Co-reporter:D. Abdullin, G. Hagelueken and O. Schiemann
Physical Chemistry Chemical Physics 2016 vol. 18(Issue 15) pp:10428-10437
Publication Date(Web):14 Mar 2016
DOI:10.1039/C6CP01307D
Pulsed electron–electron double resonance (PELDOR or DEER) in combination with site-directed spin labelling has emerged as an important method for measuring nanometer distance constraints that are used to obtain coarse-grained structures of biomolecules or to follow their conformational changes. Translating measured spin–spin distances between spin labels into structural information requires taking the conformational flexibility of spin label side chains into account. Here, we present an analysis of orientation selective PELDOR data recorded on six singly MTSSL-labelled azurin mutants. The analysis yielded conformational MTSSL ensembles, which are considerably narrower than those predicted using in silico spin labeling methods but match well with spin label conformations found in the corresponding crystal structures. The possible reasons and consequences for predicting spin label conformers in the fold of biomolecules are discussed.
Co-reporter:Andreas Meyer, Dinar Abdullin, Gregor Schnakenburg and Olav Schiemann
Physical Chemistry Chemical Physics 2016 vol. 18(Issue 13) pp:9262-9271
Publication Date(Web):01 Mar 2016
DOI:10.1039/C5CP07621H
A rigid, nitroxide substituted terpyridine ligand has been used to synthesize hetero- and homoleptic bis-terpyridine complexes of copper(II). The homoleptic complex represents a three-spin system, while the metal ion in the heteroleptic complex is in average bound to one nitroxide bearing ligand. Both complexes are used as model systems for EPR distance measurements using pulsed electron–electron double resonance (PELDOR or DEER) and relaxation induced dipolar modulation enhancement (RIDME) sequences. The results of both methods are analyzed using detailed geometric data obtained from the crystal structure of the homoleptic complex as well as information concerning ligand scrambling and the electronic structure of the copper center. In addition, both methods are compared with respect to their sensitivity, the extent of orientation selectivity and the influence of multispin effects.
Co-reporter:Andreas Meyer and Olav Schiemann
The Journal of Physical Chemistry A 2016 Volume 120(Issue 20) pp:3463-3472
Publication Date(Web):May 9, 2016
DOI:10.1021/acs.jpca.6b00716
A homoleptic bisnitroxide complex of manganese(II) was synthesized as a model system for EPR spectroscopic distance determinations involving high-spin metal ions and more than one distance. The performance of the RIDME experiment is compared with that of the more frequently used PELDOR experiment. It is shown that the PELDOR experiment yields both distances, Mn(II)–nitroxide and nitroxide–nitroxide, and that they can be separated to a certain extent, whereas the RIDME experiment yields only the Mn(II)–nitroxide distance. Both pulse sequences yield artifacts, either due to multispin effects or higher electron-spin transitions. Orientation selection is mostly introduced by the nitroxide signal and can be averaged out by variation of the observer field in the RIDME experiment. Thus, both methods might be used complementarily to obtain a reliable picture of an unknown system.
Co-reporter:Mark Kerzhner;Dinar Abdullin;Jennifer Wi&x119;cek;Dr. Hideto Matsuoka;Dr. Gregor Hagelueken;Dr. Olav Schiemann;Dr. Michael Famulok
Chemistry - A European Journal 2016 Volume 22( Issue 34) pp:12113-12121
Publication Date(Web):
DOI:10.1002/chem.201601897
Abstract
Site-directed spin labeling of RNA based on click chemistry is used in combination with pulsed electron-electron double resonance (PELDOR) to benchmark a nitroxide spin label, called here dŲ. We compare this approach with another established method that employs the rigid spin label Çm for RNA labeling. By using CD spectroscopy, thermal denaturation measurements, CW-EPR as well as PELDOR we analyzed and compared the influence of dŲ and Çm on a self-complementary RNA duplex. Our results demonstrate that the conformational diversity of dŲ is significantly reduced near the freezing temperature of a phosphate buffer, resulting in strongly orientation-selective PELDOR time traces of the dŲ-labeled RNA duplex.
