Co-reporter:Dr. Chun-Wei Lin;Bruk Mensa;Marta Barniol-Xicota; Dr. William F. DeGrado; Dr. Feng Gai
Angewandte Chemie International Edition 2017 Volume 56(Issue 19) pp:5283-5287
Publication Date(Web):2017/05/02
DOI:10.1002/anie.201701874
AbstractBecause of its importance in viral replication, the M2 proton channel of the influenza A virus has been the focus of many studies. Although we now know a great deal about the structural architecture underlying its proton conduction function, we know little about its conformational dynamics, especially those controlling the rate of this action. Herein, we employ a single-molecule fluorescence method to assess the dynamics of the inter-helical channel motion of both full-length M2 and the transmembrane domain of M2. The rate of this motion depends not only on the identity of the channel and membrane composition but also on the pH in a sigmoidal manner. For the full-length M2 channel, the rate is increased from approximately 190 μs−1 at high pH to approximately 80 μs−1 at low pH, with a transition midpoint at pH 6.1. Because the latter value is within the range reported for the conducting pKa value of the His37 tetrad, we believe that this inter-helical motion accompanies proton conduction.
Co-reporter:Rachel M. Abaskharon, Stephen P. Brown, Wenkai Zhang, Jianxin Chen, Amos B. Smith III, Feng Gai
Chemical Physics Letters 2017 Volume 683(Volume 683) pp:
Publication Date(Web):1 September 2017
DOI:10.1016/j.cplett.2017.03.064
•We demonstrate a new protein vibrational probe.•This probe is useful to study electrostatic interactions with aspartate or glutamate.•Using this probe, we show that water may exist in the interior of a small protein.Because of their negatively charged carboxylates, aspartate and glutamate are frequently found at the active or binding site of proteins. However, studying a specific carboxylate in proteins that contain multiple aspartates and/or glutamates via infrared spectroscopy is difficult due to spectral overlap. We show, herein, that isotopic-labeling of the aspartate sidechain can overcome this limitation as the resultant 13COO− asymmetric stretching vibration resides in a transparent region of the protein IR spectrum. Applicability of this site-specific vibrational probe is demonstrated by using it to assess the dynamics of an aspartate ion buried inside a small protein via two-dimensional infrared spectroscopy.Download high-res image (173KB)Download full-size image
Physical Chemistry Chemical Physics 2017 vol. 19(Issue 7) pp:5028-5036
Publication Date(Web):2017/02/15
DOI:10.1039/C6CP08924K
An infrared temperature-jump (T-jump) study by Huang et al. (Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 2788–2793) showed that the conformational relaxation kinetics of an alanine-based α-helical peptide depend not only on the final temperature (Tf) but also on the initial temperature (Ti) when Tf is fixed. Their finding indicates that the folding free energy landscape of this peptide is non-two-state like, allowing for the population of conformational ensembles with different helical lengths and relaxation times in the temperature range of the experiment. Because α-helix folding involves two fundamental events, nucleation and propagation, the results of Huang et al. thus present a unique opportunity to determine their rate constants – a long-sought goal in the study of the helix–coil transition dynamics. Herein, we capitalize on this notion and develop a coarse-grained kinetic model to globally fit the thermal unfolding curve and T-jump kinetic traces of this peptide. Using this strategy, we are able to explicitly determine the microscopic rate constants of the kinetic steps encountered in the nucleation and propagation processes. Our results reveal that the time taken to form one α-helical turn (i.e., an α-helical segment with one helical hydrogen bond) is about 315 ns, whereas the time taken to elongate this nucleus by one residue (or backbone unit) is 5.9 ns, depending on the position of the residue.
Many ions are known to affect the activity, stability, and structural integrity of proteins. Although this effect can be generally
attributed to ion-induced changes in forces that govern protein folding, delineating the underlying mechanism of action still
remains challenging because it requires assessment of all relevant interactions, such as ion–protein, ion–water, and ion–ion
interactions. Herein, we use two unnatural aromatic amino acids and several spectroscopic techniques to examine whether guanidinium
chloride, one of the most commonly used protein denaturants, and tetrapropylammonium chloride can specifically interact with
aromatic side chains. Our results show that tetrapropylammonium, but not guanidinium, can preferentially accumulate around
aromatic residues and that tetrapropylammonium undergoes a transition at ∼1.3 M to form aggregates. We find that similar to
ionic micelles, on one hand, such aggregates can disrupt native hydrophobic interactions, and on the other hand, they can
promote α-helix formation in certain peptides.
Co-reporter:Mary Rose Hilaire, Debopreeti Mukherjee, Thomas Troxler, Feng Gai
Chemical Physics Letters 2017 Volume 685(Volume 685) pp:
Publication Date(Web):1 October 2017
DOI:10.1016/j.cplett.2017.07.038
•We report the photophysical properties of 6 cyanoindoles.•The absorption spectra of 4-, 6-, and 7-cyanoindoles are significantly red-shifted from that of indole.•4-cyanoindole has the longest fluorescence lifetime in water.Several cyanotryptophans have been shown to be useful biological fluorophores. However, how their fluorescence lifetimes vary with solvent has not been examined. In this regard, herein we measure the fluorescence decay kinetics as well as the absorption and emission spectra of six cyanoindoles in different solvents. In particular, we find, among other results, that only 4-cyanoindole affords a long fluorescence lifetime and hence high quantum yield in H2O. Therefore, our measurements provide not only a guide for choosing which cyanotryptophan to use in practice but also data for computational modeling of the substitution effect on the electronic transitions of indole.Download high-res image (70KB)Download full-size image
Co-reporter:Jeffrey M. Rodgers;Rachel M. Abaskharon;Bei Ding;Jianxin Chen;Wenkai Zhang
Physical Chemistry Chemical Physics 2017 vol. 19(Issue 24) pp:16144-16150
Publication Date(Web):2017/06/21
DOI:10.1039/C7CP02442H
The CO/CN stretching vibration arising from a carbonyl/nitrile functional group in various molecular systems has been frequently used to assess, for example, local hydrogen-bonding interactions, among other applications. However, in practice it is not always easy to ascertain whether the carbonyl or nitrile group in question is engaged in such interactions. Herein, we use 4-cyanoindole and cyclopentanone as models to show that, when a fundamental CO or CN stretching mode is involved in Fermi resonance, the underlying vibrational coupling constant (W) is a convenient reporter of the hydrogen-bonding status of the corresponding carbonyl or nitrile group. Specifically, we find that for both groups a W value of 7.7 cm−1 or greater is indicative of their involvement in hydrogen-bonding interactions. Furthermore, we find that, as observed in similar studies, the Fermi resonance coupling leads to quantum beats in the two-dimensional infrared spectra of 4-cyanoindole in isopropanol, with a period of about 1.9 ps.
Physical Chemistry Chemical Physics 2016 vol. 18(Issue 14) pp:9602-9607
Publication Date(Web):08 Mar 2016
DOI:10.1039/C6CP00865H
Disulfide cleavage is one of the major causes underlying ultraviolet (UV) light-induced protein damage. While previous studies have provided strong evidence to support the notion that this process is mediated by photo-induced electron transfer from the excited state of an aromatic residue (e.g., tryptophan) to the disulfide bond, many mechanistic details are still lacking. For example, we do not know how quickly this process occurs in a protein environment. Herein, we design an experiment, which uses the unfolding kinetics of a protein as an observable, to directly assess the kinetics and mechanism of photo-induced disulfide cleavage. Our results show that this disulfide bond cleavage event takes place in ∼2 μs via a mechanism involving electron transfer from the triplet state of a tryptophan (Trp) residue to the disulfide bond. Furthermore, we find that one of the photoproducts of this reaction, a Trp-SR adduct, is formed locally, thus preventing the protein from re-cross-linking. Taken together, these findings suggest that a Trp-disulfide pair could be used as a photo-trigger to initiate protein folding dynamics and control the biological activities of disulfide-containing peptides.
Co-reporter:Wenkai Zhang, Beatrice N. Markiewicz, Rosalie S. Doerksen, Amos B. Smith, III and Feng Gai
Physical Chemistry Chemical Physics 2016 vol. 18(Issue 10) pp:7027-7034
Publication Date(Web):26 Aug 2015
DOI:10.1039/C5CP04413H
Recently it has been suggested that the CN stretching vibration of a tryptophan analog, 5-cyanotryptophan, could be used as an infrared probe of the local environment, especially the hydration status, of tryptophan residues in proteins. However, the factors that influence the frequency of this vibrational mode are not understood. To determine these factors, herein we carried out linear and nonlinear infrared measurements on the CN stretching vibration of the sidechain of 5-cyanotryptophan, 3-methyl-5-cyanoindole, in a series of protic and aprotic solvents. We found that while the CN stretching frequencies obtained in these solvents do not correlate well with any individual Kamlet–Taft solvent parameter, i.e., π* (polarizability), β (hydrogen bond accepting ability), and α (hydrogen bond donating ability), they do however, collapse on a straight line when plotted against σ = π* + β − α. This linear relationship provides a firm indication that both specific interactions, i.e., hydrogen-bonding interactions with the CN (through α) and indole N–H (through β) groups, and non-specific interactions with the molecule (through π*) work together to determine the CN stretching frequency, thus laying a quantitative framework for applying 5-cyanotryptophan to investigate the microscopic environment of proteins in a site-specific manner. Furthermore, two-dimensional and pump–probe infrared measurements revealed that a significant portion (∼31%) of the ground state bleach signal has a decay time constant of ∼12.3 ps, due to an additional vibrational relaxation channel, making it possible to use 5-cyanotryptophan to probe dynamics occurring on a timescale on the order of tens of picoseconds.
