new light on the discussion surrounding the physical basis for understanding and decomposing the volumetric properties of proteins and biomolecules in general.
Co-reporter:Mimi Gao;Melanie Berghaus;Julian vonderEcken;Dr. Stefan Raunser;Dr. Rol Winter
Angewandte Chemie 2015 Volume 127( Issue 38) pp:11240-11244
Publication Date(Web):
DOI:10.1002/ange.201504247
Abstract
Biological cells provide a large variety of rodlike filaments, including filamentous actin (F-actin), which can form meshworks and bundles. One key question remaining in the characterization of such network structures revolves around the temperature and pressure stabilities of these architectures as a way to understand why cells actively use proteins for forming them. The packing properties of F-actin in fascin- and Mg2+-induced bundles are compared, and significantly different pressure-temperature stabilities are observed because of marked differences in their nature of interaction, solvation, and packing efficiency. Moreover, differences are observed in their morphologies and disintegration scenarios. The pressure-induced dissociation of the actin bundles is reminiscent of a single unbinding transition as observed in other soft elastic manifolds.
Co-reporter:Mimi Gao;Melanie Berghaus;Julian vonderEcken;Dr. Stefan Raunser;Dr. Rol Winter
Angewandte Chemie International Edition 2015 Volume 54( Issue 38) pp:11088-11092
Publication Date(Web):
DOI:10.1002/anie.201504247
Abstract
Biological cells provide a large variety of rodlike filaments, including filamentous actin (F-actin), which can form meshworks and bundles. One key question remaining in the characterization of such network structures revolves around the temperature and pressure stabilities of these architectures as a way to understand why cells actively use proteins for forming them. The packing properties of F-actin in fascin- and Mg2+-induced bundles are compared, and significantly different pressure-temperature stabilities are observed because of marked differences in their nature of interaction, solvation, and packing efficiency. Moreover, differences are observed in their morphologies and disintegration scenarios. The pressure-induced dissociation of the actin bundles is reminiscent of a single unbinding transition as observed in other soft elastic manifolds.
Co-reporter:Johannes Möller, Sebastian Grobelny, Julian Schulze, Andre Steffen, Steffen Bieder, Michael Paulus, Metin Tolan and Roland Winter
Physical Chemistry Chemical Physics 2014 vol. 16(Issue 16) pp:7423-7429
Publication Date(Web):05 Mar 2014
DOI:10.1039/C3CP55278K
We present a study on ion specific effects on the intermolecular interaction potential V(r) of dense protein solutions under high hydrostatic pressure conditions. Small-angle X-ray scattering in combination with a liquid-state theoretical approach was used to determine the effect of structure breaking/making salt anions (Cl−, SO42−, PO43−) on the intermolecular interaction of lysozyme molecules. It was found that besides the Debye–Hückel charge screening effect, reducing the repulsiveness of the interaction potential V(r) at low salt concentrations, a specific ion effect is observed at high salt concentrations for the multivalent kosmotropic anions, which modulates also the pressure dependence of the protein–protein interaction potential. Whereas sulfate and phosphate strongly influence the pressure dependence of V(r), chloride anions do not. The strong structure-making effect of the multivalent anions, dominating for the triply charged PO43−, renders the solution structure less bulk-water-like at high salt concentrations, which leads to an altered behavior of the pressure dependence of V(r). Hence, the particular structural properties of the salt solutions are able to influence the spatial organization and the intermolecular interactions of the proteins, in particular upon compression. These results are of interest for exploring the combined effects of ionic strength, temperature and pressure on the phase behavior of protein solutions, but may also be of relevance for understanding pressure effects on the hydration behavior of biological matter under extreme environmental conditions.
Co-reporter:M. Erlkamp, S. Grobelny and R. Winter
Physical Chemistry Chemical Physics 2014 vol. 16(Issue 13) pp:5965-5976
Publication Date(Web):03 Feb 2014
DOI:10.1039/C3CP55040K
FT-IR spectroscopic, small-angle X-ray scattering and calorimetric measurements have been applied to explore the effect of the macromolecular crowder agent Ficoll on the temperature- and pressure-dependent stability diagram and folding reaction of the protein Staphylococcal Nuclease (SNase). Additionally, we compare the experimental data with approximate theoretical predictions. We found that temperature- and pressure-induced equilibrium unfolding of SNase is markedly shifted to higher temperatures and pressures in 30 wt% Ficoll solutions. The structure of the unfolded state ensemble does not seem to be strongly influenced in the presence of the crowder. Self-crowding effects have been found to become important at SNase concentrations above 10 wt% only. Our kinetic results show that the folding rate of SNase decreases markedly in the presence of Ficoll. These results indicate that besides the commonly encountered excluded volume effect, other factors need to be considered when assessing confinement effects on protein folding kinetics. Among those, crowder-induced viscosity changes seem to be prominent.
Co-reporter:Vladimir P. Voloshin, Alexandra V. Kim, Nikolai N. Medvedev, Roland Winter, Alfons Geiger
Biophysical Chemistry 2014 Volume 192() pp:1-9
Publication Date(Web):August 2014
DOI:10.1016/j.bpc.2014.05.001
•We retrieve the volumetric properties of a dissolved biomolecule by a Voronoi–Delaunay tessellation analysis of a molecular simulation run.•The impact of the solute on the local density of the solvent is short ranged.•The strong increase of the apparent volume with temperature is determined by the expansion of the extra void volume in the boundary region (the “thermal volume”).Recently a simple formalism was proposed for a quantitative analysis of interatomic voids inside a solute molecule and in the surrounding solvent. It is based on the Voronoi–Delaunay tessellation of structures, obtained in molecular simulations: successive Voronoi shells are constructed, starting from the interface between the solute molecule and the solvent, and continuing to the outside (into the solvent) as well as into the interior of the molecule. Similarly, successive Delaunay shells, consisting of Delaunay simplexes, can also be constructed. This technique can be applied to interpret volumetric data, obtained, for example, in studies of proteins in aqueous solution. In particular, it allows replacing qualitatively and descriptively introduced properties by strictly defined quantities, such as the thermal volume, by the boundary voids. The extension and the temperature behavior of the boundary region, its structure and composition are discussed in detail, using the example of a molecular dynamics model of an aqueous solution of the human amyloid polypeptide, hIAPP. We show that the impact of the solute on the local density of the solvent is short ranged, limited to the first Delaunay and the first Voronoi shell around the solute. The extra void volume, created in the boundary region between solute and solvent, determines the magnitude and the temperature dependence of the apparent volume of the solute molecule.
Co-reporter:Marie Kahse ; Mayke Werner ; Shuang Zhao ; Martin Hartmann ; Gerd Buntkowsky
The Journal of Physical Chemistry C 2014 Volume 118(Issue 37) pp:21523-21531
Publication Date(Web):August 25, 2014
DOI:10.1021/jp506544n
Mesoporous silicates (MPS) have several advantages for the immobilization of enzymes and large organic molecules. They possess well-defined pores and their surfaces can be functionalized by chemical methods. In this study, the model protein ribonuclease A (RNase A) was encapsulated in unmodified amino- and carboxy-functionalized rodlike SBA-15 with pore widths ranging from 4.0 to 5.8 nm. Differential scanning (DSC) and pressure perturbation (PPC) calorimetric techniques were employed to evaluate the stability, hydration, and volumetric properties of the confined protein. In addition, the influence of the solution pH, the surface functionalization, and cosolvents on the protein immobilization and the thermal stability of the immobilized protein are reported. The extent of stabilization depends strongly on the surface characteristics of the host, such as the charge density, and on geometric parameters, i.e., the pore size and pore volume. The addition of the chaotropic agent urea leads to an increased protein loading. Addition of the kosmotropic agent glycerol has the opposite effect. The stability of the protein RNase A confined in all the mesoporous silicates is drastically enhanced and is of the order of ΔTm ≈ 30 ± 10 °C regarding the increase in temperature stability. The highest immobilization capacity, fastest immobilization rate, and maximum thermal stability was achieved for the surface-functionalized SBA-15-COOH. The increased temperature stability is probably not only due to the entropy-driven excluded volume effect but also due to an increased hydration strength of the protein within the narrow silica pores, similar to the effects compatible osmolytes impose on protein hydration and stability. The absence of an expansivity increase of the confined protein after thermal denaturation indicates that inside the pores complete unfolding of the protein is not feasible anymore.
Co-reporter:Dr. Shobhna Kapoor;Melanie Berghaus;Saba Suladze;Daniel Prumbaum;Sebastian Grobelny;Dr. Patrick Degen;Dr. Stefan Raunser;Dr. Rol Winter
Angewandte Chemie International Edition 2014 Volume 53( Issue 32) pp:8397-8401
Publication Date(Web):
DOI:10.1002/anie.201404254
Abstract
Attractive candidates for compartmentalizing prebiotic cells are membranes comprised of single-chain fatty acids. It is generally believed that life may have originated in the depth of the protoocean, that is, under high hydrostatic pressure conditions, but the structure and physical–chemical properties of prebiotic membranes under such conditions have not yet been explored. We report the temperature- and pressure-dependent properties of membranes composed of prebiotically highly-plausible lipids and demonstrate that prebiotic membranes could not only withstand extreme temperatures, but also serve as robust models of protocells operating in extreme pressure environments. We show that pressure not only increases the stability of vesicular systems but also limits their flexibility and permeability to solutes, while still keeping the membrane in an overall fluid-like and thus functional state.
Co-reporter:Dr. Shobhna Kapoor;Melanie Berghaus;Saba Suladze;Daniel Prumbaum;Sebastian Grobelny;Dr. Patrick Degen;Dr. Stefan Raunser;Dr. Rol Winter
Angewandte Chemie 2014 Volume 126( Issue 32) pp:8537-8541
Publication Date(Web):
DOI:10.1002/ange.201404254
Abstract
Attractive candidates for compartmentalizing prebiotic cells are membranes comprised of single-chain fatty acids. It is generally believed that life may have originated in the depth of the protoocean, that is, under high hydrostatic pressure conditions, but the structure and physical–chemical properties of prebiotic membranes under such conditions have not yet been explored. We report the temperature- and pressure-dependent properties of membranes composed of prebiotically highly-plausible lipids and demonstrate that prebiotic membranes could not only withstand extreme temperatures, but also serve as robust models of protocells operating in extreme pressure environments. We show that pressure not only increases the stability of vesicular systems but also limits their flexibility and permeability to solutes, while still keeping the membrane in an overall fluid-like and thus functional state.
Co-reporter:Christian H. Hofmann, Sebastian Grobelny, Mirko Erlkamp, Roland Winter, Walter Richtering
Polymer 2014 Volume 55(Issue 8) pp:2000-2007
Publication Date(Web):10 April 2014
DOI:10.1016/j.polymer.2014.03.006
We show that the temperature-induced collapse of poly(N-isopropylacrylamide) (PNiPAm) nanogels in water/methanol mixtures can be reversed by excess hydrostatic pressure. Small angle X-ray scattering (SAXS) results reveal that first a swollen surface layer is established and then the particles swell homogeneously. A threshold pressure needed for rewelling fully collapsed nanogels indicates that hydrophobic interactions inside the nanogel have to be compensated to form a swollen surface layer. The size change is related to a change in polymer solvation detected by infrared (IR) spectroscopy. Pressure favours polymer/water hydrogen bonds to the cost of methanol/polymer bonds so that water is enriched inside the nanogel.