Co-reporter:Wolfram Ratzke, Lisa Schmitt, Hideto Matsuoka, Christoph Bannwarth, Marius Retegan, Sebastian Bange, Philippe Klemm, Frank Neese, Stefan Grimme, Olav Schiemann, John M. Lupton, and Sigurd Höger
The Journal of Physical Chemistry Letters 2016 Volume 7(Issue 22) pp:4802-4808
Publication Date(Web):October 27, 2016
DOI:10.1021/acs.jpclett.6b01907
Metal-free dual singlet–triplet organic light-emitting diode (OLED) emitters can provide direct insight into spin statistics, spin correlations and spin relaxation phenomena, through a comparison of fluorescence to phosphorescence intensity. Remarkably, such materials can also function at room temperature, exhibiting phosphorescence lifetimes of several milliseconds. Using electroluminescence, quantum chemistry, and electron paramagnetic resonance spectroscopy, we investigate the effect of the conjugation pathway on radiative and nonradiative relaxation of the triplet state in phenazine-based compounds and demonstrate that the contribution of the phenazine nπ* excited state is crucial to enabling phosphorescence.
Co-reporter:Erik Schubert, Nicole Florin, Fraser Duthie, H. Henning Brewitz, Toni Kühl, Diana Imhof, Gregor Hagelueken, Olav Schiemann
Journal of Inorganic Biochemistry 2015 Volume 148() pp:49-56
Publication Date(Web):July 2015
DOI:10.1016/j.jinorgbio.2015.05.008
•Heme binding of peptides containing heme regulatory motifs (HRM) analyzed by EPR spectroscopy•EPR spectroscopy confirms the importance of the Cys-Pro motif in the HRM•Spin states of the heme ligand depend on the peptide sequence•Expression and purification of two proteins (FeoB and GlpF) containing HRM sequences•Analysis of protein:heme binding by cw-X-Band EPR- and UV-Vis spectroscopyThe role of heme as a cofactor in enzymatic reactions has been studied for a long time and in great detail. Recently it was discovered that heme can also serve as a signalling molecule in cells but so far only few examples of this regulation have been studied. In order to discover new potentially heme-regulated proteins, we screened protein sequence databases for bacterial proteins that contain sequence features like a Cysteine–Proline (CP) motif, which is known for its heme-binding propensity. Based on this search we synthesized a series of these potential heme regulatory motifs (HRMs). We used cw EPR spectroscopy to investigate whether these sequences do indeed bind to heme and if the spin state of heme is changed upon interaction with the peptides. The corresponding proteins of two potential HRMs, FeoB and GlpF, were expressed and purified and their interaction with heme was studied by cw EPR and UV–Visible (UV–Vis) spectroscopy.Peptides and proteins containing potential heme regulatory motifs (HRMs) are investigated by cw EPR spectroscopy and UV–Vis spectroscopy. The results show that several peptides strongly bind to heme. An interaction with heme is also found for the ferrous iron transport protein FeoB from Escherichia coli.
Co-reporter:Dinar Abdullin;Nicole Florin;Dr. Gregor Hagelueken ;Dr. Olav Schiemann
Angewandte Chemie International Edition 2015 Volume 54( Issue 6) pp:1827-1831
Publication Date(Web):
DOI:10.1002/anie.201410396
Abstract
Metal ions play an important role in the catalysis and folding of proteins and oligonucleotides. Their localization within the three-dimensional fold of such biomolecules is therefore an important goal in understanding structure–function relationships. A trilateration approach for the localization of metal ions by means of long-range distance measurements based on electron paramagnetic resonance (EPR) is introduced. The approach is tested on the Cu2+ center of azurin, and factors affecting the precision of the method are discussed.
Co-reporter:Dinar Abdullin;Nicole Florin;Dr. Gregor Hagelueken ;Dr. Olav Schiemann
Angewandte Chemie 2015 Volume 127( Issue 6) pp:1847-1851
Publication Date(Web):
DOI:10.1002/ange.201410396
Abstract
Metal ions play an important role in the catalysis and folding of proteins and oligonucleotides. Their localization within the three-dimensional fold of such biomolecules is therefore an important goal in understanding structure–function relationships. A trilateration approach for the localization of metal ions by means of long-range distance measurements based on electron paramagnetic resonance (EPR) is introduced. The approach is tested on the Cu2+ center of azurin, and factors affecting the precision of the method are discussed.