Exciton coupling between two chromophores can produce a circular dichroism (CD) couplet that depends on their separation distance, among other factors. Therefore, exciton CD signals arising from aromatic sidechains, especially those of tryptophan (Trp), have been used in various protein conformational studies. However, the long-wavelength component of the commonly used CD couplet produced by a pair of Trp residues is typically located around 230 nm, thereby overlapping significantly with the protein backbone CD signal. This overlap often prevents a direct and quantitative assessment of the Trp CD couplet in question without further spectral analysis. Here, we show that this inconvenience can be alleviated by using a derivative of Trp, 5-cyanotryptophan (TrpCN), as the chromophore. Specifically, through studying a series of peptides that fold into either α-helical or ß-hairpin conformations, we demonstrate that in comparison with the Trp CD couplet, that arising from two TrpCN residues not only is significantly red-shifted but also becomes more intense due to the larger extinction coefficient of the underlying electronic transition. In addition, we show that a pair of p-cyanophenylalanines (PheCN) or a PheCN–TrpCN pair can also produce a distinct exciton CD couplet that can be useful in monitoring conformational changes in proteins.
Co-reporter:Bei Ding, Mary Rose Hilaire, and Feng Gai
The Journal of Physical Chemistry B 2016 Volume 120(Issue 23) pp:5103-5113
Publication Date(Web):May 16, 2016
DOI:10.1021/acs.jpcb.6b03199
While folding or performing functions, a protein can sample a rich set of conformational space. However, experimentally capturing all of the important motions with sufficient detail to allow a mechanistic description of their dynamics is nontrivial since such conformational events often occur over a wide range of time and length scales. Therefore, many methods have been employed to assess protein conformational dynamics, and depending on the nature of the conformational transition in question, some may be more advantageous than others. Herein, we describe our recent efforts, and also those of others, wherever appropriate, to use infrared- and fluorescence-based techniques to interrogate protein folding and functional dynamics. Specifically, we focus on discussing how to use extrinsic spectroscopic probes to enhance the structural resolution of these techniques and how to exploit various cross-linking strategies to acquire dynamic and mechanistic information that was previously difficult to attain.
Co-reporter:Jeffrey M. Rodgers; Wenkai Zhang; Christopher G. Bazewicz; Jianxin Chen; Scott H. Brewer
The Journal of Physical Chemistry Letters 2016 Volume 7(Issue 7) pp:1281-1287
Publication Date(Web):March 18, 2016
DOI:10.1021/acs.jpclett.6b00325
Varying the reduced mass of an oscillator via isotopic substitution provides a convenient means to alter its vibrational frequency and hence has found wide applications. Herein, we show that this method can also help delineate the vibrational relaxation mechanism, using four isotopomers of the unnatural amino acid p-cyano-phenylalanine (Phe-CN) as models. In water, the nitrile stretching frequencies of these isotopomers, Phe-12C14N (1), Phe-12C15N (2), Phe-13C14N (3), and Phe-13C15N (4), are found to be equally separated by ∼27 cm–1, whereas their vibrational lifetimes are determined to be 4.0 ± 0.2 (1), 2.2 ± 0.1 (2), 3.4 ± 0.2 (3), and 7.9 ± 0.5 ps (4), respectively. We find that an empirical relationship that considers the effective reduced mass of CN can accurately account for the observed frequency gaps, while the vibrational lifetime distribution, which suggests an intramolecular relaxation mechanism, can be rationalized by the order-specific density of states near the CN stretching frequency.
Co-reporter:Beatrice N. Markiewicz, Debopreeti Mukherjee, Thomas Troxler, and Feng Gai
The Journal of Physical Chemistry B 2016 Volume 120(Issue 5) pp:936-944
Publication Date(Web):January 19, 2016
DOI:10.1021/acs.jpcb.5b12233
Tryptophan (Trp) fluorescence has been widely used to interrogate the structure, dynamics, and function of proteins. In particular, it provides a convenient and site-specific means to probe a protein’s hydration status and dynamics. Herein, we show that a tryptophan analogue, 5-cyanotryptophan (TrpCN), can also be used for this purpose, but with the benefit of enhanced sensitivity to hydration. This conclusion is reached based on measurements of the static and time-resolved fluorescence properties of 5-cyanoindole, TrpCN, and TrpCN-containing peptides in different solvents, which indicate that upon dehydration the fluorescence quantum yield (QY) and lifetime (τF) of TrpCN undergo a much greater change in comparison to those of Trp. For example, in H2O the QY of TrpCN is less than 0.01, which increases to 0.11 in 1,4-dioxane. Consistently, the fluorescence decay kinetics of TrpCN in H2O are dominated by a 0.4 ns component, whereas in 1,4-dioxane the kinetics are dominated by a 6.0 ns component. The versatile utility of TrpCN as a sensitive fluorescence reporter is further demonstrated in three applications, where we used it (1) to probe the solvent property of a binary mixture consisting of dimethyl sulfoxide and H2O, (2) to monitor the binding interaction of an antimicrobial peptide with lipid membranes, and (3) to differentiate two differently hydrated environments in a folded protein.
Co-reporter:Mary Rose Mintzer, Thomas Troxler and Feng Gai
Physical Chemistry Chemical Physics 2015 vol. 17(Issue 12) pp:7881-7887
Publication Date(Web):2015/02/20
DOI:10.1039/C5CP00050E
The CN stretching frequency and fluorescence quantum yield of p-cyanophenylalanine are sensitive to environment. As such, this unnatural amino acid has found broad applications, ranging from studying how proteins fold to determining the local electric field of membranes. Herein, we demonstrate that the fluorescence of p-cyanophenylalanine can be quenched by selenomethionine through an electron transfer process occurring at short distances, thus further expanding its spectroscopic utility. Using this fluorophore–quencher pair, we are able to show that short polyproline peptides (1–4 prolines) are not rigid; instead, they sample a bimodal conformational distribution.
We expand the spectroscopic utility of a well-known infrared and fluorescence probe, p-cyanophenylalanine, by showing that it can also serve as a pH sensor. This new application is based on the notion that the fluorescence quantum yield of this unnatural amino acid, when placed at or near the N-terminal end of a polypeptide, depends on the protonation status of the N-terminal amino group of the peptide. Using this pH sensor, we are able to determine the N-terminal pKa values of nine tripeptides and also the membrane penetration kinetics of a cell-penetrating peptide. Taken together, these examples demonstrate the applicability of using this unnatural amino acid fluorophore to study pH-dependent biological processes or events that accompany a pH change.
Co-reporter:Rachel M. Abaskharon; Robert M. Culik; G. Andrew Woolley
The Journal of Physical Chemistry Letters 2015 Volume 6(Issue 3) pp:521-526
Publication Date(Web):January 22, 2015
DOI:10.1021/jz502654q
The attempt frequency or prefactor (k0) of the transition-state rate equation of protein folding kinetics has been estimated to be on the order of 106 s–1, which is many orders of magnitude smaller than that of chemical reactions. Herein we use the mini-protein Trp-cage to show that it is possible to significantly increase the value of k0 for a protein folding reaction by rigidifying the transition state. This is achieved by reducing the conformational flexibility of a key structural element (i.e., an α-helix) formed in the transition state via photoisomerization of an azobenzene cross-linker. We find that this strategy not only decreases the folding time of the Trp-cage peptide by more than an order of magnitude (to ∼100 ns at 25 °C) but also exposes parallel folding pathways, allowing us to provide, to the best of our knowledge, the first quantitative assessment of the curvature of the transition-state free-energy surface of a protein.
Co-reporter:Lev Chuntonov, Ileana M. Pazos, Jianqiang Ma, and Feng Gai
The Journal of Physical Chemistry B 2015 Volume 119(Issue 12) pp:4512-4520
Publication Date(Web):March 4, 2015
DOI:10.1021/acs.jpcb.5b00745
It has recently been shown that the ester carbonyl stretching vibration can be used as a sensitive probe of local electrostatic field in molecular systems. To further characterize this vibrational probe and extend its potential applications, we studied the kinetics of chemical exchange between differently hydrogen-bonded (H-bonded) ester carbonyl groups of methyl acetate (MA) and ethyl acetate (EA) in methanol. We found that, while both MA and EA can form zero, one, or two H-bonds with the solvent, the population of the 2hb state in MA is significantly smaller than that in EA. Using a combination of linear and nonlinear infrared measurements and numerical simulations, we further determined the rate constants for the exchange between these differently H-bonded states. We found that for MA the chemical exchange reaction between the two dominant states (i.e., 0hb and 1hb states) has a relaxation rate constant of 0.14 ps–1, whereas for EA the three-state chemical exchange reaction occurs in a predominantly sequential manner with the following relaxation rate constants: 0.11 ps–1 for exchange between 0hb and 1hb states and 0.12 ps–1 for exchange between 1hb and 2hb states.
Co-reporter:Mary Rose Hilaire; Rachel M. Abaskharon
The Journal of Physical Chemistry Letters 2015 Volume 6(Issue 13) pp:2546-2553
Publication Date(Web):June 11, 2015
DOI:10.1021/acs.jpclett.5b00957
The effect of macromolecular crowding on the structure, dynamics, and reactivity of biomolecules is well established and the relevant research has been extensively reviewed. Herein, we focus our discussion on crowding effects arising from small cosolvent molecules and densely packed surface conditions. In addition, we highlight recent efforts that capitalize on the excluded volume effect for various tailored biochemical and biophysical applications. Specifically, we discuss how a targeted increase in local mass density can be exploited to gain insight into the folding dynamics of the protein of interest and how confinement via reverse micelles can be used to study a range of biophysical questions, from protein hydration dynamics to amyloid formation.
Site-selective isotopic labeling of amide carbonyls offers a nonperturbative means to introduce a localized infrared probe into proteins. Although this strategy has been widely used to investigate various biological questions, the dependence of the underlying amide I vibrational frequency on electric fields (or Stark tuning rate) has not been fully determined, which prevents it from being used in a quantitative manner in certain applications. Herein, through the use of experiments and molecular dynamics simulations, the Stark tuning rate of the amide I vibration of an isotopically labeled backbone carbonyl in a transmembrane α-helix is determined to be approximately 1.4 cm−1/(MV/cm). This result provides a quantitative basis for using this vibrational model to assess local electric fields in proteins, among other applications. For instance, by using this value, we are able to show that the backbone region of a dipeptide has a surprisingly low dielectric constant.