Co-reporter:Shobhna Kapoor ; Alexander Werkmüller ; Roger S. Goody Δ; Herbert Waldmann
Journal of the American Chemical Society 2013 Volume 135(Issue 16) pp:6149-6156
Publication Date(Web):April 5, 2013
DOI:10.1021/ja312671j
Proteins attached to the plasma membrane frequently encounter mechanical stresses, including high hydrostatic pressure (HHP) stress. Signaling pathways involving membrane-associated small GTPases (e.g., Ras) have been identified as critical loci for pressure perturbation. However, the impact of mechanical stimuli on biological outputs is still largely terra incognita. The present study explores the effect of HHP on the membrane association, dissociation, and intervesicle transfer process of N-Ras by using a FRET-based assay to obtain the kinetic parameters and volumetric properties along the reaction path of these processes. Notably, membrane association is fostered upon pressurization. Conversely, depending on the nature and lateral organization of the lipid membrane, acceleration or retardation is observed for the dissociation step. In addition, HHP can be inferred as a positive regulator of N-Ras clustering, in particular in heterogeneous membranes. The susceptibility of membrane interaction to pressure raises the idea of a role of lipidated signaling molecules as mechanosensors, transducing mechanical stimuli to chemical signals by regulating their membrane binding and dissociation. Finally, our results provide first insights into the influence of pressure on membrane-associated Ras-controlled signaling events in organisms living under extreme environmental conditions such as those that are encountered in the deep sea and sub-seafloor environments, where pressures reach the kilobar (100 MPa) range.
Co-reporter:Janine Seeliger, Kathrin Estel, Nelli Erwin and Roland Winter
Physical Chemistry Chemical Physics 2013 vol. 15(Issue 23) pp:8902-8907
Publication Date(Web):04 Mar 2013
DOI:10.1039/C3CP44412K
Owing to the presence of various types of osmolytes in the cellular environment, this study focuses on the impact of stabilizing (TMAO and betaine) as well as destabilizing (urea) cosolvents on the aggregation and fibrillation reaction of the highly amyloidogenic islet amyloid polypeptide (IAPP). IAPP is associated with type-2 diabetes mellitus and is responsible for the disease accompanying β-cell membrane permeabilization and final β-cell loss. To reveal the impact of the cosolvents on the aggregation kinetics, conformational and morphological changes upon IAPP fibrillation, Thioflavin T fluorescence spectroscopy, atomic force microscopy and attenuated total reflection Fourier-transform infrared spectroscopy were applied. For TMAO, and less pronounced for betaine, a decrease of the growth rate of fibrils is observed, whereas the lag phase remains essentially unchanged, indicating the ability of the compatible solutes to stabilize large oligomeric and protofibrillar structures and therefore hamper fibril elongation. Conversely, urea displays concentration-dependent prolongation of the lag phase, indicating stabilization of IAPP in its unfolded monomeric state, hence leading to retardation of IAPP nuclei formation. Mixtures of urea with TMAO, and to a lesser extent with betaine, exhibit a counteractive effect. TMAO is able to fully compensate the prolonged lag phase induced by urea. This strongly matches the findings of a counteraction of TMAO and urea in protein folding and unfolding experiments. The data also reveal that the influence of these cosolvents is only on the aggregation kinetics without markedly changing the final IAPP fibrillar morphology, i.e., the solution structure and cosolvent composition essentially affect the kinetics of the fibrillation process only.
Co-reporter:Sebastian Grobelny, Christian H. Hofmann, Mirko Erlkamp, Felix A. Plamper, Walter Richtering and Roland Winter
Soft Matter 2013 vol. 9(Issue 25) pp:5862-5866
Publication Date(Web):20 May 2013
DOI:10.1039/C3SM27653H
We investigated thermosensitive poly(N-isopropylacrylamide) microgels by high-pressure small angle X-ray scattering and Fourier-transform infrared spectroscopy below and above the collapse temperature. The measurements reveal little pressure-induced deswelling below the volume phase transition temperature and clear re-swelling of the collapsed gels at temperatures above the VPTT.
Co-reporter:Yong Zhai ; Dr. Rol Winter
ChemPhysChem 2013 Volume 14( Issue 2) pp:386-393
Publication Date(Web):
DOI:10.1002/cphc.201200767
Abstract
FT-IR spectroscopic and thermodynamic measurements were designed to explore the effect of a macromolecular crowder, dextran, on the temperature and pressure-dependent phase diagram of the protein Ribonuclease A (RNase A), and we compare the experimental data with approximate theoretical predictions based on configuration entropy. Exploring the crowding effect on the pressure-induced unfolding of proteins provides insight in protein stability and folding under cell-like dense conditions, since pressure is a fundamental thermodynamic variable linked to molecular volume. Moreover, these studies are of relevance for understanding protein stability in deep-sea organisms, which have to cope with pressures in the kbar range. We found that not only temperature-induced equilibrium unfolding of RNase A, but also unfolding induced by pressure is markedly prohibited in the crowded dextran solutions, suggesting that crowded environments such as those found intracellularly, will also oppress high-pressure protein unfolding. The FT-IR spectroscopic measurements revealed a marked increase in unfolding pressure of 2 kbar in the presence of 30 wt % dextran. Whereas the structural changes upon thermal unfolding of the protein are not significantly influenced in the presence of the crowding agent, through stabilization by dextran the pressure-unfolded state of the protein retains more ordered secondary structure elements, which seems to be a manifestation of the entropic destabilization of the unfolded state by crowding.
Co-reporter:Yong Zhai ; Dr. Rol Winter
ChemPhysChem 2013 Volume 14( Issue 2) pp:
Publication Date(Web):
DOI:10.1002/cphc.201390006
Co-reporter:Alexer Werkmüller;Dr. Gemma Triola; Dr. Herbert Waldmann; Dr. Rol Winter
ChemPhysChem 2013 Volume 14( Issue 16) pp:3698-3705
Publication Date(Web):
DOI:10.1002/cphc.201300617
Abstract
Plasma-membrane-associated Ras proteins typically control signal transduction processes. As nanoclustering and membrane viscosity sensing provide plausible signaling mechanisms, determination of the rotational and translational dynamics of membrane-bound Ras isoforms can help to link their dynamic mobility to their function. Herein, by using time-resolved fluorescence anisotropy and correlation spectroscopic measurements, we obtain the rotational-correlation time and the translational diffusion coefficient of lipidated boron-dipyrromethene-labeled Ras, both in bulk Ras and upon membrane binding. The results show that the second lipidation motif of N-Ras triggers dimer formation in bulk solution, whereas K-Ras4B is monomeric. Upon membrane binding, an essentially free rotation of the G-domain is observed, along with a high lateral mobility; the latter is essentially limited by the viscosity of the membrane and by lipid-mediated electrostatic interactions. This high diffusional mobility warrants rapid recognition–binding sequences in the membrane-bound state, thereby facilitating efficient interactions between the Ras proteins and scaffolding or effector proteins. The lipid-like rapid lateral diffusion observed here complies with in vivo data.
Co-reporter:Alexer Werkmüller;Dr. Gemma Triola; Dr. Herbert Waldmann; Dr. Rol Winter
ChemPhysChem 2013 Volume 14( Issue 16) pp:
Publication Date(Web):
DOI:10.1002/cphc.201390077
Co-reporter:Katrin Weise ; Shobhna Kapoor ; Alexander Werkmüller ; Simone Möbitz ; Gunther Zimmermann ; Gemma Triola ; Herbert Waldmann
Journal of the American Chemical Society 2012 Volume 134(Issue 28) pp:11503-11510
Publication Date(Web):June 21, 2012
DOI:10.1021/ja305518h
K-Ras4B is a small GTPase whose selective membrane localization and clustering into microdomains are mediated by its polybasic farnesylated C-terminus. The importance of the subcellular distribution for the signaling activity of K-Ras4B became apparent from recent in vivo studies, showing that the delta subunit of cGMP phosphodiesterase (PDEδ), which possesses a hydrophobic prenyl-binding pocket, is able to function as a potential binding partner for farnesylated proteins, thereby leading to a modulation of the spatiotemporal organization of K-Ras. Even though PDEδ has been suggested to serve as a cytosolic carrier for Ras, the functional transport mechanism still remains largely elusive. In this study, the effect of PDEδ on the interaction of GDP- and GTP-loaded K-Ras4B with neutral and anionic model biomembranes has been investigated by a combination of different spectroscopic and imaging techniques. The results show that PDEδ is not able to extract K-Ras4B from membranes. Rather, the K-Ras4B/PDEδ complex formed in bulk solution turned out to be unstable in the presence of heterogeneous membranes, resulting in a release of farnesylated K-Ras4B upon membrane contact. With the additional observation of enhanced membrane affinity for the K-Ras4B/PDEδ complex, a molecular mechanism for the PDEδ−K-Ras4B-membrane interaction could be proposed. This includes an effective delivery of PDEδ-solubilized K-Ras4B to the plasma membrane, probably through cytoplasmic diffusion, the dissociation of the K-Ras4B/PDEδ complex upon plasma membrane contact, and finally the membrane binding of released farnesylated K-Ras4B that leads to K-Ras4B-enriched microdomain formation.
Co-reporter:Martin A. Schroer, Metin Tolan and Roland Winter
Physical Chemistry Chemical Physics 2012 vol. 14(Issue 26) pp:9486-9491
Publication Date(Web):02 May 2012
DOI:10.1039/C2CP41041A
Using small-angle X-ray scattering data of concentrated solutions of the protein lysozyme taken at different pressures and temperatures, the isothermal pressure derivative and the isobaric temperature derivative of the structure factor S(q) were determined. The pressure derivative of S(q) allows us to test various models for the triplet correlation function g3. Significant differences were found in comparison to simple liquids reflecting the more complex interaction potential in dense protein solutions.
Co-reporter:Florian Evers, Christoph Jeworrek, Katrin Weise, Metin Tolan and Roland Winter
Soft Matter 2012 vol. 8(Issue 7) pp:2170-2175
Publication Date(Web):05 Jan 2012
DOI:10.1039/C2SM06835D
In reference to the complexity and heterogeneity of cellular membranes, the structure and lateral ordering of lipid monolayers and bilayers composed of multi-component lipid mixtures have been investigated and compared in the present study. These complex model biomembrane systems represent valuable model systems, e.g. for studies of lipid–peptide interactions, where an integrated in situ multi-technique approach using both monolayer and bilayer techniques is required. A zwitterionic (3 components) and an anionic (5 components) heterogeneous model membrane system have been characterized that consist of saturated and unsaturated phospholipids as well as cholesterol. Lipid monolayers were analyzed by surface X-ray scattering techniques, and both the vertical structure (electron density profile) and the degree of in-plane ordering were determined as a function of surface pressure and temperature. The corresponding structure and lateral organisation of the bilayer membranes were characterized by atomic force microscopy. Both monolayers and bilayers reveal ordered domain formation, the monolayer ones being of much smaller size and different temperature stability, however. Furthermore, the charge density of the lipid monolayer has a drastic influence on the size of ordered domains as well as the intermolecular distances of the lipid molecules.