Co-reporter:Marius I. Arz;Martin Straßmann;Andreas Meyer;Dr. Gregor Schnakenburg;Dr. Olav Schiemann;Dr. Alexer C. Filippou
Chemistry - A European Journal 2015 Volume 21( Issue 35) pp:12509-12516
Publication Date(Web):
DOI:10.1002/chem.201502199
Abstract
One-electron oxidation of the disilicon(0) compound Si2(Idipp)2 (1, Idipp=1,3-bis(2,6-diisopropylphenyl)imidazolin-2-ylidene) with [Fe(C5Me5)2][B(ArF)4] (ArF=C6H3-3,5-(CF3)2) affords selectively the green radical salt [Si2(Idipp)2][B(ArF)4] (1-[B(ArF)4). Oxidation of the centrosymmetric 1 occurs reversibly at a low redox potential (E1/2=−1.250 V vs. Fc+/Fc), and is accompanied by considerable structural changes as shown by single-crystal X-ray structural analysis of 1-B(ArF)4. These include a shortening of the SiSi bond, a widening of the Si-Si-CNHC angles, and a lowering of the symmetry, leading to a quite different conformation of the NHC substituents at the two inequivalent Si sites in 1+. Comparative quantum chemical calculations of 1 and 1+ indicate that electron ejection occurs from the symmetric (n+) combination of the Si lone pairs (HOMO). EPR studies of 1-B(ArF)4 in frozen solution verified the inequivalency of the two Si sites observed in the solid-state, and point in agreement with the theoretical results to an almost equal distribution of the spin density over the two Si atoms, leading to quite similar 29Si hyperfine coupling tensors in 1+. EPR studies of 1-B(ArF)4 in liquid solution unraveled a topomerization with a low activation barrier that interconverts the two Si sites in 1+.
Co-reporter:Dinar Abdullin, Fraser Duthie, Andreas Meyer, Elisa S. Müller, Gregor Hagelueken, and Olav Schiemann
The Journal of Physical Chemistry B 2015 Volume 119(Issue 43) pp:13534-13542
Publication Date(Web):May 22, 2015
DOI:10.1021/acs.jpcb.5b02118
EPR-based nanometre distance measurements are becoming ever more important in structural biology. Usually the distance constraints are measured between two nitroxide spin labels. Yet, distance measurements between a metal center and spin labels enable, e.g., the localization of metal ions within the tertiary fold of biomolecules. Therefore, it is important to find methods that provide such distance information quickly, with high precision and reliability. In the present study, two methods, pulsed electron–electron double resonance (PELDOR) and relaxation-induced dipolar modulation enhancement (RIDME), are compared on the heme-containing and spin-labeled cytochrome P450cam. Special emphasis is put on the optimization of the dead-time free RIDME experiment and several ways of data analysis. It turned out that RIDME appears to be better suited for distance measurements involving metal ions like low-spin Fe3+ than PELDOR.
Co-reporter:Nitin C. Kunjir, Gunnar W. Reginsson, Olav Schiemann and Snorri Th. Sigurdsson
Physical Chemistry Chemical Physics 2013 vol. 15(Issue 45) pp:19673-19685
Publication Date(Web):17 Oct 2013
DOI:10.1039/C3CP52789A
Trityl based spin labels are emerging as a complement to nitroxides in nanometer distance measurements using EPR methods. The narrow spectral width of the trityl radicals prompts us to ask the question at which distance between these spin centers, the pseudo-secular part of the dipolar coupling and spin density delocalization have to be taken into account. For this, two trityl–trityl and one trityl–nitroxide model compounds were synthesized with well-defined interspin distances. Continuous wave (CW) EPR, double quantum coherence (DQC) and pulsed electron–electron double resonance (PELDOR) spectra were acquired from these compounds at commercial X-band frequencies. The data analysis shows that two of the compounds, with distances of up to 25 Å, fall into the strong coupling regime and that precise distances can only be obtained if both the spin density delocalization and the pseudo-secular part of the dipolar coupling are included in the analysis.
Co-reporter:Gunnar W. Reginsson;Nitin C. Kunjir; Snorri Th. Sigurdsson; Olav Schiemann
Chemistry - A European Journal 2012 Volume 18( Issue 43) pp:13580-13584
Publication Date(Web):
DOI:10.1002/chem.201203014
Co-reporter:Hexian Huang;Christos Pliotas;James H. Naismith;Ian R. Booth;Richard Ward;Susan S. Black;Akiko Rasmussen;Emma Branigan;Gregor Hagelueken
PNAS 2012 Volume 109 (Issue 40 ) pp:
Publication Date(Web):2012-10-02
DOI:10.1073/pnas.1202286109
The heptameric mechanosensitive channel of small conductance (MscS) provides a critical function in Escherichia coli where it opens in response to increased bilayer tension. Three approaches have defined different closed and open structures
of the channel, resulting in mutually incompatible models of gating. We have attached spin labels to cysteine mutants on key
secondary structural elements specifically chosen to discriminate between the competing models. The resulting pulsed electron–electron
double resonance (PELDOR) spectra matched predicted distance distributions for the open crystal structure of MscS. The fit
for the predictions by structural models of MscS derived by other techniques was not convincing. The assignment of MscS as
open in detergent by PELDOR was unexpected but is supported by two crystal structures of spin-labeled MscS. PELDOR is therefore
shown to be a powerful experimental tool to interrogate the conformation of transmembrane regions of integral membrane proteins.