Co-reporter:Beatrice N. Markiewicz, Rolando Oyola, Deguo Du, and Feng Gai
Biochemistry 2014 Volume 53(Issue 7) pp:
Publication Date(Web):February 5, 2014
DOI:10.1021/bi401568a
Protein and peptide aggregation is an important issue both in vivo and in vitro. Herein, we examine the aggregation behaviors of two well-studied β-hairpins, Trpzip1 and Trpzip2. Previous studies suggested that Trpzip2 remains monomeric up to a concentration of ∼15 mM whereas Trpzip1 readily aggregates at micromolar concentrations at acidic or neutral pH. This disparity is puzzling considering that these two peptides differ only in their turn sequences (i.e., GN vs NG). We hypothesize that these peptides can aggregate from their folded states via native edge-to-edge interactions and that the Lys8 residue in Trpzip2 is a more effective aggregation gatekeeper, because of a more favorable orientation. In support of this hypothesis, we find that increasing the pH to 13 or replacing Lys8 with a hydrophobic and photolabile Lys analogue, Lys(nvoc), leads to a significant increase in the aggregation propensity of Trpzip2, and that the aggregation of this Trpzip2 mutant can be reversed upon restoring the native Lys side chain via photocleavage of the nvoc moiety. In addition, we find that while both Trpzip1 and Trpzip2 form parallel β-sheet aggregates, the Lys(nvoc) Trpzip2 mutant forms antiparallel β-sheets and more stable fibrils. Taken together, these findings provide another example showing how sensitive peptide and protein aggregation is to minor sequence variation and that it is possible to use a photolabile non-natural amino acid, such as Lys(nvoc), to tune the rate of peptide aggregation and to control fibrillar structure.
Co-reporter:Ileana M. Pazos;Dr. Ayanjeet Ghosh;Dr. Matthew J. Tucker;Dr. Feng Gai
Angewandte Chemie International Edition 2014 Volume 53( Issue 24) pp:6080-6084
Publication Date(Web):
DOI:10.1002/anie.201402011
Abstract
The ability to quantify the local electrostatic environment of proteins and protein/peptide assemblies is key to gaining a microscopic understanding of many biological interactions and processes. Herein, we show that the ester carbonyl stretching vibration of two non-natural amino acids, L-aspartic acid 4-methyl ester and L-glutamic acid 5-methyl ester, is a convenient and sensitive probe in this regard, since its frequency correlates linearly with the local electrostatic field for both hydrogen-bonding and non-hydrogen-bonding environments. We expect that the resultant frequency–electric-field map will find use in various applications. Furthermore, we show that, when situated in a non-hydrogen-bonding environment, this probe can also be used to measure the local dielectric constant (ε). For example, its application to amyloid fibrils formed by Aβ16–22 revealed that the interior of such β-sheet assemblies has an ε value of approximately 5.6.
Co-reporter:Ayanjeet Ghosh, Matthew J. Tucker, and Feng Gai
The Journal of Physical Chemistry B 2014 Volume 118(Issue 28) pp:7799-7805
Publication Date(Web):April 8, 2014
DOI:10.1021/jp411901m
It is well known that histidine is involved in many biological functions due to the structural versatility of its side chain. However, probing the conformational transitions of histidine in proteins, especially those occurring on an ultrafast time scale, is difficult. Herein we show, using a histidine dipeptide as a model, that it is possible to probe the tautomer and protonation status of a histidine residue by measuring the two-dimensional infrared (2D IR) spectrum of its amide I vibrational transition. Specifically, for the histidine dipeptide studied, the amide unit of the histidine gives rise to three spectrally resolvable amide I features at approximately 1630, 1644, and 1656 cm–1, respectively, which, based on measurements at different pH values and frequency calculations, are assigned to a τ tautomer (1630 cm–1 component) and a π tautomer with a hydrated (1644 cm–1 component) or dehydrated (1656 cm–1 component) amide. Because of the intrinsic ultrafast time resolution of 2D IR spectroscopy, we believe that the current approach, when combined with the isotope editing techniques, will be useful in revealing the structural dynamics of key histidine residues in proteins that are important for function.
Co-reporter:Robert M. Culik, Rachel M. Abaskharon, Ileana M. Pazos, and Feng Gai
The Journal of Physical Chemistry B 2014 Volume 118(Issue 39) pp:11455-11461
Publication Date(Web):September 12, 2014
DOI:10.1021/jp508056w
Trifluoroethanol (TFE) is commonly used to induce protein secondary structure, especially α-helix formation. Due to its amphiphilic nature, however, TFE can also self-associate to form micellelike, nanometer-sized clusters. Herein, we hypothesize that such clusters can act as nanocrowders to increase protein folding rates via the excluded volume effect. To test this hypothesis, we measure the conformational relaxation kinetics of an intrinsically disordered protein, the phosphorylated kinase inducible domain (pKID), which forms a helix–turn–helix in TFE solutions. We find that the conformational relaxation rate of pKID displays a rather complex dependence on TFE percentage (v/v): while it first decreases between 0 and 5%, between 5 and 15% the rate increases and then remains relatively unchanged between 15 and 30% and finally decreases again at higher percentages (i.e., 50%). This trend coincides with the fact that TFE clustering is maximized in the range of 15–30%, thus providing validation of our hypothesis. Another line of supporting evidence comes from the observation that the relaxation rate of a monomeric helical peptide, which due to its predominantly local interactions in the folded state is less affected by crowding, does not show a similar TFE dependence.
Co-reporter:Beatrice N. Markiewicz;Robert M. Culik
Science China Chemistry 2014 Volume 57( Issue 12) pp:1615-1624
Publication Date(Web):2014 December
DOI:10.1007/s11426-014-5225-5
Chemical cross-linking provides an effective avenue to reduce the conformational entropy of polypeptide chains and hence has become a popular method to induce or force structural formation in peptides and proteins. Recently, other types of molecular constraints, especially photoresponsive linkers and functional groups, have also found increased use in a wide variety of applications. Herein, we provide a concise review of using various forms of molecular strategies to constrain proteins, thereby stabilizing their native states, gaining insight into their folding mechanisms, and/or providing a handle to trigger a conformational process of interest with light. The applications discussed here cover a wide range of topics, ranging from delineating the details of the protein folding energy landscape to controlling protein assembly and function.
Co-reporter:Beatrice N. Markiewicz, Lijiang Yang, Robert M. Culik, Yi Qin Gao, and Feng Gai
The Journal of Physical Chemistry B 2014 Volume 118(Issue 12) pp:3317-3325
Publication Date(Web):March 10, 2014
DOI:10.1021/jp500774q
Understanding the structural nature of the free energy bottleneck(s) encountered in protein folding is essential to elucidating the underlying dynamics and mechanism. For this reason, several techniques, including Φ-value analysis, have previously been developed to infer the structural characteristics of such high free-energy or transition states. Herein we propose that one (or few) appropriately placed backbone and/or side chain cross-linkers, such as disulfides, could be used to populate a thermodynamically accessible conformational state that mimics the folding transition state. Specifically, we test this hypothesis on a model β-hairpin, Trpzip4, as its folding mechanism has been extensively studied and is well understood. Our results show that cross-linking the two β-strands near the turn region increases the folding rate by an order of magnitude, to about (500 ns)−1, whereas cross-linking the termini results in a hyperstable β-hairpin that has essentially the same folding rate as the uncross-linked peptide. Taken together, these findings suggest that cross-linking is not only a useful strategy to manipulate folding free energy barriers, as shown in other studies, but also, in some cases, it can be used to stabilize a folding transition state analogue and allow for direct assessment of the folding process on the downhill side of the free energy barrier. The calculated free energy landscape of the cross-linked Trpzip4 also supports this picture. An empirical analysis further suggests, when folding of β-hairpins does not involve a significant free energy barrier, the folding time (τ) follows a power law dependence on the number of hydrogen bonds to be formed (nH), namely, τ = τ0nHα, with τ0 = 20 ns and α = 2.3.
Journal of the American Chemical Society 2013 Volume 135(Issue 20) pp:7668-7673
Publication Date(Web):May 5, 2013
DOI:10.1021/ja401473m
Protein folding involves a large number of sequential molecular steps or conformational substates. Thus, experimental characterization of the underlying folding energy landscape for any given protein is difficult. Herein, we present a new method that can be used to determine the major characteristics of the folding energy landscape in question, e.g., to distinguish between activated and barrierless downhill folding scenarios. This method is based on the idea that the conformational relaxation kinetics of different folding mechanisms at a given final condition will show different dependences on the initial condition. We show, using both simulation and experiment, that it is possible to differentiate between disparate kinetic folding models by comparing temperature jump (T-jump) relaxation traces obtained with a fixed final temperature and varied initial temperatures, which effectively varies the initial potential (VIP) of the system of interest. We apply this method (hereafter refer to as VIPT-jump) to two model systems, tryptophan zipper (Trpzip)-2c and BBL, and our results show that BBL exhibits characteristics of barrierless downhill folding, whereas Trpzip-2c folding encounters a free energy barrier. In addition, using the T-jump data of BBL we are able to provide, via Langevin dynamics simulations, a realistic estimate of its conformational diffusion coefficient.
Co-reporter:Ileana M. Pazos, Rachel M. Roesch, Feng Gai
Chemical Physics Letters 2013 Volume 563() pp:93-96
Publication Date(Web):20 March 2013
DOI:10.1016/j.cplett.2013.02.015
To expand the spectroscopic utility of the non-natural amino acid p-cyanophenylalanine (PheCN), we examine the quenching efficiencies of a series of commonly encountered anions toward its fluorescence. We find that iodide exhibits an unusually large Stern–Volmer quenching constant, making it a convenient choice in PheCN fluorescence quenching studies. Indeed, using the villin headpiece subdomain as a testbed we demonstrate that iodide quenching of PheCN fluorescence offers a convenient means to reveal protein conformational heterogeneity. Furthermore, we show that the amino group of PheCN strongly quenches its fluorescence, suggesting that PheCN could be used as a local pH sensor.Graphical abstractHighlights► Iodide is an efficient quencher of PheCN fluorescence. ► Phosphate does not quench fluorescence. ► Quenching of PheCN fluorescence is a useful tool to examine conformational heterogeneity of proteins.