Co-reporter:Yong Zhai, Parkson Lee-Gau Chong, Leeandrew Jacques-Asa Taylor, Mirko Erlkamp, Sebastian Grobelny, Claus Czeslik, Erik Watkins, and Roland Winter
Langmuir 2012 Volume 28(Issue 11) pp:5211-5217
Publication Date(Web):February 21, 2012
DOI:10.1021/la300142r
The polar lipid fraction E (PLFE) is a major tetraether lipid component in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Using differential scanning and pressure perturbation calorimetry as well as ultrasound velocity and density measurements, we have determined the compressibilities and volume fluctuations of PLFE liposomes derived from different cell growth temperatures (Tg = 68, 76, and 81 °C). The compressibility and volume fluctuation values of PLFE liposomes, which are substantially less than those detected from diester lipid membranes (e.g., DPPC), exhibit small but significant differences with Tg. Among the three Tgs employed, 76 °C leads to the least compressible and most tightly packed PLFE membranes. This temperature is within the range for optimal cell growth (75–80 °C). It is known that a decrease in Tg decreases the number of cyclopentane rings in archael tetraether lipids. Thus, our data enable us to present the new view that membrane packing in PLFE liposomes varies with the number of cyclopentane rings in a nonlinear manner, reaching maximal tightness when the tetraether lipids are derived from cells grown at optimal Tgs. In addition, we have studied the effects of pressure on total layer thickness, d, and neutron scattering length density, ρn, of a silicon–D2O interface that is covered with a PLFE membrane using neutron reflectometry (NR). At 55 °C, d and ρn are found to be rather insensitive to pressure up to 1800 bar, suggesting minor changes of the thickness of the membrane’s hydrophobic core and headgroup orientation upon compression only.
Co-reporter:Janine Seeliger;Dr. Florian Evers;Christoph Jeworrek;Shobhna Kapoor;Dr. Katrin Weise;Erika Andreetto;Dr. Metin Tolan;Dr. Aphrodite Kapurniotu;Dr. Rol Winter
Angewandte Chemie 2012 Volume 124( Issue 3) pp:703-707
Publication Date(Web):
DOI:10.1002/ange.201105877
Co-reporter:Janine Seeliger;Dr. Florian Evers;Christoph Jeworrek;Shobhna Kapoor;Dr. Katrin Weise;Erika Andreetto;Dr. Metin Tolan;Dr. Aphrodite Kapurniotu;Dr. Rol Winter
Angewandte Chemie International Edition 2012 Volume 51( Issue 3) pp:679-683
Publication Date(Web):
DOI:10.1002/anie.201105877
Co-reporter:Shobhna Kapoor;Gemma Triola;Ingrid R. Vetter;Mirko Erlkamp;Herbert Waldmann
PNAS 2012 Volume 109 (Issue 2 ) pp:
Publication Date(Web):2012-01-10
DOI:10.1073/pnas.1110553109
Regulation of protein function is often linked to a conformational switch triggered by chemical or physical signals. To evaluate
such conformational changes and to elucidate the underlying molecular mechanisms of subsequent protein function, experimental
identification of conformational substates and characterization of conformational equilibria are mandatory. We apply pressure
modulation in combination with FTIR spectroscopy to reveal equilibria between spectroscopically resolved substates of the
lipidated signaling protein N-Ras. Pressure has the advantage that its thermodynamic conjugate is volume, a parameter that
is directly related to structure. The conformational dynamics of N-Ras in its different nucleotide binding states in the absence
and presence of a model biomembrane was probed by pressure perturbation. We show that not only nucleotide binding but also
the presence of the membrane has a drastic effect on the conformational dynamics and selection of conformational substates
of the protein, and a new substate appearing upon membrane binding could be uncovered. Population of this new substate is
accompanied by structural reorientations of the G domain, as also indicated by complementary ATR-FTIR and IRRAS measurements.
These findings thus illustrate that the membrane controls signaling conformations by acting as an effective interaction partner,
which has consequences for the G-domain orientation of membrane-associated N-Ras, which in turn is known to be critical for
its effector and modulator interactions. Finally, these results provide insights into the influence of pressure on Ras-controlled
signaling events in organisms living under extreme environmental conditions as they are encountered in the deep sea where
pressures reach the kbar range.
Co-reporter:Shobhna Kapoor;Katrin Weise;Mirko Erlkamp;Gemma Triola
European Biophysics Journal 2012 Volume 41( Issue 10) pp:801-813
Publication Date(Web):2012 October
DOI:10.1007/s00249-012-0841-5
Ras proteins are proto-oncogenes that function as molecular switches linking extracellular stimuli with an overlapping but distinctive range of biological outcomes. Although modulatable interactions between the membrane and the Ras C-terminal hypervariable region (HVR) harbouring the membrane anchor motifs enable signalling specificity to be determined by their location, it is becoming clear that the spatial orientation of different Ras proteins is also crucial for their functions. To reveal the orientation of the G-domain at membranes, we conducted an extensive study on different Ras isoforms anchored to model raft membranes. The results show that the G-domain mediates the Ras–membrane interaction by inducing different sets of preferred orientations in the active and inactive states with largely parallel orientation relative to the membrane of most of the helices. The distinct locations of the different isoforms, exposing them to different effectors and regulators, coupled with different G-domain-membrane orientation, suggests synergy between this type of recognition motif and the specificity conferred by the HVR, thereby validating the concept of isoform specificity in Ras.
Co-reporter:Christoph Jeworrek, Sebastian Uelner and Roland Winter
Soft Matter 2011 vol. 7(Issue 6) pp:2709-2719
Publication Date(Web):24 Jan 2011
DOI:10.1039/C0SM01255F
Mixtures of the short-chain 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) and the long-chain 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) have emerged as a powerful system to study membrane-associated, biologically relevant macromolecules and assemblies. Whereas the temperature-dependent behavior of various mixtures of this system has been studied in detail, almost nothing is known about the pressure-dependent phase behavior of the system. Here we are using small-angle X-ray scattering (SAXS) and Fourier-transform infrared (FTIR) spectroscopy to study the p,T-phase diagram of the system DMPC/DHPC (at a molar ratio of 3.2). The results reveal the existence of further high-pressure phases of the system and are prerequisite for understanding of the structure and dynamics of biomolecules in bicellar environments at high-pressure conditions, such as for high-pressure NMR studies of proteins embedded in bicellar structures. By combining time-resolved SAXS (trSAXS) with the pressure-jump relaxation technique we were also able to reveal a detailed picture of the kinetic processes involved in the phase transitions between lamellar, bicellar and nematic phases. Generally, the transitions follow a two-step mechanism consisting of a fast (subsecond) component involving conformational transitions of the lipid chains, which are followed by slower relaxational processes of the lipid mesophase structures including changes in their hydration properties. Depending on the lipid phases involved, the direction of the pressure-jump and the pressure-jump amplitude, this kinetic trapping lasts for a few seconds up to a maximum of several minutes.
Co-reporter:Maximilian N. Andrews, Roland Winter
Biophysical Chemistry 2011 Volume 156(Issue 1) pp:43-50
Publication Date(Web):June 2011
DOI:10.1016/j.bpc.2010.12.007
Conformational properties of the full-length human and rat islet amyloid polypeptide 1–37 (amyloidogenic hIAPP and non-amyloidogenic rIAPP, respectively) were studied at 310 and 330 K by MD simulations both for the cysteine (reduced IAPP) and cystine (oxidized IAPP) moieties. At all temperatures studied, IAPP does not adopt a well-defined conformation and is essentially random coil in solution, although transient helices appear forming along the peptide between residues 8 and 22, particularly in the reduced form. Above the water percolation transition (at 320 K), the reduced hIAPP moiety presents a considerably diminished helical content remaining unstructured, while the natural cystine moiety reaches a rather compact state, presenting a radius of gyration that is almost 10% smaller and characterized by intrapeptide H-bonds that form many β-bridges in the C-terminal region. This compact conformation presents a short end-to-end distance and seems to form through the formation of β-sheet conformations in the C-terminal region with a minimization of the Y/F distances in a two-step mechanism: the first step taking place when the Y37/F23 distance is ~ 1.1 nm, and subsequently Y37/F15 reaches its minimum of ~ 0.86 nm. rIAPP, which does not aggregate, also presents transient helical conformations. A particularly stable helix is located in proximity of the C-terminal region, starting from residues L27 and P28. Our MD simulations show that P28 in rIAPP influences the secondary structure of IAPP by stabilizing the peptide in helical conformations. When this helix is not present, the peptide presents bends or H-bonded turns at P28 that seem to inhibit the formation of the β-bridges seen in hIAPP. Conversely, hIAPP is highly disordered in the C-terminal region, presenting transient isolated β-strand conformations, particularly at higher temperatures and when the natural disulfide bond is present. Such conformational differences found in our simulations could be responsible for the different aggregational propensities of the two different homologues. In fact, the fragment 30–37, which is identical in both homologues, is known to aggregate in vitro, hence the overall sequence must be responsible for the amyloidogenicity of hIAPP. The increased helicity in rIAPP induced by the serine-to-proline variation at residue 28 seems to be a plausible inhibitor of its aggregation.Graphical abstractResearch Highlights► Comparison between monomeric hIAPP and rIAPP at 310 and 330 K. ► Collapse to compact conformation of oxidized hIAPP at 330 K. ► Effect of the disulfide bridge on the conformation of IAPP. ► Effect of proline on the secondary structure of IAPP. ► Effect of in silico mutations on the conformation of hIAPP and rIAPP.
Co-reporter:Martin A. Schroer;Yong Zhai;D. C. Florian Wiel;Christoph J. Sahle;Dr. Julia Nase;Dr. Michael Paulus;Dr. Metin Tolan;Dr. Rol Winter
Angewandte Chemie International Edition 2011 Volume 50( Issue 48) pp:11413-11416
Publication Date(Web):
DOI:10.1002/anie.201104380
Co-reporter:Yong Zhai, Linus Okoro, Alan Cooper, Roland Winter
Biophysical Chemistry 2011 156(1) pp: 13-23
Publication Date(Web):
DOI:10.1016/j.bpc.2010.12.010
Co-reporter:Martin A. Schroer;Yong Zhai;D. C. Florian Wiel;Christoph J. Sahle;Dr. Julia Nase;Dr. Michael Paulus;Dr. Metin Tolan;Dr. Rol Winter
Angewandte Chemie 2011 Volume 123( Issue 48) pp:11615-11618
Publication Date(Web):
DOI:10.1002/ange.201104380
Co-reporter:Christoph Jeworrek, Florian Evers, Mirko Erlkamp, Sebastian Grobelny, Metin Tolan, Parkson Lee-Gau Chong, and Roland Winter
Langmuir 2011 Volume 27(Issue 21) pp:13113-13121
Publication Date(Web):September 12, 2011
DOI:10.1021/la202027s
We report X-ray reflectivity (XRR) and grazing incidence X-ray diffraction (GIXD) measurements of archaeal bipolar tetraether lipid monolayers at the air–water interface. Specifically, Langmuir films made of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius grown at three different temperatures, i.e., 68, 76, and 81 °C, were examined. The dependence of the structure and packing properties of PLFE monolayers on surface pressure were analyzed in a temperature range between 10 and 50 °C at different pH values. Additionally, the interaction of PLFE monolayers (using lipids derived from cells grown at 76 °C) with the ion channel peptide gramicidin was investigated as a function of surface pressure. A total monolayer thickness of approximately 30 Å was found for all monolayers, hinting at a U-shaped conformation of the molecules with both head groups in contact with the interface. The monolayer thickness increased with rising film pressure and decreased with increasing temperature. At 10 and 20 °C, large, highly crystalline domains were observed by GIXD, whereas at higher temperatures no distinct crystallinity could be observed. For lipids derived from cells grown at higher temperatures, a slightly more rigid structure in the lipid dibiphytanyl chains was observed. A change in the pH of the subphase had an influence only on the structure of the lipid head groups. The addition of gramicidin to an PLFE monolayer led to a more disordered state as observed by XRR. In GIXD measurements, no major changes in lateral organization could be observed, except for a decrease of the size of crystalline domains, indicating that gramicidin resides mainly in the disordered areas of the monolayer and causes local membrane perturbation, only.