Co-reporter:Robert M. Culik, Srinivas Annavarapu, Vikas Nanda, Feng Gai
Chemical Physics 2013 Volume 422() pp:131-134
Publication Date(Web):30 August 2013
DOI:10.1016/j.chemphys.2013.01.021
Abstract
Using the miniprotein Trp-cage as a model, we show that D-amino acids can be used to facilitate the delineation of protein folding mechanism. Specifically, we study the folding–unfolding kinetics of three Trp-cage mutants where the native glycine residue near the C-terminus of the α-helix is replaced by a D-amino acid. A previous study showed that these mutations increase the Trp-cage stability, due to a terminal capping effect. Our results show that the stabilizing effect of D-asparagine and d-glutamine originates almost exclusively from a decrease in the unfolding rate, while the D-alanine mutation results in a similar decrease in the unfolding rate, but it also increases the folding rate. Together, these results support a folding mechanism wherein the α-helix formation in the transition state is nucleated at the N-terminus, whereas those long-range native interactions stabilizing this helix are developed at the downhill side of the folding free energy barrier.
The Journal of Physical Chemistry A 2013 Volume 117(Issue 29) pp:6164-6170
Publication Date(Web):May 6, 2013
DOI:10.1021/jp4003643
For fluorescence-based single-molecule studies, photobleaching of the dye reporter often limits the time window over which individual molecules can be followed. As such, many strategies, for example, using a cocktail of chemical reagents, have been developed to decrease the rate of photobleaching. Herein, we introduce a new and highly effective method to enhance the photostability of one of the commonly used fluorescent dyes, rhodamine 6G (R6G). We show that micrometer-sized polydimethylsiloxane (PDMS) wells, when the PDMS surface is properly treated, not only provide a confined environment for single-molecule detection but can also significantly increase the survival time of individual R6G molecules before photobleaching. Moreover, our results suggest, consistent with several previous studies, that R6G photobleaching involves a radical state.
Co-reporter:Beatrice N. Markiewicz, Hyunil Jo, Robert M. Culik, William F. DeGrado, and Feng Gai
The Journal of Physical Chemistry B 2013 Volume 117(Issue 47) pp:14688-14696
Publication Date(Web):November 8, 2013
DOI:10.1021/jp409334h
Internal friction arising from local steric hindrance and/or the excluded volume effect plays an important role in controlling not only the dynamics of protein folding but also conformational transitions occurring within the native state potential well. However, experimental assessment of such local friction is difficult because it does not manifest itself as an independent experimental observable. Herein, we demonstrate, using the miniprotein trp-cage as a testbed, that it is possible to selectively increase the local mass density in a protein and hence the magnitude of local friction, thus making its effect directly measurable via folding kinetic studies. Specifically, we show that when a helix cross-linker, m-xylene, is placed near the most congested region of the trp-cage it leads to a significant decrease in both the folding rate (by a factor of 3.8) and unfolding rate (by a factor of 2.5 at 35 °C) but has little effect on protein stability. Thus, these results, in conjunction with those obtained with another cross-linked trp-cage and two uncross-linked variants, demonstrate the feasibility of using a nonperturbing cross-linker to help quantify the effect of internal friction. In addition, we estimate that a m-xylene cross-linker could lead to an increase in the roughness of the folding energy landscape by as much as 0.4–1.0kBT.
Co-reporter:Robert M. Culik ; Hyunil Jo ; William F. DeGrado
Journal of the American Chemical Society 2012 Volume 134(Issue 19) pp:8026-8029
Publication Date(Web):April 27, 2012
DOI:10.1021/ja301681v
Thioamides are sterically almost identical to their oxoamide counterparts, but they are weaker hydrogen bond acceptors. Therefore, thioamide amino acids are excellent candidates for perturbing the energetics of backbone–backbone H-bonds in proteins and hence should be useful in elucidating protein folding mechanisms in a site-specific manner. Herein, we validate this approach by applying it to probe the dynamic role of interstrand H-bond formation in the folding kinetics of a well-studied β-hairpin, tryptophan zipper. Our results show that reducing the strength of the peptide’s backbone–backbone H-bonds, except the one directly next to the β-turn, does not change the folding rate, suggesting that most native interstrand H-bonds in β-hairpins are formed only after the folding transition state.
Co-reporter:Arnaldo L. Serrano, Osman Bilsel, and Feng Gai
The Journal of Physical Chemistry B 2012 Volume 116(Issue 35) pp:10631-10638
Publication Date(Web):August 14, 2012
DOI:10.1021/jp211296e
The villin headpiece subdomain (HP35) has become one of the most widely used model systems in protein folding studies, due to its small size and ultrafast folding kinetics. Here, we use HP35 as a test bed to show that the fluorescence decay kinetics of an unnatural amino acid, p-cyanophenylalanine (PheCN), which are modulated by a nearby quencher (e.g., tryptophan or 7-azatryptophan) through the mechanism of fluorescence resonance energy transfer (FRET), can be used to detect protein conformational heterogeneity. This method is based on the notion that protein conformations having different donor–acceptor distances and interconverting slowly compared to the fluorescence lifetime of the donor (PheCN) would exhibit different donor fluorescence lifetimes. Our results provide strong evidence suggesting that the native free energy basin of HP35 is populated with conformations that differ mostly in the position and mean helicity of the C-terminal helix. This finding is consistent with several previous experimental and computational studies. Moreover, this result holds strong implications for computational investigation of the folding mechanism of HP35.
The Journal of Physical Chemistry B 2012 Volume 116(Issue 41) pp:12473-12478
Publication Date(Web):September 22, 2012
DOI:10.1021/jp307414s
While the thermodynamic effects of trimethylamine oxide (TMAO), urea, and guanidine hydrochloride (GdnHCl) on protein stability are well understood, the underlying mechanisms of action are less well characterized and, in some cases, even under debate. Herein, we employ the stretching vibration of two infrared (IR) reporters, i.e., nitrile (C≡N) and carbonyl (C═O), to directly probe how these cosolvents mediate the ability of water to form hydrogen bonds with the solute of interest, e.g., a peptide. Our results show that these three agents, despite having different effects on protein stability, all act to decrease the strength of the hydrogen bonds formed between water and the infrared probe. While the behavior of TMAO appears to be consistent with its protein-protecting ability, those of urea and GdnHCl are inconsistent with their role as protein denaturants. The latter is of particular interest as it provides strong evidence indicating that although urea and GdnHCl can perturb the hydrogen-bonding property of water their protein-denaturing ability does not arise from a simple indirect mechanism.
There is growing demand for novel methods that could render the controlled disassembly of higher-order structures formed, for example, by peptides. Herein, we demonstrate such a method based on the application of a photocaged variant of the amino acid lysine, namely, lys(Nvoc). Specifically, we introduce lys(Nvoc) into the primary sequence of the amyloidogenic peptide, Aβ16–22, at a position where the native side chain is known to play a key role in fibril formation via hydrophobic interactions. Both AFM and infrared spectroscopic measurements indicate that the resultant Aβ16–22 mutant is able to form fibrils whereas, more importantly, the fibrils thus formed can be completely disassembled upon irradiation with near-UV light, which cleaves the photolabile Nvoc moiety and triggers the restoration of the lysine side chain. These results suggest that the generation of a single charge in a highly hydrophobic region of the fibrils is sufficient to promote their dissociation. Thus, we envisage that the current approach will find useful applications wherein controlled structural disassembly or content release is required.
Co-reporter:Lin Guo, Kathryn B. Smith-Dupont, and Feng Gai
Biochemistry 2011 Volume 50(Issue 12) pp:
Publication Date(Web):February 21, 2011
DOI:10.1021/bi102068j
Recently, we have shown that association with an antimicrobial peptide (AMP) can drastically alter the diffusion behavior of the constituent lipids in model membranes (Biochemistry 49, 4672−4678). In particular, we found that the diffusion time of a tracer fluorescent lipid through a confocal volume measured via fluorescence correlation spectroscopy (FCS) is distributed over a wide range of time scales, indicating the formation of stable and/or transient membrane species that have different mobilities. A simple estimate, however, suggested that the slow diffusing species are too large to be attributed to AMP oligomers or pores that are tightly bound to a small number of lipids. Thus, we tentatively ascribed them to membrane domains and/or clusters that possess distinctively different diffusion properties. In order to further substantiate our previous conjecture, herein we study the diffusion behavior of the membrane-bound peptide molecules using the same AMPs and model membranes. Our results show, in contrast to our previous findings, that the diffusion times of the membrane-bound peptides exhibit a much narrower distribution that is more similar to that of the lipids in peptide-free membranes. Thus, taken together, these results indicate that while AMP molecules prompt domain formation in membranes, they are not tightly associated with the lipid domains thus formed. Instead, they are likely located at the boundary regions separating various domains and acting as mobile fences.
Co-reporter:Matthias M. Waegele, Robert M. Culik, and Feng Gai
The Journal of Physical Chemistry Letters 2011 Volume 2(Issue 20) pp:2598-2609
Publication Date(Web):September 23, 2011
DOI:10.1021/jz201161b
Elucidating the underlying molecular mechanisms of protein folding and function is a very exciting and active research area, but poses significant challenges. This is due in part to the fact that existing experimental techniques are incapable of capturing snapshots along the “reaction coordinate” in question with both sufficient spatial and temporal resolutions. In this regard, recent years have seen increased interests and efforts in the development and employment of site-specific probes to enhance the structural sensitivity of spectroscopic techniques in conformational and dynamical studies of biological molecules. In particular, the spectroscopic and chemical properties of nitriles, thiocyanates, and azides render these groups attractive for the interrogation of complex biochemical constructs and processes. Here, we review their signatures in vibrational, fluorescence, and NMR spectra and their utility in the context of elucidating chemical structure and dynamics of protein and DNA molecules.
Co-reporter:Julie M. G. Rogers, Alexei L. Polishchuk, Lin Guo, Jun Wang, William F. DeGrado, and Feng Gai
Langmuir 2011 Volume 27(Issue 7) pp:3815-3821
Publication Date(Web):March 14, 2011
DOI:10.1021/la200480d
The structure and function of the influenza A M2 proton channel have been the subject of intensive investigations in recent years because of their critical role in the life cycle of the influenza virus. Using a truncated version of the M2 proton channel (i.e., M2TM) as a model, here we show that fluctuations in the fluorescence intensity of a dye reporter that arise from both fluorescence quenching via the mechanism of photoinduced electron transfer (PET) by an adjacent tryptophan (Trp) residue and local motions of the dye molecule can be used to probe the conformational dynamics of membrane proteins. Specifically, we find that the dynamics of the conformational transition between the N-terminal open and C-terminal open states of the M2TM channel occur on a timescale of about 500 μs and that the binding of either amantadine or rimantadine does not inhibit the pH-induced structural equilibrium of the channel. These results are consistent with the direct occluding mechanism of inhibition which suggests that the antiviral drugs act by sterically occluding the channel pore.
Co-reporter:Arnaldo L. Serrano, Matthew J. Tucker, and Feng Gai
The Journal of Physical Chemistry B 2011 Volume 115(Issue 22) pp:7472-7478
Publication Date(Web):May 13, 2011
DOI:10.1021/jp200628b
The nucleation event in α-helix formation is a fundamental process in protein folding. However, determining how quickly it takes place based on measurements of the relaxation dynamics of helical peptides is difficult because such relaxations invariably contain contributions from various structural transitions such as from helical to nonhelical states and helical to partial-helical conformations. Herein, we measure the temperature-jump (T-jump) relaxation kinetics of three model peptides that fold into a single-turn α-helix, using time-resolved infrared spectroscopy, aiming to provide a direct assessment of the helix nucleation rate. The α-helical structure of these peptides is stabilized by a covalent cross-linker formed between the side chains of two residues at the i and i + 4 positions. If we assume that this cross-linker mimics the structural constraint arising from a strong side chain–side chain interaction (e.g., a salt bridge) in proteins, these peptides would represent good models for studying the nucleation process of an α-helix in a protein environment. Indeed, we find that the T-jump induced relaxation rate of these peptides is approximately (0.6 μs)−1 at room temperature, which is slower than that of commonly studied alanine-based helical peptides but faster than that of a naturally occurring α-helix whose folded state is stabilized by a series of side chain–side chain interactions. Taken together, our results put an upper limit of about 1 μs for the helix nucleation time at 20 °C and suggest that the subsequent propagation steps occur with a time constant of about 240 ns.
Co-reporter:Geronda Montalvo ; Matthias M. Waegele ; Scott Shandler ; Feng Gai ;William F. DeGrado
Journal of the American Chemical Society 2010 Volume 132(Issue 16) pp:5616-5618
Publication Date(Web):April 7, 2010
DOI:10.1021/ja100459a
Synthetic foldamers consisting of β-amino acids offer excellent model systems for examining the effect of backbone flexibility on the dynamics of protein folding. Herein, we study the folding−unfolding kinetics of a β-peptide that folds into a 14-helical structure in water. We find that the T-jump induced relaxation kinetics of this peptide occur on the nanosecond time scale and are noticeably slower than those of alanine-based α-helical peptides, and additionally, the relaxation rates show a weaker dependence on temperature. These differences appear to indicate that the folding energy landscapes of these peptides are different. In addition, we find that the amide I′ band of this β-peptide exhibits a sharp feature at ∼1612 cm−1, which we believe is a distinct infrared reporter of 14-helix.
The helical hairpin motif plays a key role as a receptor site in DNA binding and protein−protein interactions. Thus, various helical hairpins have recently been developed to assess the factors that control the DNA and/or protein binding affinities of this structural motif and to form synthetic templates for protein and drug design. In addition, several lines of evidence suggest that rapid acquisition of a helical hairpin structure from the unfolded ensemble may guide the rapid formation of helical proteins. Despite its importance as a crucial structural element in protein folding and binding, the folding mechanism of the helical hairpin motif has not been thoroughly studied. Herein, we investigate the structural determinants of the folding kinetics of a naturally occurring helical hairpin (porcine PYY) that is free of disulfide bonds and metal ion-induced cross-links using an infrared temperature-jump technique. It is found that mutations in the turn region predominantly increase the barrier of folding irrespective of the temperature, whereas the effect of mutations that perturb the hydrophobic interactions between the two helices is temperature-dependent. At low temperatures, deletion of hydrophobic side chains is found to predominantly affect the unfolding rate, while the opposite is observed at high temperatures. These results are interpreted in terms of a folding mechanism in which the turn is formed in the transition state and also based on the assumption that cross-strand hydrophobic contacts exist in the thermally unfolded state of PYY.
Co-reporter:Hyunil Jo, Robert M. Culik, Ivan V. Korendovych, William F. DeGrado, and Feng Gai
Biochemistry 2010 Volume 49(Issue 49) pp:
Publication Date(Web):November 15, 2010
DOI:10.1021/bi101711a
The nitrile stretching vibration is increasingly used as a sensitive infrared probe of local protein environments. However, site-specific incorporation of a nitrile moiety into proteins is difficult. Here we show that various aromatic nitriles can be easily incorporated into peptides and proteins via either thiol alkylation or arylation reaction.
Co-reporter:Kathryn B. Smith-Dupont, Lin Guo and Feng Gai
Biochemistry 2010 Volume 49(Issue 22) pp:
Publication Date(Web):May 10, 2010
DOI:10.1021/bi100426p
Many antimicrobial peptides (AMPs) function by forming various oligomeric structures and/or pores upon binding to bacterial membranes. Because such peptide aggregates are capable of inducing membrane thinning and membrane permeabilization, we expected that AMP binding would also affect the diffusivity or mobility of the lipid molecules in the membrane. Herein, we show that measurements of the diffusion times of individual lipids through a confocal volume via fluorescence correlation spectroscopy (FCS) provide a sensitive means of probing the underlying AMP−membrane interactions. In particular, results obtained with two well-studied AMPs, magainin 2 and mastoparan X, and two model membranes indicate that this method is capable of revealing structural information, especially the heterogeneity of the peptide−membrane system, that is otherwise difficult to obtain using common ensemble methods. Moreover, because of the high sensitivity of FCS, this method allows examination of the effect of AMPs on the membrane structure at very low peptide/lipid ratios.
Co-reporter:Arnaldo L. Serrano, Thomas Troxler, Matthew J. Tucker, Feng Gai
Chemical Physics Letters 2010 Volume 487(4–6) pp:303-306
Publication Date(Web):5 March 2010
DOI:10.1016/j.cplett.2010.01.058
Abstract
The non-natural amino acid p-cyanophenylalanine (PheCN) has recently emerged as a useful fluorescent probe of proteins; however, its photophysical properties have not been systematically examined. Herein, we measure the fluorescence quantum yield and the fluorescence lifetime of PheCN in a series of solvents. It is found that the fluorescence lifetime of PheCN shows a linear dependence on the Kamlet–Taft parameter α of the protic solvents used, indicating that the solute-solvent hydrogen bonding interactions mediate the non-radiative decay rate. Thus, results of this study provide a basis for quantitative application of PheCN fluorescence in protein conformational studies.
Fluorescence resonance energy transfer (FRET) provides a powerful means to study protein conformational changes. However, the incorporation of an exogenous FRET pair into a protein could lead to undesirable structural perturbations of the native fold. One of the viable strategies to minimizing such perturbations is to use non-natural amino acid-based FRET pairs. Previously, we showed that p-cyanophenylalanine (PheCN) and tryptophan (Trp) constitute such a FRET pair, useful for monitoring protein folding–unfolding transitions. Here we further show that 7-azatryptophan (7AW) and 5-hydroxytryptophan (5HW) can also serve as a FRET acceptor to PheCN, and the resultant FRET pairs offer certain advantages over PheCN–Trp. For example, the fluorescence spectrum of 7AW is sufficiently separated from that of PheCN, making it straightforward to decompose the FRET spectrum into donor and acceptor contributions. Moreover, we show that PheCN, Trp, and 7AW can be used together to form a multi-FRET system, allowing more structural information to be extracted from a single FRET experiment. The applicability of these FRET systems is demonstrated in a series of studies where they are employed to monitor the urea-induced unfolding transitions of the villin headpiece subdomain (HP35), a designed ββα motif (BBA5), and the human Pin1 WW domain.
Co-reporter:Michelle R. Bunagan ; Jianmin Gao ; Jeffery W. Kelly
Journal of the American Chemical Society 2009 Volume 131(Issue 21) pp:7470-7476
Publication Date(Web):May 8, 2009
DOI:10.1021/ja901860f
Backbone−backbone hydrogen bonds are a common feature of native protein structures, yet their thermodynamic and kinetic influence on folding has long been debated. This is reflected by the disparity between current protein folding models, which place hydrogen bond formation at different stages along the folding trajectory. For example, previous studies have suggested that the denatured state of the villin headpiece subdomain contains a residual helical structure that may provide a bias toward the folded state by confining the conformational search associated with its folding. Although helical hydrogen bonds clearly stabilize the folded state, here we show, using an amide-to-ester mutation strategy, that the formation of backbone hydrogen bonds within helices is not rate-limiting in the folding of the subdomain, thereby suggesting that such hydrogen bonds are unlikely to be formed en route from the denatured to the transition state. On the other hand, elimination of hydrogen bonds within the turn region elicits a slower folding rate, consistent with the hypothesis that these residues are involved in the formation of a folding nucleus. While illustrating a potentially conserved aspect of helix-turn-helix folding, our results further underscore the inherent importance of turns in protein supersecondary structure formation.