Co-reporter:Stefan Gruzielanek Dr.;Yong Zhai ;Rol Winter Dr.
ChemPhysChem 2010 Volume 11( Issue 9) pp:2016-2020
Publication Date(Web):
DOI:10.1002/cphc.202000074
Abstract
The influence of pressure on the nucleation rate of insulin under fibril-forming conditions was studied and subsequently analysed using classical nucleation theory. The aim was a better understanding and quantification of the influence of pressure on protein aggregation/fibrillation reactions. The application of pressure has a drastic accelerating effect on the nucleation and growth process of insulin fibrils. We show that this effect arises from a volume decrease upon nucleus formation, due to formation of a less hydrated and more compact transition state that can be quantified extending nucleation theory by a pressure–volume term. Conversely, the absolute values of the lag time and the critical size of the nucleus cannot be satisfactorily described by the classical nucleation theory, which might be due to the presence of secondary effects, such as parallel aggregation pathways or fragmentation processes.
Co-reporter:Katrin Weise Dr.;Diana Radovan Dr.;Andrea Gohlke;Norbert Opitz Dr.;Rol Winter Dr.
ChemBioChem 2010 Volume 11( Issue 9) pp:1280-1290
Publication Date(Web):
DOI:10.1002/cbic.201000039
Abstract
Type II diabetes mellitus (T2DM) is associated with β-cell failure, which correlates with the formation of pancreatic islet amyloid deposits. The human islet amyloid polypeptide (hIAPP) is the major component of islet amyloid and undergoes structural changes followed by self-association and pathological tissue deposition during aggregation in T2DM. There is clear evidence that the aggregation process is accelerated in the presence of particular lipid membranes. Whereas hIAPP aggregation has been extensively studied in homogeneous model membrane systems, especially negatively charged lipid bilayers, information on the interaction of hIAPP with heterogeneous model raft membranes has been missing until now. In the present study, we focus on the principles of aggregation and amyloid formation of hIAPP in the presence of model raft membranes. Time-lapse tapping mode AFM and confocal fluorescence microscopy experiments followed membrane permeabilization and localization of hIAPP in the raft membrane. Together with the ThT and WST-1 assay, the data revealed elevated cytotoxicity of hIAPP oligomers on INS-1E cells.
Co-reporter:Daniel Sellin, Li-Mei Yan, Aphrodite Kapurniotu, Roland Winter
Biophysical Chemistry 2010 150(1–3) pp: 73-79
Publication Date(Web):
DOI:10.1016/j.bpc.2010.01.006
Co-reporter:Matthias Pühse, Martina Keerl, Christine Scherzinger, Walter Richtering, Roland Winter
Polymer 2010 Volume 51(Issue 16) pp:3653-3659
Publication Date(Web):22 July 2010
DOI:10.1016/j.polymer.2010.06.011
We investigated the hydration and swelling properties of poly(N-isopropylacrylamide) and poly(N-isopropylacrylamide)-poly(N,N-diethylacrylamide) derived microgels by Fourier transform infrared- (FTIR-) spectroscopy in a wide region of the temperature–pressure plane. These systems are known to show a swollen-to-collapsed-transition upon temperature elevation. Our data reveal that pressure favours the swollen, hydrated state over the collapsed state in all systems investigated. A detailed analysis of the fractions of the respective IR sensitive amide-I′-subbands allowed the calculation of ΔGo and ΔVo for the pressure-induced swelling process as well as evaluation of various intra- and intermolecular hydrogen bonding connectivities in the different systems. In fact, considerable differences exist between different polymer or microgel types with regards to their hydrogen bonding pattern as a function of temperature and pressure, and the microgels may even exhibit a biphasic swelling behavior. Notably, the thermodynamic parameters derived reveal to be in the same order of magnitude as measured for the pressure and cold denaturation of proteins.
Co-reporter:Roland Winter and Christoph Jeworrek
Soft Matter 2009 vol. 5(Issue 17) pp:3157-3173
Publication Date(Web):07 May 2009
DOI:10.1039/B901690B
Besides temperature, hydrostatic pressure has been used as a physical-chemical parameter for studying the energetics and phase behavior of membrane systems. First we review some theoretical aspects of lipid self-assembly. Then, the temperature and pressure dependent structure and phase behavior of lipid bilayers, differing in chain configuration, headgroup structure and composition as revealed by using thermodynamic, spectroscopic and scattering experiments is discussed. We also report on the lateral organization of phase-separated lipid membranes and model raft mixtures as well as the influence of peptide and protein incorporation on membrane structure and dynamics upon pressurization. Also the effect of other additives, such as ions, cholesterol, and anaesthetics is discussed. Furthermore, we introduce pressure as a kinetic variable. Applying the pressure-jump relaxation technique in combination with time-resolved synchrotron X-ray diffraction, the kinetics of various lipid phase transformations was investigated. Finally, also new data on pressure effects on membrane mimetics, such as surfactants and microemulsions, are presented.
Co-reporter:Rajesh Mishra Dr.;Daniel Sellin;Diana Radovan;Andrea Gohlke ;Rol Winter Dr.
ChemBioChem 2009 Volume 10( Issue 3) pp:445-449
Publication Date(Web):
DOI:10.1002/cbic.200800762
Co-reporter:Rajesh Mishra Dr.;Matthias Geyer Dr.;Rol Winter Dr.
ChemBioChem 2009 Volume 10( Issue 11) pp:1769-1772
Publication Date(Web):
DOI:10.1002/cbic.200900237
Co-reporter:Diana Radovan, Vytautas Smirnovas and Roland Winter
Biochemistry 2008 Volume 47(Issue 24) pp:
Publication Date(Web):May 23, 2008
DOI:10.1021/bi800503j
Type II diabetes mellitus is a disease which is characterized by peripheral insulin resistance coupled with a progressive loss of insulin secretion that is associated with a decrease in pancreatic islet β-cell mass and the deposition of amyloid in the extracellular matrix of β-cells, which lead to islet cell death. The principal component of the islet amyloid is a pancreatic hormone called islet amyloid polypeptide (IAPP). High-pressure coupled with FT-IR spectroscopic and AFM studies were carried out to elucidate further information about the aggregation pathway as well as the aggregate structures of IAPP. To this end, a comparative fibrillation study of IAPP fragments was carried out as well. As high hydrostatic pressure (HHP) is acting to weaken or even prevent hydrophobic self-organization and electrostatic interactions, application of HHP has been used as a measure to reveal the importance of these interactions in the fibrillation process of IAPP and its fragments. IAPP preformed fibrils exhibit a strong polymorphism with heterogeneous structures, a large population of which are rather sensitive to high hydrostatic pressure, thus indicating a high percentage of ionic and hydrophobic interactions and loose packing of these species. Conversely, fragments 1−19 and 1−29 are resistant to pressure treatment, suggesting more densely packed aggregate structures with less void volume and strong cooperative hydrogen bonding. Furthermore, the FT-IR data indicate that fragment 1−29 has intermolecular β-sheet conformational properties different from those of fragment 1−19, the latter exhibiting polymorphic behavior with more disordered structures and less strongly hydrogen bonded fibrillar assemblies. The data also suggest that hydrophobic interactions and/or less efficient packing of amino acids 30−37 region leads to the marked pressure sensitivity observed for full-length IAPP.
Co-reporter:Christoph Jeworrek, Matthias Pühse and Roland Winter
Langmuir 2008 Volume 24(Issue 20) pp:11851-11859
Publication Date(Web):September 4, 2008
DOI:10.1021/la801947v
By using the pressure-jump relaxation technique in combination with time-resolved synchrotron small-angle X-ray diffraction (TRSAXS), the kinetics of lipid phase transformations of ternary lipid mixtures serving as model systems of heterogeneous raftlike membranes were investigated. To this end, we first established the temperature−pressure phase diagram of a model lipid raft mixture, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)/cholesterol (1:2:1), using Fourier transform infrared spectroscopy and SAXS, covering the pressure range from 1 bar to 10 kbar at temperatures in the range from 7 to 80 °C. We then studied the kinetics of interlamellar phase transitions of the ternary lipid system involving transitions from the fluidlike (liquid-disordered, ld) phase to the liquid-ordered (lo)/liquid-disordered (ld) two-phase coexistence region as well as between the two- and three-phase coexistence regions of the system, where also solid-ordered phases (so) are involved. The phase transition from the all-fluid ld phase to the lo+ld two-phase coexistence region turns out to be rather rapid. Phases appear or disappear within the 25 ms time resolution of the technique, followed by a slow lattice relaxation process, which, depending on the pressure-jump amplitude, takes several seconds. Contrary to many one-component phospholipid phase transitions, the kinetics of the ld ↔ lo+ld transition follows a similar time scale and mechanism for the pressurization and depressurization direction. A similar behavior is observed for the phase transition kinetics of the so+lo+ld ↔ lo+ld transformation and even for the so+lo+ld ↔ ld transformation, jumping across the lo+ld two-phase region. All transitions are fully reversible, and no intermediate states are populated. As indicated by the complex relaxation profiles observed, the overall rates observed seem to reflect the effect of coupling of various dynamical processes through the transformation, involving fast conformational changes in the sub-millisecond time regime and slow relaxation of the lattices growing, probably being largely controlled by the transport and redistribution of water into and in the new phases of the multilamellar vesicle assemblies.
Co-reporter:Rajesh Mishra Dr. ;Rol Winter Dr.
Angewandte Chemie 2008 Volume 120( Issue 35) pp:6618-6621
Publication Date(Web):
DOI:10.1002/ange.200802027
Co-reporter:Rajesh Mishra Dr. ;Rol Winter Dr.
Angewandte Chemie International Edition 2008 Volume 47( Issue 35) pp:6518-6521
Publication Date(Web):
DOI:10.1002/anie.200802027
Co-reporter:Rol Winter ;Ralf Ludwig
ChemPhysChem 2008 Volume 9( Issue 18) pp:2635-2636
Publication Date(Web):
DOI:10.1002/cphc.200800769
No abstract is available for this article.