Co-reporter:Matthias M. Waegele, Matthew J. Tucker, Feng Gai
Chemical Physics Letters 2009 Volume 478(4–6) pp:249-253
Publication Date(Web):27 August 2009
DOI:10.1016/j.cplett.2009.07.058
Abstract
The nitrile (CN) stretching vibration is sensitive to environment, making nitrile-derivatized amino acids an increasingly utilized tool to study various biological processes. Herein, we show that the bandwidth of the CN stretching vibration of 5-cyanotryptophan is particularly sensitive to water, rendering it an attractive infrared probe of local hydration status. We confirm the utility of this probe in biological applications by using it to examine how the hydration status of individual tryptophan sidechains of an antimicrobial peptide, indolicidin, changes upon peptide binding to model membranes. Furthermore, we show that p-cyanophenylalanine and 5-cyanotryptophan constitute a useful fluorescence energy transfer pair.
Co-reporter:Smita Mukherjee, Pramit Chowdhury and Feng Gai
The Journal of Physical Chemistry B 2009 Volume 113(Issue 2) pp:531-535
Publication Date(Web):December 19, 2008
DOI:10.1021/jp809817s
It is well-known that water plays a crucial role in the folding, dynamics, and function of proteins. Here we provide further evidence showing that the aggregation kinetics of peptides also depend strongly on their hydration status. Using reverse micelles as a tool to modulate the accessible number of water molecules and infrared spectroscopy and transmission electron microscopy as means to monitor aggregate formation, we show that the rate of aggregation of two amyloid forming peptides increases significantly under conditions where limited hydration of the peptide molecule is expected to occur. These results not only are in accord with recent computer simulations indicating that the expulsion of interfacial water molecules is a key event in the dimerization/oligmerization of amyloid β (Aβ) peptides but also have implications for amyloid formation in vivo where molecular crowding is expected to influence the solvation status of proteins.
Co-reporter:Lin Guo, Jaeheung Park, Taegon Lee, Pramit Chowdhury, Manho Lim and Feng Gai
The Journal of Physical Chemistry B 2009 Volume 113(Issue 17) pp:6158-6163
Publication Date(Web):April 6, 2009
DOI:10.1021/jp900009x
We show that the equilibrium unfolding transition of horse carbonmonoxy myoglobin monitored by the stretching vibration of the CO ligand, a local environmental probe, is very sharp and, thus, quite different from those measured by global conformational reporters. In addition, the denatured protein exhibits an A0-like CO band. We hypothesize that this sharp transition reports penetration of water into the heme pocket of the protein. Parallel experiments on horse apomyoglobin, wherein an environment-sensitive fluorescent probe, nile red, was used, also reveals a similar putative hydration event. Given the importance of dehydration in protein folding and also the recent debate over the interpretation of probe-dependent unfolding transitions, these results have strong implications on the mechanism of protein folding.
Co-reporter:Julie M. Glasscock, Yongjin Zhu, Pramit Chowdhury, Jia Tang and Feng Gai
Biochemistry 2008 Volume 47(Issue 42) pp:
Publication Date(Web):September 25, 2008
DOI:10.1021/bi8012406
Previously, we have shown that p-cyanophenylalanine (PheCN) and tryptophan (Trp) constitute an efficient fluorescence resonance energy transfer (FRET) pair that has several advantages over commonly used dye pairs. Here, we aim to examine the general applicability of this FRET pair in protein folding−unfolding studies by applying it to the urea-induced unfolding transitions of two small proteins, the villin headpiece subdomain (HP35) and the lysin motif (LysM) domain. Depending on whether PheCN is exposed to solvent, we are able to extract either qualitative information about the folding pathway, as demonstrated by HP35, which has been suggested to unfold in a stepwise manner, or quantitative thermodynamic and structural information, as demonstrated by LysM, which has been shown to be an ideal two-state folder. Our results show that the unfolding transition of HP35 reported by FRET occurs at a denaturant concentration lower than that measured by circular dichroism (CD) and that the loop linking helix 2 and helix 3 remains compact in the denatured state, which are consistent with the notion that HP35 unfolds in discrete steps and that its unfolded state contains residual structures. On the other hand, our FRET results on the LysM domain allow us to develop a model for extracting structural and thermodynamic parameters about its unfolding, and we find that our results are in agreement with those obtained by other methods. Given the fact that PheCN is a non-natural amino acid and, thus, amenable to incorporation into peptides and proteins via existing peptide synthesis and protein expression methods, we believe that the FRET method demonstrated here is widely applicable to protein conformational studies, especially to the study of relatively small proteins.
Co-reporter:Smita Mukherjee, Pramit Chowdhury, Michelle R. Bunagan and Feng Gai
The Journal of Physical Chemistry B 2008 Volume 112(Issue 30) pp:9146-9150
Publication Date(Web):July 9, 2008
DOI:10.1021/jp801721p
The folding mechanism and dynamics of a helical protein may strongly depend on how quickly its constituent α-helices can fold independently. Thus, our understanding of the protein folding problem may be greatly enhanced by a systematic survey of the folding rates of individual α-helical segments derived from their parent proteins. As a first step, we have studied the relaxation kinetics of the central helix (L9:41-74) of the ribosomal protein L9 from the bacterium Bacillus stearothermophilus, in response to a temperature-jump (T-jump) using infrared spectroscopy. L9:41-74 has been shown to exhibit unusually high helicity in aqueous solution due to a series of side chain-side chain interactions, most of which are electrostatic in nature, while still remaining monomeric over a wide concentration range. Thus, this peptide represents an excellent model system not only for examining how the folding rate of naturally occurring helices differs from that of the widely studied alanine-based peptides, but also for estimating the folding speed limit of (small) helical proteins. Our results show that the T-jump induced relaxation rate of L9:41-74 is significantly slower than that of alanine-based peptides. For example, at 11 °C its relaxation time constant is about 2 μs, roughly seven times slower than that of SPE5, an alanine-rich peptide of similar chain length. In addition, our results show that the folding rate of a truncated version of L9:41-74 is even slower. Taken together, these results suggest that individual α-helical segments in proteins may fold on a time scale that is significantly slower than the folding time of alanine-based peptides. Furthermore, we argue that the relaxation rate of L9:41-74 measured between 8 and 45 °C provides a realistic estimate of the ultimate folding rate of (small) helical proteins over this temperature range.
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
Co-reporter:Yongjin Zhu, Xiaoran Fu, Ting Wang, Atsuo Tamura, Shoji Takada, Jeffery G. Saven, Feng Gai
Chemical Physics 2004 Volume 307(2–3) pp:99-109
Publication Date(Web):27 December 2004
DOI:10.1016/j.chemphys.2004.05.008
Abstract
Many simple, single-domain proteins fold via first order kinetics, indicative of a single, dominant free energy barrier. Because folding usually involves the burial of hydrophobic side chains, the acquisition of native structure may be associated with a decrease in the heat capacity of the system. If the transition state ensemble involves the burial of hydrophobic residues, the folding rates show a well-known concave downward dependence on temperature, exhibiting a maximum folding rate with respect to temperature. Within the framework of transition state theory, the maximum folding rate for a specific native structure depends simply on the entropic barrier as well as the heat capacity of activation. The latter is related to the mean hydrophobicity when the protein is largely unfrustrated with regard to its stabilizing interactions. As an example, here we show that the maximum folding rate of the three-helix bundle structure of 1prb7–53, the GA module of an albumin binding domain, can indeed be fine-tuned using computational design methods to identify and design structurally consistent mutations that modulate its hydrophobic content. Specifically, we find that the logarithm of the maximal folding rate depends linearly on the mean hydrophobic content of the designed sequences, where faster folding correlates with higher mean hydrophobicity.
Both turn sequence and interstrand hydrophobic side-chain–side-chain interaction have been suggested to be important determinants
of β-hairpin stability. However, their roles in controlling the folding dynamics of β-hairpins have not been clearly determined.
Herein, we investigated the structural stability and folding kinetics of a series of tryptophan zippers by static IR and CD
spectroscopies and the IR temperature jump method. Our results support a β-hairpin folding mechanism wherein the rate-limiting
event corresponds to the formation of the turn. We find that the logarithm of the folding rate depends linearly on the entropic
change associated with the turn formation, where faster folding correlates with lower entropic cost. Moreover, a stronger
turn-promoting sequence increases the stability of a β-hairpin primarily by increasing its folding rate, whereas a stronger
hydrophobic cluster increases the stability of a β-hairpin primarily by decreasing its unfolding rate.
Co-reporter:Zelleka Getahun;Cheng-Yen Huang;Yongjin Zhu;Jason W. Klemke;William F. DeGrado
PNAS 2002 Volume 99 (Issue 5 ) pp:2788-2793
Publication Date(Web):2002-03-05
DOI:10.1073/pnas.052700099
The helix-coil transition kinetics of an α-helical peptide were investigated by time-resolved infrared spectroscopy coupled
with laser-induced temperature-jump initiation method. Specific isotope labeling of the amide carbonyl groups with 13C at selected residues was used to obtain site-specific information. The relaxation kinetics following a temperature jump,
obtained by probing the amide I′ band of the peptide backbone, exhibit nonexponential behavior and are sensitive to both initial
and final temperatures. These data are consistent with a conformation diffusion process on the folding energy landscape, in
accord with a recent molecular dynamics simulation study.
Co-reporter:Cheng Zhu, Ziwei Dai, Huanhuan Liang, Tao Zhang, Feng Gai, Luhua Lai
Biophysical Journal (5 November 2013) Volume 105(Issue 9) pp:
Publication Date(Web):5 November 2013
DOI:10.1016/j.bpj.2013.09.014
De novo protein design offers a unique means to test and advance our understanding of how proteins fold. However, most current design methods are native structure eccentric and folding kinetics has rarely been considered in the design process. Here, we show that a de novo designed mini-protein DS119, which folds into a βαβ structure, exhibits unusually slow and concentration-dependent folding kinetics. For example, the folding time for 50 μM of DS119 was estimated to be ∼2 s. Stopped-flow fluorescence resonance energy transfer experiments further suggested that its folding was likely facilitated by a transient dimerization process. Taken together, these results highlight the need for consideration of the entire folding energy landscape in de novo protein design and provide evidence suggesting nonnative interactions can play a key role in protein folding.