Co-reporter:Simmon Hofstetter, Christian Denter, Roland Winter, Lynn M. McMullen, Michael G. Gänzle
Journal of Microbiological Methods (October 2012) Volume 91(Issue 1) pp:93-100
Publication Date(Web):1 October 2012
DOI:10.1016/j.mimet.2012.07.023
A method for measuring the fluidity of inner membranes of populations of endospores of Clostridium spp. with a fluorescent dye was developed. Cells of Clostridium beijerinckii ATCC 8260 and Clostridium sporogenes ATCC 7955 were allowed to sporulate in the presence of 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) on a soil-based media. Labeling of endospores with LAURDAN did not affect endospore viability. Removal of the outer membranes of endospores was done using a chemical treatment and confirmed using transmission electron microscopy (TEM). Two-photon confocal laser scanning microscopy (CLSM), and generalized polarization (GP) measurements were used to assess fluorescence of endospores. Lipid composition analysis of cells and endospores was done to determine whether differences in GP values are attributable to differences in membrane composition. Removal of the outer membranes of endospores did not significantly impact GP values. Decoated, labeled endospores of C. sporogenes ATCC 7955 and C. beijerinckii ATCC 8260 exhibited GP values of 0.77 ± 0.031 and 0.74 ± 0.027 respectively. Differences in ratios of fatty acids between cells and endospores are unlikely to be responsible for high GP values observed in endospores. These GP values indicate high levels of lipid order and the exclusion of water from within inner membranes of endospores.Highlights► A method for measuring membrane fluidity of endospores was developed. ► The fluorescence method can be applied to endospore populations in solution. ► Outer membranes of endospores do not significantly interfere with this method. ► We report that the inner membranes of endospores are highly ordered.
Co-reporter:Shobhna Kapoor, Alexander Werkmüller, Christian Denter, Yong Zhai, Jonas Markgraf, Katrin Weise, Norbert Opitz, Roland Winter
Biochimica et Biophysica Acta (BBA) - Biomembranes (April 2011) Volume 1808(Issue 4) pp:
Publication Date(Web):April 2011
DOI:10.1016/j.bbamem.2011.01.011
By using Fourier transform infrared (FT-IR) spectroscopy in combination with differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), ultrasound velocimetry, Laurdan fluorescence spectroscopy, fluorescence microscopy and atomic force microscopy (AFM), the temperature and pressure dependent phase behavior of the five-component anionic model raft lipid mixture DOPC/DOPG/DPPC/DPPG/cholesterol (20:5:45:5:25 mol%) was investigated. A temperature range from 5 to 65 °C and a pressure range up to 16 kbar were covered to establish the temperature–pressure phase diagram of this heterogeneous model biomembrane system. Incorporation of 10–20 mol% PG still leads to liquid-ordered (lo)-liquid-disordered (ld) phase coexistence regions over a wide range of temperatures and pressures. Compared to the corresponding neutral model raft mixture (DOPC/DPPC/Chol 25:50:25 mol%), the p,T-phase diagram is - as expected and in accordance with the Gibbs phase rule - more complex, the phase sequence as a function of temperature and pressure is largely similar, however. This anionic heterogeneous model membrane system will serve as a more realistic model biomembrane system to study protein interactions with anionic lipid bilayers displaying liquid-disordered/liquid-ordered domain coexistence over a wide range of the temperature–pressure plane, thus allowing also studies of biologically relevant systems encountered under extreme environmental conditions.Research Highlights► The T,p-phase diagram of an anionic model raft mixture has been determined. ► A temperature range from 5 to 65 °C and a pressure range up to 16 kbar were covered. ► Biological studies under extreme environmental conditions are feasible, now.
Co-reporter:Diana Radovan, Norbert Opitz, Roland Winter
FEBS Letters (6 May 2009) Volume 583(Issue 9) pp:1439-1445
Publication Date(Web):6 May 2009
DOI:10.1016/j.febslet.2009.03.059
Type II diabetes mellitus (T2DM) is a disease characterized by progressive deposition of amyloid in the extracellular matrix of β-cells. We investigated the interaction of the islet amyloid polypeptide (IAPP) with lipid model raft mixtures and INS-1E cells using fluorescence microscopy techniques. Following preferential partitioning of IAPP into the fluid lipid phase, the membrane suffers irreversible damage and predominantly circularly-shaped lipid-containing IAPP amyloid is formed. Interaction studies with the pancreatic β-cell line INS-1E revealed that growing IAPP fibrils also incorporate substantial amounts of cellular membranes in vivo. Additionally, the inhibitory effect of the red wine compound resveratrol on IAPP fibril formation has been studied, alluding to its potential use in developing therapeutic strategies against T2DM.
Co-reporter:Johannes Möller, Martin A. Schroer, Mirko Erlkamp, Sebastian Grobelny, Michael Paulus, Sebastian Tiemeyer, Florian J. Wirkert, Metin Tolan, Roland Winter
Biophysical Journal (6 June 2012) Volume 102(Issue 11) pp:
Publication Date(Web):6 June 2012
DOI:10.1016/j.bpj.2012.04.043
Understanding the intermolecular interaction potential, V(r), of proteins under the influence of temperature, pressure, and salt concentration is essential for understanding protein aggregation, crystallization, and protein phase behavior in general. Here, we report small-angle x-ray scattering studies on dense lysozyme solutions of high ionic strength as a function of temperature and pressure. We show that the interaction potential changes in a nonlinear fashion over a wide range of temperatures, salt, and protein concentrations. Neither temperature nor protein and salt concentration lead to marked changes in the pressure dependence of V(r), indicating that changes of the water structure dominate the pressure dependence of the intermolecular forces. Furthermore, by analysis of the temperature, pressure, and ionic strength dependence of the normalized second virial coefficient, b2, we show that the interaction can be fine-tuned by pressure, which can be used to optimize b2 values for controlled protein crystallization.
Co-reporter:Christoph Jeworrek, Florian Evers, Jörg Howe, Klaus Brandenburg, Metin Tolan, Roland Winter
Biophysical Journal (4 May 2011) Volume 100(Issue 9) pp:
Publication Date(Web):4 May 2011
DOI:10.1016/j.bpj.2011.03.019
We report x-ray reflectivity and grazing incidence x-ray diffraction measurements of lipopolysaccharide (LPS) monolayers at the water-air interface. Our investigations reveal that the structure and lateral ordering of the LPS molecules is very different from phospholipid systems and can be modulated by the ionic strength of the aqueous subphase in an ion-dependent manner. Our findings also indicate differential effects of monovalent and divalent ions on the two-dimensional ordering of lipid domains. Na+ ions interact unspecifically with LPS molecules based on their ability to efficiently screen the negative charges of the LPS molecules, whereas Ca2+ ions interact specifically by cross-linking adjacent molecules in the monolayer. At low lateral pressures, Na+ ions present in the subphase lead to a LPS monolayer structure ordered over large areas with high compressibility, nearly hexagonal packing of the hydrocarbon chains, and high density in the LPS headgroup region. At higher film pressures, the LPS monolayer becomes more rigid and results in a less perfect, oblique packing of the LPS hydrocarbon chains as well as a smaller lateral size of highly ordered domains on the monolayer. Furthermore, associated with the increased surface pressure, a conformational change of the sugar headgroups occurs, leading to a thickening of the entire LPS monolayer structure. The effect of Ca2+ ions in the subphase is to increase the rigidity of the LPS monolayer, leading to an oblique packing of the hydrocarbon chains already at low film pressures, an upright orientation of the sugar moieties, and much smaller sizes of ordered domains in the plane of the monolayer. In the presence of both Na+- and Ca2+ ions in the subphase, the screening effect of Na+ is predominant at low film pressures, whereas, at higher film pressures, the structure and lateral organization of LPS molecules is governed by the influence of Ca2+ ions. The unspecific charge-screening effect of the Na+ ions on the conformation of the sugar moiety becomes less dominant at biologically relevant lateral pressures.
Co-reporter:Suman Jha, Daniel Sellin, Ralf Seidel, Roland Winter
Journal of Molecular Biology (26 June 2009) Volume 389(Issue 5) pp:907-920
Publication Date(Web):26 June 2009
DOI:10.1016/j.jmb.2009.04.077
Human islet amyloid polypeptide (hIAPP), which is considered the primary culprit for β-cell loss in type 2 diabetes mellitus patients, is synthesized in β-cells of the pancreas from its precursor pro-islet amyloid polypeptide (proIAPP), which may be important in early intracellular amyloid formation as well. We compare the amyloidogenic propensities and conformational properties of proIAPP and hIAPP in the presence of negatively charged lipid membranes, which have been discussed as loci of initiation of the fibrillation reaction. Circular dichroism studies verify the initial secondary structures of proIAPP and hIAPP to be predominantly unordered with small amounts of ordered secondary structure elements, and exhibit minor differences between these two peptides only. Using attenuated total reflection–Fourier transform infrared spectroscopy and thioflavin T fluorescence spectroscopy, as well as atomic force microscopy, we show that in the presence of negatively charged membranes, proIAPP exhibits a much higher amyloidogenic propensity than in bulk solvent. Compared to hIAPP, it is still much less amyloidogenic, however. Although differences in the secondary structures of the aggregated species of hIAPP and proIAPP at the lipid interface are small, they are reflected in morphological changes. Unlike hIAPP, proIAPP forms essentially oligomeric-like structures at the lipid interface. Besides the interaction with anionic membranes [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) + x1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]], interaction with zwitterionic homogeneous (DOPC) and heterogeneous (1,2-dipalmitoyl-sn-glycero-3-phosphocholine:DOPC:cholesterol 1:2:1 model raft mixture) membranes has also been studied. Both peptides do not aggregate significantly at DOPC bilayers. In the presence of the model raft membrane, hIAPP aggregates markedly as well. Conversely, proIAPP clusters into less ordered structures and to a minor extent at raft membranes only. The addition of proIAPP to hIAPP retards the hIAPP fibrillation process also in the presence of negatively charged lipid bilayers. In excess proIAPP, increased aggregation levels are finally observed, however, which could be attributed to seed-induced cofibrillation of proIAPP.
Co-reporter:Andrea Gohlke, Gemma Triola, Herbert Waldmann, Roland Winter
Biophysical Journal (19 May 2010) Volume 98(Issue 10) pp:
Publication Date(Web):19 May 2010
DOI:10.1016/j.bpj.2010.02.005
Ras GTPases play a crucial role in signal transduction cascades involved in cell differentiation and proliferation, and membrane binding is essential for their proper function. To determine the influence of the nature of the lipid anchor motif and the difference between the active (GTP) and inactive (GDP) forms of N-Ras on partitioning and localization in the lipid membrane, five different N-Ras constructs with different lipid anchors and nucleotide loading (Far/Far (GDP), HD/Far (GDP), HD/HD (GDP), Far (GDP), and HD/Far (GppNHp)) were synthesized. Using the surface plasmon resonance technique, we were able to follow the insertion and dissociation process of the lipidated proteins into and out of model membranes consisting of pure liquid-ordered (lo) or liquid-disordered (ld) phase and a heterogeneous two-phase mixture, i.e., a raft mixture with lo + ld phase coexistence. In addition, we examined the influence of negatively charged headgroups and stored curvature elastic stress on the binding properties of the lipidated N-Ras proteins. In most cases, significant differences were found for the various anchor motifs. In general, N-Ras proteins insert preferentially into a fluidlike, rather than a rigid, ordered lipid bilayer environment. Electrostatic interactions with lipid headgroups or stored curvature elastic stress of the membrane seem to have no drastic effect on the binding and dissociation processes of the lipidated proteins. The monofarnesylated N-Ras exhibits generally the highest association rate and fastest dissociation process in fluidlike membranes. Double lipidation, especially including farnesylation, of the protein leads to drastically reduced initial binding rates but strong final association. The change in the nucleotide loading of the natural N-Ras HD/Far induces a slightly different binding and dissociation kinetics, as well as stability of association, and seems to influence the tendency to segregate laterally in the membrane plane. The GDP-bound inactive form of N-Ras with an HD/Far anchor shows stronger membrane association, which might be due to a more pronounced tendency to self-assemble in the membrane matrix than is seen with the active GTP-bound form.