Co-reporter:Lin Guo, Pramit Chowdhury, Julie M. Glasscock, Feng Gai
Journal of Molecular Biology (31 December 2008) Volume 384(Issue 5) pp:1029-1036
Publication Date(Web):31 December 2008
DOI:10.1016/j.jmb.2008.03.006
It is generally believed that unfolded or denatured proteins show random-coil statistics and hence their radius of gyration simply scales with solvent quality (or concentration of denaturant). Indeed, nearly all proteins studied thus far have been shown to undergo a gradual and continuous expansion with increasing concentration of denaturant. Here, we use fluorescence correlation spectroscopy (FCS) to show that while protein A, a multi-domain and predominantly helical protein, expands gradually and continuously with increasing concentration of guanidine hydrochloride (GdnHCl), the F(ab′)2 fragment of goat anti-rabbit antibody IgG, a multi-subunit all β-sheet protein does not show such continuous expansion behavior. Instead, it first expands and then contracts with increasing concentration of GdnHCl. Even more striking is the fact that the hydrodynamic radius of the most expanded F(ab′)2 ensemble, observed at 3–4 M GdnHCl, is ∼ 3.6 times that of the native protein. Further FCS measurements involving urea and NaCl show that the unusually expanded F(ab′)2 conformations might be due to electrostatic repulsions. Taken together, these results suggest that specific interactions need to be considered while assessing the conformational and statistical properties of unfolded proteins, particularly under conditions of low solvent quality.
Co-reporter:Jia Tang, Seung-Gu Kang, Jeffery G. Saven, Feng Gai
Journal of Molecular Biology (29 May 2009) Volume 389(Issue 1) pp:90-102
Publication Date(Web):29 May 2009
DOI:10.1016/j.jmb.2009.03.074
Metals are the most commonly encountered protein cofactors, and they play important structural and functional roles in biology. In many cases, metal binding provides a major driving force for a polypeptide chain to fold. While there are many studies on the structure, stability, and function of metal-binding proteins, there are few studies focusing on understanding the kinetic mechanism of metal-induced folding. Herein, the Zn2+-induced folding kinetics of a small zinc-binding protein are studied; the CH11 peptide is derived from the first cysteine/histidine-rich region (CH1 domain) of the protein interaction domains of the transcriptional coregulator CREB-binding protein. Computational design is used to introduce tryptophan and histidine mutations that are structurally consistent with CH11; these mutants are studied using stopped-flow tryptophan fluorescence experiments. The Zn2+-induced CH11 folding kinetics are consistent with two parallel pathways, where the initial binding of Zn2+ occurs at two sites. However, the initially formed Zn2+-bound complexes can proceed either directly to the folded state where zinc adopts a tetrahedral coordination or to an off-pathway misligated intermediate. While elimination of those ligands responsible for misligation simplifies the folding kinetics, it also leads to a decrease in the zinc binding constant. Therefore, these results suggest why these nonnative zinc ligands in the CH11 motif are conserved in several distantly related organisms and why the requirement for function can lead to kinetic frustration in folding. In addition, the loop closure rate of the CH11 peptide is determined based on the proposed model and temperature-dependent kinetic measurements.
Co-reporter:Smita Mukherjee, Matthias M. Waegele, Pramit Chowdhury, Lin Guo, Feng Gai
Journal of Molecular Biology (16 October 2009) Volume 393(Issue 1) pp:227-236
Publication Date(Web):16 October 2009
DOI:10.1016/j.jmb.2009.08.016
Macromolecular crowding is one of the key characteristics of the cellular environment and is therefore intimately coupled to the process of protein folding in vivo. While previous studies have provided invaluable insight into the effect of crowding on the stability and folding rate of protein tertiary structures, very little is known about how crowding affects protein folding dynamics at the secondary structure level. In this study, we examined the thermal stability and folding–unfolding kinetics of three small folding motifs (i.e., a 34-residue α-helix, a 34-residue cross-linked helix–turn–helix, and a 16-residue β-hairpin) in the presence of two commonly used crowding agents, Dextran 70 (200 g/L) and Ficoll 70 (200 g/L). We found that these polymers do not induce any appreciable changes in the folding kinetics of the two helical peptides, which is somewhat surprising as the helix-coil transition kinetics have been shown to depend on viscosity. Also to our surprise and in contrast to what has been observed for larger proteins, we found that crowding leads to an appreciable decrease in the folding rate of the shortest β-hairpin peptide, indicating that besides the excluded volume effect, other factors also need to be considered when evaluating the net effect of crowding on protein folding kinetics. A model considering both the static and the dynamic effects arising from the presence of the crowding agent is proposed to rationalize these results.
Biophysical Journal (16 June 2010) Volume 98(Issue 12) pp:
Publication Date(Web):16 June 2010
DOI:10.1016/j.bpj.2010.03.050
Lateral diffusion of cell membrane constituents is a prerequisite for many biological functions. However, the diffusivity (or mobility) of a membrane-bound species can be influenced by many factors. To provide a better understanding of how the conformation and location of a membrane-bound biological molecule affect its mobility, herein we study the diffusion properties of a pH low insertion peptide (pHLIP) in model membranes using fluorescence correlation spectroscopy. It is found that when the pHLIP peptide is located on the membrane surface, its lateral diffusion is characterized by a distribution of diffusion times, the characteristic of which depends on the peptide/lipid ratio. Whereas, under conditions where pHLIP adopts a well-defined transmembrane α-helical conformation the peptide still exhibits heterogeneous diffusion, the distribution of diffusion times is found to be independent of the peptide/lipid ratio. Taken together, these results indicate that the mobility of a membrane-bound species is sensitive to its conformation and location and that diffusion measurement could provide useful information regarding the conformational distribution of membrane-bound peptides. Furthermore, the observation that the mobility of a membrane-bound species depends on its concentration may have important implications for diffusion-controlled reactions taking place in membranes.
Biophysical Journal (10 May 2016) Volume 110(Issue 9) pp:
Publication Date(Web):10 May 2016
DOI:10.1016/j.bpj.2016.03.030
As judged by a single publication metric, the activity in the protein folding field has been declining over the past 5 years, after enjoying a decade-long growth. Does this development indicate that the field is sunsetting or is this decline only temporary? Upon surveying a small territory of its landscape, we find that the protein folding field is still quite active and many important findings have emerged from recent experimental studies. However, it is also clear that only continued development of new techniques and methods, especially those enabling dissection of the fine details and features of the protein folding energy landscape, will fuel this old field to move forward.
Co-reporter:Pramit Chowdhury, Wei Wang, Stacey Lavender, Michelle R. Bunagan, ... Feng Gai
Journal of Molecular Biology (1 June 2007) Volume 369(Issue 2) pp:462-473
Publication Date(Web):1 June 2007
DOI:10.1016/j.jmb.2007.03.042
Members of the serine proteinase inhibitor (serpin) family play important roles in the inflammatory and coagulation cascades. Interaction of a serpin with its target proteinase induces a large conformational change, resulting in insertion of its reactive center loop (RCL) into the main body of the protein as a new strand within β-sheet A. Intermolecular insertion of the RCL of one serpin molecule into the β-sheet A of another leads to polymerization, a widespread phenomenon associated with a general class of diseases known as serpinopathies. Small peptides are known to modulate the polymerization process by binding within β-sheet A. Here, we use fluorescence correlation spectroscopy (FCS) to probe the mechanism of peptide modulation of α1-antitrypsin (α1-AT) polymerization and depolymerization, and employ a statistical computationally-assisted design strategy (SCADS) to identify new tetrapeptides that modulate polymerization. Our results demonstrate that peptide-induced depolymerization takes place via a heterogeneous, multi-step process that begins with internal fragmentation of the polymer chain. One of the designed tetrapeptides is the most potent antitrypsin depolymerizer yet found.
Co-reporter:Deguo Du, Michelle R. Bunagan, Feng Gai
Biophysical Journal (1 December 2007) Volume 93(Issue 11) pp:
Publication Date(Web):1 December 2007
DOI:10.1529/biophysj.107.108548
The formation of the monomeric α-helix represents one of the simplest scenarios in protein folding; however, our current understanding of the folding dynamics of the α-helix motif is mainly based on studies of alanine-rich model peptides. To examine the effect of peptide sequence on the folding kinetics of α-helices, we studied the relaxation kinetics of a 21-residue helical peptide, Conantokin-T (Con-T), using time-resolved infrared spectroscopy in conjunction with a laser-induced temperature jump technique. Con-T is a neuroactive peptide containing a large number of charged residues that is found in the venom of the piscivorous cone snail Conus tulipa. The temperature-jump relaxation kinetics of Con-T is distinctly slower than that of previously studied alanine-based peptides, suggesting that the folding time of α-helices is sequence-dependent. Furthermore, it appears that the slower folding of Con-T can be attributed to the fact that its helical conformation is stabilized by charge-charge interactions or salt bridges. Although this finding contradicts an earlier molecular dynamics simulation, it also has implications for existing models of protein folding.
Physical Chemistry Chemical Physics 2017 - vol. 19(Issue 7) pp:NaN5036-5036
Publication Date(Web):2017/01/30
DOI:10.1039/C6CP08924K
An infrared temperature-jump (T-jump) study by Huang et al. (Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 2788–2793) showed that the conformational relaxation kinetics of an alanine-based α-helical peptide depend not only on the final temperature (Tf) but also on the initial temperature (Ti) when Tf is fixed. Their finding indicates that the folding free energy landscape of this peptide is non-two-state like, allowing for the population of conformational ensembles with different helical lengths and relaxation times in the temperature range of the experiment. Because α-helix folding involves two fundamental events, nucleation and propagation, the results of Huang et al. thus present a unique opportunity to determine their rate constants – a long-sought goal in the study of the helix–coil transition dynamics. Herein, we capitalize on this notion and develop a coarse-grained kinetic model to globally fit the thermal unfolding curve and T-jump kinetic traces of this peptide. Using this strategy, we are able to explicitly determine the microscopic rate constants of the kinetic steps encountered in the nucleation and propagation processes. Our results reveal that the time taken to form one α-helical turn (i.e., an α-helical segment with one helical hydrogen bond) is about 315 ns, whereas the time taken to elongate this nucleus by one residue (or backbone unit) is 5.9 ns, depending on the position of the residue.
Co-reporter:Jeffrey M. Rodgers, Rachel M. Abaskharon, Bei Ding, Jianxin Chen, Wenkai Zhang and Feng Gai
Physical Chemistry Chemical Physics 2017 - vol. 19(Issue 24) pp:NaN16150-16150
Publication Date(Web):2017/06/05
DOI:10.1039/C7CP02442H
The CO/CN stretching vibration arising from a carbonyl/nitrile functional group in various molecular systems has been frequently used to assess, for example, local hydrogen-bonding interactions, among other applications. However, in practice it is not always easy to ascertain whether the carbonyl or nitrile group in question is engaged in such interactions. Herein, we use 4-cyanoindole and cyclopentanone as models to show that, when a fundamental CO or CN stretching mode is involved in Fermi resonance, the underlying vibrational coupling constant (W) is a convenient reporter of the hydrogen-bonding status of the corresponding carbonyl or nitrile group. Specifically, we find that for both groups a W value of 7.7 cm−1 or greater is indicative of their involvement in hydrogen-bonding interactions. Furthermore, we find that, as observed in similar studies, the Fermi resonance coupling leads to quantum beats in the two-dimensional infrared spectra of 4-cyanoindole in isopropanol, with a period of about 1.9 ps.
Physical Chemistry Chemical Physics 2016 - vol. 18(Issue 14) pp:NaN9607-9607
Publication Date(Web):2016/03/08
DOI:10.1039/C6CP00865H
Disulfide cleavage is one of the major causes underlying ultraviolet (UV) light-induced protein damage. While previous studies have provided strong evidence to support the notion that this process is mediated by photo-induced electron transfer from the excited state of an aromatic residue (e.g., tryptophan) to the disulfide bond, many mechanistic details are still lacking. For example, we do not know how quickly this process occurs in a protein environment. Herein, we design an experiment, which uses the unfolding kinetics of a protein as an observable, to directly assess the kinetics and mechanism of photo-induced disulfide cleavage. Our results show that this disulfide bond cleavage event takes place in ∼2 μs via a mechanism involving electron transfer from the triplet state of a tryptophan (Trp) residue to the disulfide bond. Furthermore, we find that one of the photoproducts of this reaction, a Trp-SR adduct, is formed locally, thus preventing the protein from re-cross-linking. Taken together, these findings suggest that a Trp-disulfide pair could be used as a photo-trigger to initiate protein folding dynamics and control the biological activities of disulfide-containing peptides.
Co-reporter:Wenkai Zhang, Beatrice N. Markiewicz, Rosalie S. Doerksen, Amos B. Smith, III and Feng Gai
Physical Chemistry Chemical Physics 2016 - vol. 18(Issue 10) pp:NaN7034-7034
Publication Date(Web):2015/08/26
DOI:10.1039/C5CP04413H
Recently it has been suggested that the CN stretching vibration of a tryptophan analog, 5-cyanotryptophan, could be used as an infrared probe of the local environment, especially the hydration status, of tryptophan residues in proteins. However, the factors that influence the frequency of this vibrational mode are not understood. To determine these factors, herein we carried out linear and nonlinear infrared measurements on the CN stretching vibration of the sidechain of 5-cyanotryptophan, 3-methyl-5-cyanoindole, in a series of protic and aprotic solvents. We found that while the CN stretching frequencies obtained in these solvents do not correlate well with any individual Kamlet–Taft solvent parameter, i.e., π* (polarizability), β (hydrogen bond accepting ability), and α (hydrogen bond donating ability), they do however, collapse on a straight line when plotted against σ = π* + β − α. This linear relationship provides a firm indication that both specific interactions, i.e., hydrogen-bonding interactions with the CN (through α) and indole N–H (through β) groups, and non-specific interactions with the molecule (through π*) work together to determine the CN stretching frequency, thus laying a quantitative framework for applying 5-cyanotryptophan to investigate the microscopic environment of proteins in a site-specific manner. Furthermore, two-dimensional and pump–probe infrared measurements revealed that a significant portion (∼31%) of the ground state bleach signal has a decay time constant of ∼12.3 ps, due to an additional vibrational relaxation channel, making it possible to use 5-cyanotryptophan to probe dynamics occurring on a timescale on the order of tens of picoseconds.
Co-reporter:Mary Rose Mintzer, Thomas Troxler and Feng Gai
Physical Chemistry Chemical Physics 2015 - vol. 17(Issue 12) pp:NaN7887-7887
Publication Date(Web):2015/02/20
DOI:10.1039/C5CP00050E
The CN stretching frequency and fluorescence quantum yield of p-cyanophenylalanine are sensitive to environment. As such, this unnatural amino acid has found broad applications, ranging from studying how proteins fold to determining the local electric field of membranes. Herein, we demonstrate that the fluorescence of p-cyanophenylalanine can be quenched by selenomethionine through an electron transfer process occurring at short distances, thus further expanding its spectroscopic utility. Using this fluorophore–quencher pair, we are able to show that short polyproline peptides (1–4 prolines) are not rigid; instead, they sample a bimodal conformational distribution.
Co-reporter:Beatrice N. Markiewicz, Thomas Lemmin, Wenkai Zhang, Ismail A. Ahmed, Hyunil Jo, Giacomo Fiorin, Thomas Troxler, William F. DeGrado and Feng Gai
Physical Chemistry Chemical Physics 2016 - vol. 18(Issue 41) pp:NaN28950-28950
Publication Date(Web):2016/09/22
DOI:10.1039/C6CP03426H
The M2 proton channel of the influenza A virus has been the subject of extensive studies because of its critical role in viral replication. As such, we now know a great deal about its mechanism of action, especially how it selects and conducts protons in an asymmetric fashion. The conductance of this channel is tuned to conduct protons at a relatively low biologically useful rate, which allows acidification of the viral interior of a virus entrapped within an endosome, but not so great as to cause toxicity to the infected host cell prior to packaging of the virus. The dynamic, structural and chemical features that give rise to this tuning are not fully understood. Herein, we use a tryptophan (Trp) analog, 5-cyanotryptophan, and various methods, including linear and nonlinear infrared spectroscopies, static and time-resolved fluorescence techniques, and molecular dynamics simulations, to site-specifically interrogate the structure and hydration dynamics of the Trp41 gate in the transmembrane domain of the M2 proton channel. Our results suggest that the Trp41 sidechain adopts the t90 rotamer, the χ2 dihedral angle of which undergoes an increase of approximately 35° upon changing the pH from 7.4 to 5.0. Furthermore, we find that Trp41 is situated in an environment lacking bulk-like water, and somewhat surprisingly, the water density and dynamics do not show a measurable difference between the high (7.4) and low (5.0) pH states. Since previous studies have shown that upon channel opening water flows into the cavity above the histidine tetrad (His37), the present finding thus provides evidence indicating that the lack of sufficient water molecules near Trp41 needed to establish a continuous hydrogen bonding network poses an additional energetic bottleneck for proton conduction.
.jpg)
![Ethanaminium,2-[[(dodecyloxy)hydroxyphosphinyl]oxy]-N,N,N-trimethyl-, inner salt](http://img.cochemist.com/ccimg/29600/29557-51-5.png)
![Ethanaminium,2-[[(dodecyloxy)hydroxyphosphinyl]oxy]-N,N,N-trimethyl-, inner salt](http://img.cochemist.com/ccimg/29600/29557-51-5_b.png)
![Hexadecanoic acid,1,1'-[1-[[[(2-aminoethoxy)hydroxyphosphinyl]oxy]methyl]-1,2-ethanediyl] ester](http://img.cochemist.com/ccimg/3100/3026-45-7.png)
![Hexadecanoic acid,1,1'-[1-[[[(2-aminoethoxy)hydroxyphosphinyl]oxy]methyl]-1,2-ethanediyl] ester](http://img.cochemist.com/ccimg/3100/3026-45-7_b.png)
![3,5,9-Trioxa-4-phosphapentacosan-1-aminium,4-hydroxy-N,N,N-trimethyl-10-oxo-7-[(1-oxohexadecyl)oxy]-, inner salt, 4-oxide](http://img.cochemist.com/ccimg/2700/2644-64-6.png)
![3,5,9-Trioxa-4-phosphapentacosan-1-aminium,4-hydroxy-N,N,N-trimethyl-10-oxo-7-[(1-oxohexadecyl)oxy]-, inner salt, 4-oxide](http://img.cochemist.com/ccimg/2700/2644-64-6_b.png)
![2-Amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid](http://img.cochemist.com/ccimg/1200/1137-00-4.png)
![2-Amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid](http://img.cochemist.com/ccimg/1200/1137-00-4_b.png)
![3,5,8-Trioxa-4-phosphahexacos-17-en-1-aminium,4-hydroxy-N,N,N-trimethyl-9-oxo-7-[[(1-oxohexadecyl)oxy]methyl]-, inner salt,4-oxide, (7R,17Z)-](http://img.cochemist.com/ccimg/26900/26853-31-6.png)
![3,5,8-Trioxa-4-phosphahexacos-17-en-1-aminium,4-hydroxy-N,N,N-trimethyl-9-oxo-7-[[(1-oxohexadecyl)oxy]methyl]-, inner salt,4-oxide, (7R,17Z)-](http://img.cochemist.com/ccimg/26900/26853-31-6_b.png)