Co-reporter:Parkson Lee-Gau Chong, Michael Sulc, Roland Winter
Biophysical Journal (17 November 2010) Volume 99(Issue 10) pp:
Publication Date(Web):17 November 2010
DOI:10.1016/j.bpj.2010.09.061
Bipolar tetraether lipids (BTLs) are abundant in crenarchaeota, which thrive in both thermophilic and nonthermophilic environments, with wide-ranging growth temperatures (4–108°C). BTL liposomes can serve as membrane models to explore the role of BTLs in the thermal stability of the plasma membrane of crenarchaeota. In this study, we focus on the liposomes made of the polar lipid fraction E (PLFE). PLFE is one of the main BTLs isolated from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Using molecular acoustics (ultrasound velocimetry and densimetry), pressure perturbation calorimetry, and differential scanning calorimetry, we have determined partial specific adiabatic and isothermal compressibility, their respective compressibility coefficients, partial specific volume, and relative volume fluctuations of PLFE large unilamellar vesicles (LUVs) over a wide range of temperatures (20–85°C). The results are compared with those obtained from liposomes made of dipalmitoyl-L-α-phosphatidylcholine (DPPC), a conventional monopolar diester lipid. We found that, in the entire temperature range examined, compressibilities of PLFE LUVs are low, comparable to those found in gel state of DPPC. Relative volume fluctuations of PLFE LUVs at any given temperature examined are 1.6–2.2 times more damped than those found in DPPC LUVs. Both compressibilities and relative volume fluctuations in PLFE LUVs are much less temperature-sensitive than those in DPPC liposomes. The isothermal compressibility coefficient (βTlipid) of PLFE LUVs changes from 3.59 × 10−10 Pa−1 at 25°C to 4.08 × 10−10 Pa−1 at 78°C. Volume fluctuations of PLFE LUVs change only 0.25% from 30°C to 80°C. The highly damped volume fluctuations and their low temperature sensitivity, echo that PLFE liposomes are rigid and tightly packed. To our knowledge, the data provide a deeper understanding of lipid packing in PLFE liposomes than has been previously reported, as well as a molecular explanation for the low solute permeation and limited membrane lateral motion. The obtained results may help to establish new strategies for rational design of stable BTL-based liposomes for drug/vaccine delivery.
Co-reporter:Martin A. Schroer, Michael Paulus, Christoph Jeworrek, Christina Krywka, Saskia Schmacke, Yong Zhai, D. C. Florian Wieland, Christoph J. Sahle, Michael Chimenti, Catherine A. Royer, Bertrand Garcia-Moreno, Metin Tolan, Roland Winter
Biophysical Journal (17 November 2010) Volume 99(Issue 10) pp:
Publication Date(Web):17 November 2010
DOI:10.1016/j.bpj.2010.09.046
A structural interpretation of the thermodynamic stability of proteins requires an understanding of the structural properties of the unfolded state. High-pressure small-angle x-ray scattering was used to measure the effects of temperature, pressure, denaturants, and stabilizing osmolytes on the radii of gyration of folded and unfolded state ensembles of staphylococcal nuclease. A set of variants with the internal Val-66 replaced with Ala, Tyr, or Arg was used to examine how changes in the volume and polarity of an internal microcavity affect the dimensions of the native state and the pressure sensitivity of the ensemble. The unfolded state ensembles achieved for these proteins with high pressure were more compact than those achieved at high temperature, and were all very sensitive to the presence of urea and glycerol. Substitutions at the hydrophobic core detectably altered the conformation of the protein, even in the folded state. The introduction of a charged residue, such as Arg, inside the hydrophobic interior of a protein could dramatically alter the structural properties, even those of the unfolded state. The data suggest that a charge at an internal position can interfere with the formation of transient hydrophobic clusters in the unfolded state, and ensure that the pressure-unfolded form of a protein occupies the maximum volume possible. Only at high temperatures does the radius of gyration of the unfolded state ensemble approach the value for a statistical random coil.
Co-reporter:Christopher Rosin, Mirko Erlkamp, Julian von der Ecken, Stefan Raunser, Roland Winter
Biophysical Journal (16 December 2014) Volume 107(Issue 12) pp:
Publication Date(Web):16 December 2014
DOI:10.1016/j.bpj.2014.11.006
Actin is the main component of the microfilament system in eukaryotic cells and can be found in distinct morphological states. Global (G)-actin is able to assemble into highly organized, supramolecular cellular structures known as filamentous (F)-actin and bundled (B)-actin. To evaluate the structure and stability of G-, F-, and B-actin over a wide range of temperatures and pressures, we used Fourier transform infrared spectroscopy in combination with differential scanning and pressure perturbation calorimetry, small-angle x-ray scattering, laser confocal scanning microscopy, and transmission electron microscopy. Our analysis was designed to provide new (to our knowledge) insights into the stabilizing forces of actin self-assembly and to reveal the stability of the actin polymorphs, including in conditions encountered in extreme environments. In addition, we sought to explain the limited pressure stability of actin self-assembly observed in vivo. G-actin is not only the least temperature-stable but also the least pressure-stable actin species. Under abyssal conditions, where temperatures as low as 1–4°C and pressures up to 1 kbar are reached, G-actin is hardly stable. However, the supramolecular assemblies of actin are stable enough to withstand the extreme conditions usually encountered on Earth. Beyond ∼3–4 kbar, filamentous structures disassemble, and beyond ∼4 kbar, complete dissociation of F-actin structures is observed. Between ∼1 and 2 kbar, some disordering of actin assemblies commences, in agreement with in vivo observations. The limited pressure stability of the monomeric building block seems to be responsible for the suppression of actin assembly in the kbar pressure range.
Co-reporter:Benjamin Sperlich, Shobhna Kapoor, Herbert Waldmann, Roland Winter, Katrin Weise
Biophysical Journal (12 July 2016) Volume 111(Issue 1) pp:
Publication Date(Web):12 July 2016
DOI:10.1016/j.bpj.2016.05.042
K-Ras4B is a membrane-bound small GTPase with a prominent role in cancer development. It contains a polybasic farnesylated C-terminus that is required for the correct localization and clustering of K-Ras4B in distinct membrane domains. PDEδ and the Ca2+-binding protein calmodulin (CaM) are known to function as potential binding partners for farnesylated Ras proteins. However, they differ in the number of interaction sites with K-Ras4B, leading to different modes of interaction, and thus affect the subcellular distribution of K-Ras4B in different ways. Although it is clear that Ca2+-bound CaM can play a role in the dynamic spatial cycle of K-Ras4B in the cell, the exact molecular mechanism is only partially understood. In this biophysical study, we investigated the effect of Ca2+/CaM on the interaction of GDP- and GTP-loaded K-Ras4B with heterogeneous model biomembranes by using a combination of different spectroscopic and imaging techniques. The results show that Ca2+/CaM is able to extract K-Ras4B from negatively charged membranes in a nucleotide-independent manner. Moreover, the data demonstrate that the complex of Ca2+/CaM and K-Ras4B is stable in the presence of anionic membranes and shows no membrane binding. Finally, the influence of Ca2+/CaM on the interaction of K-Ras4B with membranes is compared with that of PDEδ, which was investigated in a previous study. Although both CaM and PDEδ exhibit a hydrophobic binding pocket for farnesyl, they have different effects on membrane binding of K-Ras4B and hence should be capable of regulating K-Ras4B plasma membrane localization in the cell.
Co-reporter:Janine Seeliger, Katrin Weise, Norbert Opitz, Roland Winter
Journal of Molecular Biology (10 August 2012) Volume 421(Issues 2–3) pp:348-363
Publication Date(Web):10 August 2012
DOI:10.1016/j.jmb.2012.01.048
Fibrillar aggregates of the islet amyloid polypeptide (IAPP) and amyloid-β (Aβ) are known to deposit at pancreatic β-cells and neuronal cells and are associated with the cell degenerative diseases type-2 diabetes mellitus (T2DM) and Alzheimer's disease (AD), respectively. Since IAPP is secreted by β-cells and a membrane-damaging effect of IAPP has been discussed as a reason for β-cell dysfunction and the development of T2DM, studies of the interaction of IAPP with the β-cell membrane are of high relevance for gaining a molecular-level understanding of the underlying mechanism. Recently, it has also been shown that patients suffering from T2DM exhibit an increased risk to develop AD and vice versa, and a molecular link between AD and T2DM has been suggested. In this study, membrane lipids from the rat insulinoma-derived INS-1E β-cell line were isolated, and their interaction with the amyloidogenic peptides IAPP and Aβ and a mixture of both peptides has been studied. To yield insight into the associated peptides' conformational changes and their effect on the membrane integrity during aggregation, we have carried out attenuated total reflection Fourier transform infrared spectroscopy, fluorescence microscopy, and atomic force microscopy experiments. The IAPP–Aβ heterocomplexes formed were shown to adsorb, aggregate, and permeabilize the isolated β-cell membrane significantly slower than pure IAPP, however, at a rate that is much faster than that of pure Aβ. In addition, it could be shown that isolated β-cell membranes cause similar effects on the kinetics of IAPP and IAPP–Aβ fibril formation as anionic heterogeneous model membranes.Download high-res image (199KB)Download full-size imageHighlights► Isolation and characterization of membrane lipids from a β-cell line of rat. ► Interaction studies of IAPP, Aβ, and both peptides with isolated β-cell membranes. ► Detection of IAPP–Aβ heterocomplex formation in the peptide mixture. ► Different properties of the heterocomplexes compared to the individual peptides. ► IAPP–Aβ heterocomplexes still permeabilize the isolated β-cell membranes, at a drastically reduced rate, however.
Co-reporter:Elena Decaneto, Saba Suladze, Christopher Rosin, Martina Havenith, Wolfgang Lubitz, Roland Winter
Biophysical Journal (1 December 2015) Volume 109(Issue 11) pp:
Publication Date(Web):1 December 2015
DOI:10.1016/j.bpj.2015.10.023
Membrane type 1-matrix metalloproteinase (MT1-MMP or MMP-14) is a zinc-transmembrane metalloprotease involved in the degradation of extracellular matrix and tumor invasion. While changes in solvation of MT1-MMP have been recently studied, little is known about the structural and energetic changes associated with MT1-MMP while interacting with substrates. Steady-state kinetic and thermodynamic data (including activation energies and activation volumes) were measured over a wide range of temperatures and pressures by means of a stopped-flow fluorescence technique. Complementary temperature- and pressure-dependent Fourier-transform infrared measurements provided corresponding structural information of the protein. MT1-MMP is stable and active over a wide range of temperatures (10–55°C). A small conformational change was detected at 37°C, which is responsible for the change in activity observed at the same temperature. Pressure decreases the enzymatic activity until complete inactivation occurs at 2 kbar. The inactivation is associated with changes in the rate-limiting step of the reaction caused by additional hydration of the active site upon compression and/or minor conformational changes in the active site region. Based on these data, an energy and volume diagram could be established for the various steps of the enzymatic reaction.
Co-reporter:Saba Suladze, Marie Kahse, Nelli Erwin, Daniel Tomazic, Roland Winter
Methods (1 April 2015) Volume 76() pp:67-77
Publication Date(Web):1 April 2015
DOI:10.1016/j.ymeth.2014.08.007
Pressure perturbation calorimetry (PPC) is an efficient technique to study the volumetric properties of biomolecules in solution. In PPC, the coefficient of thermal expansion of the partial volume of the biomolecule is deduced from the heat consumed or produced after small isothermal pressure-jumps. The expansion coefficient strongly depends on the interaction of the biomolecule with the solvent or cosolvent as well as on its packing and internal dynamic properties. This technique, complemented with molecular acoustics and densimetry, provides valuable insights into the basic thermodynamic properties of solvation and volume effects accompanying interactions, reactions and phase transitions of biomolecular systems. After outlining the principles of the technique, we present representative examples on protein folding, including effects of cosolvents and crowding, together with a discussion of the interpretation, and further applications.
Co-reporter:Katrin Weise ; Shobhna Kapoor ; Christian Denter ; Jörg Nikolaus ; Norbert Opitz ; Sebastian Koch ; Gemma Triola ; Andreas Herrmann ; Herbert Waldmann
Journal of the American Chemical Society () pp:
Publication Date(Web):December 9, 2010
DOI:10.1021/ja107532q
The K-Ras4B GTPase is a major oncoprotein whose sig-naling activity depends on its correct localization to negatively charged subcellular membranes and nanoclustering in membrane microdomains. Selective localization and clustering are mediated by the polybasic farnesylated C-terminus of K-Ras4B, but the mechanisms and molecular determinants involved are largely unknown. In a combined chemical biological and biophysical approach we investigated the partitioning of semisynthetic fully functional lipidated K-Ras4B proteins into heterogeneous anionic model membranes and membranes composed of viral lipid extracts. Independent of GDP/GTP-loading, K-Ras4B is preferentially localized in liquid-disordered (ld) lipid domains and forms new protein-containing fluid domains that are recruiting multivalent acidic lipids by an effective, electrostatic lipid sorting mechanism. In addition, GDP-GTP exchange and, thereby, Ras activation results in a higher concentration of activated K-Ras4B in the nanoscale signaling platforms. Conversely, palmitoylated and farnesylated N-Ras proteins partition into the ld phase and concentrate at the ld/lo phase boundary of heterogeneous membranes. Next to the lipid anchor system, the results reveal an involvement of the G-domain in the membrane interaction process by determining minor but yet significant structural reorientations of the GDP/GTP-K-Ras4B proteins at lipid interfaces. A molecular mechanism for isoform-specific Ras signaling from separate membrane microdomains is postulated from the results of this study.
Co-reporter:S. R. Al-Ayoubi, P. H. Schummel, M. Golub, J. Peters and R. Winter
Physical Chemistry Chemical Physics 2017 - vol. 19(Issue 22) pp:NaN14237-14237
Publication Date(Web):2017/04/20
DOI:10.1039/C7CP00705A
We studied the effects of temperature and hydrostatic pressure on the dynamical properties and folding stability of highly concentrated lysozyme solutions in the absence and presence of the osmolytes trimethylamine-N-oxide (TMAO) and urea. Elastic incoherent neutron scattering (EINS) was applied to determine the mean-squared displacement (MSD) of the protein's hydrogen atoms to yield insights into the effects of these cosolvents on the averaged sub-nanosecond dynamics in the pressure range from ambient up to 4000 bar. To evaluate the additional effect of self-crowding, two protein concentrations (80 and 160 mg mL−1) were used. We observed a distinct effect of TMAO on the internal hydrogen dynamics, namely a reduced mobility. Urea, on the other hand, revealed no marked effect and consequently, no counteracting effect in an urea–TMAO mixture was observed. Different from the less concentrated protein solution, no significant effect of pressure on the MSD was observed for 160 mg mL−1 lysozyme. The EINS experiments were complemented by Fourier-transform infrared (FTIR) spectroscopy measurements, which led to additional insights into the folding stability of lysozyme under the various environmental conditions. We observed a stabilization of the protein in the presence of the compatible osmolyte TMAO and a destabilization in the presence of urea against temperature and pressure for both protein concentrations. Additionally, we noticed a slight destabilizing effect upon self-crowding at very high protein concentration (160 mg mL−1), which is attributable to transient destabilizing intermolecular interactions. Furthermore, a pressure–temperature diagram could be obtained for lysozyme at these high protein concentrations that mimics densely packed intracellular conditions.
Co-reporter:Martin A. Schroer, Metin Tolan and Roland Winter
Physical Chemistry Chemical Physics 2012 - vol. 14(Issue 26) pp:NaN9491-9491
Publication Date(Web):2012/05/02
DOI:10.1039/C2CP41041A
Using small-angle X-ray scattering data of concentrated solutions of the protein lysozyme taken at different pressures and temperatures, the isothermal pressure derivative and the isobaric temperature derivative of the structure factor S(q) were determined. The pressure derivative of S(q) allows us to test various models for the triplet correlation function g3. Significant differences were found in comparison to simple liquids reflecting the more complex interaction potential in dense protein solutions.
Co-reporter:Janine Seeliger, Kathrin Estel, Nelli Erwin and Roland Winter
Physical Chemistry Chemical Physics 2013 - vol. 15(Issue 23) pp:NaN8907-8907
Publication Date(Web):2013/03/04
DOI:10.1039/C3CP44412K
Owing to the presence of various types of osmolytes in the cellular environment, this study focuses on the impact of stabilizing (TMAO and betaine) as well as destabilizing (urea) cosolvents on the aggregation and fibrillation reaction of the highly amyloidogenic islet amyloid polypeptide (IAPP). IAPP is associated with type-2 diabetes mellitus and is responsible for the disease accompanying β-cell membrane permeabilization and final β-cell loss. To reveal the impact of the cosolvents on the aggregation kinetics, conformational and morphological changes upon IAPP fibrillation, Thioflavin T fluorescence spectroscopy, atomic force microscopy and attenuated total reflection Fourier-transform infrared spectroscopy were applied. For TMAO, and less pronounced for betaine, a decrease of the growth rate of fibrils is observed, whereas the lag phase remains essentially unchanged, indicating the ability of the compatible solutes to stabilize large oligomeric and protofibrillar structures and therefore hamper fibril elongation. Conversely, urea displays concentration-dependent prolongation of the lag phase, indicating stabilization of IAPP in its unfolded monomeric state, hence leading to retardation of IAPP nuclei formation. Mixtures of urea with TMAO, and to a lesser extent with betaine, exhibit a counteractive effect. TMAO is able to fully compensate the prolonged lag phase induced by urea. This strongly matches the findings of a counteraction of TMAO and urea in protein folding and unfolding experiments. The data also reveal that the influence of these cosolvents is only on the aggregation kinetics without markedly changing the final IAPP fibrillar morphology, i.e., the solution structure and cosolvent composition essentially affect the kinetics of the fibrillation process only.
Co-reporter:Johannes Möller, Sebastian Grobelny, Julian Schulze, Andre Steffen, Steffen Bieder, Michael Paulus, Metin Tolan and Roland Winter
Physical Chemistry Chemical Physics 2014 - vol. 16(Issue 16) pp:NaN7429-7429
Publication Date(Web):2014/03/05
DOI:10.1039/C3CP55278K
We present a study on ion specific effects on the intermolecular interaction potential V(r) of dense protein solutions under high hydrostatic pressure conditions. Small-angle X-ray scattering in combination with a liquid-state theoretical approach was used to determine the effect of structure breaking/making salt anions (Cl−, SO42−, PO43−) on the intermolecular interaction of lysozyme molecules. It was found that besides the Debye–Hückel charge screening effect, reducing the repulsiveness of the interaction potential V(r) at low salt concentrations, a specific ion effect is observed at high salt concentrations for the multivalent kosmotropic anions, which modulates also the pressure dependence of the protein–protein interaction potential. Whereas sulfate and phosphate strongly influence the pressure dependence of V(r), chloride anions do not. The strong structure-making effect of the multivalent anions, dominating for the triply charged PO43−, renders the solution structure less bulk-water-like at high salt concentrations, which leads to an altered behavior of the pressure dependence of V(r). Hence, the particular structural properties of the salt solutions are able to influence the spatial organization and the intermolecular interactions of the proteins, in particular upon compression. These results are of interest for exploring the combined effects of ionic strength, temperature and pressure on the phase behavior of protein solutions, but may also be of relevance for understanding pressure effects on the hydration behavior of biological matter under extreme environmental conditions.
Co-reporter:M. Erlkamp, S. Grobelny and R. Winter
Physical Chemistry Chemical Physics 2014 - vol. 16(Issue 13) pp:NaN5976-5976
Publication Date(Web):2014/02/03
DOI:10.1039/C3CP55040K
FT-IR spectroscopic, small-angle X-ray scattering and calorimetric measurements have been applied to explore the effect of the macromolecular crowder agent Ficoll on the temperature- and pressure-dependent stability diagram and folding reaction of the protein Staphylococcal Nuclease (SNase). Additionally, we compare the experimental data with approximate theoretical predictions. We found that temperature- and pressure-induced equilibrium unfolding of SNase is markedly shifted to higher temperatures and pressures in 30 wt% Ficoll solutions. The structure of the unfolded state ensemble does not seem to be strongly influenced in the presence of the crowder. Self-crowding effects have been found to become important at SNase concentrations above 10 wt% only. Our kinetic results show that the folding rate of SNase decreases markedly in the presence of Ficoll. These results indicate that besides the commonly encountered excluded volume effect, other factors need to be considered when assessing confinement effects on protein folding kinetics. Among those, crowder-induced viscosity changes seem to be prominent.
Co-reporter:Trung Quan Luong and Roland Winter
Physical Chemistry Chemical Physics 2015 - vol. 17(Issue 35) pp:NaN23278-23278
Publication Date(Web):2015/08/12
DOI:10.1039/C5CP03529E
We investigated the combined effects of cosolvents and pressure on the hydrolysis of a model peptide catalysed by α-chymotrypsin. The enzymatic activity was measured in the pressure range from 0.1 to 200 MPa using a high-pressure stopped-flow systems with 10 ms time resolution. A kosmotropic (trimethalymine-N-oxide, TMAO) and chaotropic (urea) cosolvent and mixtures thereof were used as cosolvents. High pressure enhances the hydrolysis rate as a consequence of a negative activation volume, ΔV#, which, depending on the cosolvent system, amounts to −2 to −4 mL mol−1. A more negative activation volume can be explained by a smaller compression of the ES complex relative to the transition state. Kinetic constants, such as kcat and the Michaelis constant KM, were determined for all solution conditions as a function of pressure. With increasing pressure, kcat increases by about 35% and its pressure dependence by a factor of 1.9 upon addition of 2 M urea, whereas 1 M TMAO has no significant effect on kcat and its pressure dependence. Similarly, KM increases upon addition of urea 6-fold. Addition of TMAO compensates the urea-effect on kcat and KM to some extent. The maximum rate of the enzymatic reaction increases with increasing pressure in all solutions except in the TMAO:urea 1:2 mixture, where, remarkably, pressure is found to have no effect on the rate of the enzymatic reaction anymore. Our data clearly show that compatible solutes can easily override deleterious effects of harsh environmental conditions, such as high hydrostatic pressures in the 100 MPa range, which is the maximum pressure encountered in the deep biosphere on Earth.
Co-reporter:Janine Seeliger, Nelli Erwin, Christopher Rosin, Marie Kahse, Katrin Weise and Roland Winter
Physical Chemistry Chemical Physics 2015 - vol. 17(Issue 11) pp:NaN7513-7513
Publication Date(Web):2015/02/12
DOI:10.1039/C4CP05845C
Not only drastic temperature- but also pressure-induced perturbations of membrane organization pose a serious challenge to the biological cell. Although high hydrostatic pressure significantly influences the structural properties and thus functional characteristics of cells, this has not prevented life from invading the high pressure habitats of marine depths where pressures up to the 100 MPa level are encountered. Here, the temperature- and pressure-dependent structure and phase behavior of giant plasma membrane vesicles have been explored in the absence and presence of membrane proteins using a combined spectroscopic and microscopic approach. Demixing into extended liquid-ordered and liquid-disordered domains is observed over a wide range of temperatures and pressures. Only at pressures beyond 200 MPa a physiologically unfavorable all gel-like ordered lipid phase is reached at ambient temperature. This is in fact the pressure range where the membrane–protein function has generally been observed to cease, thereby shedding new light on the possible origin of this observation.
Co-reporter:Vladimir P. Voloshin, Nikolai N. Medvedev, Nikolai Smolin, Alfons Geiger and Roland Winter
Physical Chemistry Chemical Physics 2015 - vol. 17(Issue 13) pp:NaN8508-8508
Publication Date(Web):2015/02/09
DOI:10.1039/C5CP00251F
Understanding the physical basis of the structure, stability and function of proteins in solution, including extreme environmental conditions, requires knowledge of their temperature and pressure dependent volumetric properties. One physical–chemical property of proteins that is still little understood is their partial molar volume and its dependence on temperature and pressure. We used molecular dynamics simulations of aqueous solutions of a typical monomeric folded protein, staphylococcal nuclease (SNase), to study and analyze the pressure dependence of the apparent volume, Vapp, and its components by the Voronoi–Delaunay method. We show that the strong decrease of Vapp with pressure (βT = 0.95 × 10−5 bar−1, in very good agreement with the experimental value) is essentially due to the compression of the molecular volume, VM, ultimately, of its internal voids, VemptyM. Changes of the intrinsic volume (defined as the Voronoi volume of the molecule), the contribution of the solvent to the apparent volume, and of the contribution of the boundary voids between the protein and the solvent have also been studied and quantified in detail. The pressure dependences of the volumetric characteristics obtained are compared with the temperature dependent behavior of these quantities and with corresponding results for a natively unfolded polypeptide.
Co-reporter:Christopher Rosin, Paul Hendrik Schummel and Roland Winter
Physical Chemistry Chemical Physics 2015 - vol. 17(Issue 13) pp:NaN8337-8337
Publication Date(Web):2014/11/07
DOI:10.1039/C4CP04431B
We studied the effects of kosmotropic and chaotropic cosolvents, trimethylamine-N-oxide (TMAO) and urea, as well as crowding agents (dextran) on the polymerization reaction of actin. Time-lapse fluorescence intensity and anisotropy experiments were carried out to yield information about the kinetics of the polymerization process. To also quantitatively describe the effects, cosolvents and crowding impose on the underlying rate constants of the G-to-F-transformation, an integrative stochastic simulation model was applied. Drastic and diverse changes in the lag phase and association rates as well as the critical actin concentration were observed under different solvent conditions. The association rate constant is drastically increased by TMAO but decreased by urea. In mixtures of these osmolytes, TMAO counteracts not only the deleterious effect of urea on protein structure and stability, but also on the protein–protein interactions in the course of actin polymerization. Owing to the excluded volume effect, cell-like macromolecular crowding conditions increase the nucleation and association rates by one order of magnitude. Our results clearly reveal the pronounced sensitivity of the actin polymerization reaction to changes in cosolvent conditions and the presence of macromolecular crowding, and suggest that such effects should be taken into account in any discussion of the actin polymerization reaction in vivo.
Co-reporter:Julian Schulze, Johannes Möller, Jonathan Weine, Karin Julius, Nico König, Julia Nase, Michael Paulus, Metin Tolan and Roland Winter
Physical Chemistry Chemical Physics 2016 - vol. 18(Issue 21) pp:NaN14256-14256
Publication Date(Web):2016/05/05
DOI:10.1039/C6CP01791F
We present results from small-angle X-ray scattering and turbidity measurements on the effect of high hydrostatic pressure on the phase behavior of dense lysozyme solutions in the liquid–liquid phase separation region, and characterize the underlying intermolecular protein–protein interactions as a function of temperature and pressure under charge-screening conditions (0.5 M NaCl). A reentrant liquid–liquid phase separation region is observed at elevated pressures, which may originate in the pressure dependence of the solvent-mediated protein–protein interaction. A temperature-pressure-concentration phase diagram was constructed for highly concentrated lysozyme solutions over a wide range of temperatures, pressures and protein concentrations including the critical region of the liquid–liquid miscibility gap.
Co-reporter:Nelli Erwin, Benjamin Sperlich, Guillaume Garivet, Herbert Waldmann, Katrin Weise and Roland Winter
Physical Chemistry Chemical Physics 2016 - vol. 18(Issue 13) pp:NaN8962-8962
Publication Date(Web):2016/02/26
DOI:10.1039/C6CP00563B
In a combined chemical-biological and biophysical approach we explored the membrane partitioning of the lipidated signaling proteins N-Ras and K-Ras4B into membrane systems of different complexity, ranging from one-component lipid bilayers and anionic binary and ternary heterogeneous membrane systems even up to partitioning studies on protein-free and protein-containing giant plasma membrane vesicles (GPMVs). To yield a pictorial view of the localization process, imaging using confocal laser scanning and atomic force microscopy was performed. The results reveal pronounced isoform-specific differences regarding the lateral distribution and formation of protein-rich membrane domains. Line tension is one of the key parameters controlling not only the size and dynamic properties of segregated lipid domains but also the partitioning process of N-Ras that acts as a lineactant. The formation of N-Ras protein clusters is even recorded for almost vanishing hydrophobic mismatch. Conversely, for K-Ras4B, selective localization and clustering are electrostatically mediated by its polybasic farnesylated C-terminus. The formation of K-Ras4B clusters is also observed for the multi-component GPMV membrane, i.e., it seems to be a general phenomenon, largely independent of the details of the membrane composition, including the anionic charge density of lipid headgroups. Our data indicate that unspecific and entropy-driven membrane-mediated interactions play a major role in the partitioning behavior, thus relaxing the need for a multitude of fine-tuned interactions. Such a scenario seems also to be reasonable recalling the high dynamic nature of cellular membranes. Finally, we note that even relatively simple models of heterogeneous membranes are able to reproduce many of the properties of much more complex biological membranes.
Co-reporter:Nelli Erwin, Satyajit Patra and Roland Winter
Physical Chemistry Chemical Physics 2016 - vol. 18(Issue 43) pp:NaN30028-30028
Publication Date(Web):2016/10/17
DOI:10.1039/C6CP06553H
The ubiquitous Ca2+-sensing protein calmodulin (CaM) interacts with more than 300 diverse target proteins that are involved in numerous signaling pathways in eukaryotic cells. This unique promiscuous target binding behavior and the underlying functional versatility of CaM is a result of its structural flexibility. CaM spans multiple conformational substates in solution providing adaptable binding surfaces for different target proteins. The conformational space of this protein needs to be explored to shed more light on the mechanism of target recognition and protein function. Here, we used pressure modulation in combination with FTIR spectroscopy to populate and probe otherwise transient low-lying excited conformational substates of CaM close in energy to its ground state, which are supposed to be functionally relevant in recognition and ligand binding events. The pressure-induced conformational changes of CaM were studied in its Ca2+-free and Ca2+-bound state and in the presence of the hypervariable region (HVR) of the signaling peptide K-Ras4B as a binding partner. We demonstrate that the conformational dynamics of CaM is vastly affected by binding of both Ca2+ ions and the lipidated signaling peptide K-Ras4B. Moreover, we could uncover conformational substates of CaM by pressure perturbation that are partially unfolded and more solvated and conceivably facilitate target recognition by exposing the required binding surfaces.
Co-reporter:Nikolai Smolin, Vladimir P. Voloshin, Alexey V. Anikeenko, Alfons Geiger, Roland Winter and Nikolai N. Medvedev
Physical Chemistry Chemical Physics 2017 - vol. 19(Issue 9) pp:NaN6357-6357
Publication Date(Web):2017/01/13
DOI:10.1039/C6CP07903B
We performed all-atom MD simulations of the protein SNase in aqueous solution and in the presence of two major osmolytes, trimethylamine-N-oxide (TMAO) and urea, as cosolvents at various concentrations and compositions and at different pressures and temperatures. The distributions of the cosolvent molecules and their orientation in the surroundings of the protein were analyzed in great detail. The distribution of urea is largely conserved near the protein. It varies little with pressure and temperature, and does practically not depend on the addition of TMAO. The slight decrease with temperature of the number of urea molecules that are in contact with the SNase molecule is consistent with the view that the interaction of the protein with urea is mainly of enthalpic nature. Most of the TMAO molecules tend to be oriented to the protein by its methyl groups, a small amount of these molecules contact the protein by its oxygen, forming hydrogen bonds with the protein, only. Unlike urea, the fraction of TMAO in the hydration shell of SNase slightly increases with temperature (a signature of a prevailing hydrophobic interaction between TMAO and SNase), and decreases significantly upon the addition of urea. This behavior reflects the diverse nature of the interaction of the two osmolytes with the protein. Using the Voronoi volume of the atoms of the solvent molecules (water, urea, TMAO), we compared the fraction of the volume occupied by a given type of solvent molecule in the hydration shell and in the bulk solvent. The volume fraction of urea in the hydration shell is more than two times larger than in the bulk, whereas the volume fraction of TMAO in the hydration shell is only slightly larger in the binary solvent (TMAO + water) and becomes even less than in the bulk in the ternary solvent (TMAO + water + urea). Thus, TMAO tends to be excluded from the hydration shell of the protein. The behavior of the two cosolvents in the vicinity of the protein does not change much with pressure (from 1 to 5000 bar) and temperature (from 280 to 330 K). This is also in line with the conception of the “osmophobic effect” of TMAO to protect proteins from denaturation also at harsh environmental conditions. We also calculated the volumetric parameters of SNase and found that the cosolvents have a small but significant effect on the apparent volume and its contributions, i.e. the intrinsic, molecular and thermal volumes.
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