Co-reporter:Richard Ward, Christos Pliotas, Emma Branigan, Christian Hacker, Akiko Rasmussen, Gregor Hagelueken, Ian R. Booth, Samantha Miller, John Lucocq, James H. Naismith, Olav Schiemann
Biophysical Journal (18 February 2014) Volume 106(Issue 4) pp:
Publication Date(Web):18 February 2014
DOI:10.1016/j.bpj.2014.01.008
Mechanosensitive channel proteins are important safety valves against osmotic shock in bacteria, and are involved in sensing touch and sound waves in higher organisms. The mechanosensitive channel of small conductance (MscS) has been extensively studied. Pulsed electron-electron double resonance (PELDOR or DEER) of detergent-solubilized protein confirms that as seen in the crystal structure, the outer ring of transmembrane helices do not pack against the pore-forming helices, creating an apparent void. The relevance of this void to the functional form of MscS in the bilayer is the subject of debate. Here, we report PELDOR measurements of MscS reconstituted into two lipid bilayer systems: nanodiscs and bicelles. The distance measurements from multiple mutants derived from the PELDOR data are consistent with the detergent-solution arrangement of the protein. We conclude, therefore, that the relative positioning of the transmembrane helices is preserved in mimics of the cell bilayer, and that the apparent voids are not an artifact of detergent solution but a property of the protein that will have to be accounted for in any molecular mechanism of gating.
Co-reporter:Andreas Meyer; Gregor Schnakenburg; Robert Glaum
Inorganic Chemistry () pp:
Publication Date(Web):August 14, 2015
DOI:10.1021/acs.inorgchem.5b01157
The surprisingly complicated crystal structure of (bis(terpyridine))copper(II) tetraphenylborate [Cu(tpy)2](BPh4)2 (tpy = 2,2′:6′,2″-terpyridine) consists of six crystallographically independent [Cu(tpy)2]2+ complexes. At ambient temperature, five out of six [CuIIN6] chromophores appear to be compressed octahedra, while at 100 K, four exhibit elongated and only two compressed octahedral geometry. Temperature dependent single crystal UV/vis (100, 298 K) and EPR measurements (20, 100, 298 K) as well as AOM calculations suggest that the octahedra which show apparently compressed octahedral geometry (XRD) result from dynamic Jahn–Teller behavior of elongated octahedra [CuIIN6]. The detailed correlation of structural and spectroscopic data allows an understanding of the strongly solvent-dependent structures of the [Cu(tpy)2]2+ complex in solution.
Co-reporter:Andreas Meyer, Dinar Abdullin, Gregor Schnakenburg and Olav Schiemann
Physical Chemistry Chemical Physics 2016 - vol. 18(Issue 13) pp:NaN9271-9271
Publication Date(Web):2016/03/01
DOI:10.1039/C5CP07621H
A rigid, nitroxide substituted terpyridine ligand has been used to synthesize hetero- and homoleptic bis-terpyridine complexes of copper(II). The homoleptic complex represents a three-spin system, while the metal ion in the heteroleptic complex is in average bound to one nitroxide bearing ligand. Both complexes are used as model systems for EPR distance measurements using pulsed electron–electron double resonance (PELDOR or DEER) and relaxation induced dipolar modulation enhancement (RIDME) sequences. The results of both methods are analyzed using detailed geometric data obtained from the crystal structure of the homoleptic complex as well as information concerning ligand scrambling and the electronic structure of the copper center. In addition, both methods are compared with respect to their sensitivity, the extent of orientation selectivity and the influence of multispin effects.
Co-reporter:Nitin C. Kunjir, Gunnar W. Reginsson, Olav Schiemann and Snorri Th. Sigurdsson
Physical Chemistry Chemical Physics 2013 - vol. 15(Issue 45) pp:NaN19685-19685
Publication Date(Web):2013/10/17
DOI:10.1039/C3CP52789A
Trityl based spin labels are emerging as a complement to nitroxides in nanometer distance measurements using EPR methods. The narrow spectral width of the trityl radicals prompts us to ask the question at which distance between these spin centers, the pseudo-secular part of the dipolar coupling and spin density delocalization have to be taken into account. For this, two trityl–trityl and one trityl–nitroxide model compounds were synthesized with well-defined interspin distances. Continuous wave (CW) EPR, double quantum coherence (DQC) and pulsed electron–electron double resonance (PELDOR) spectra were acquired from these compounds at commercial X-band frequencies. The data analysis shows that two of the compounds, with distances of up to 25 Å, fall into the strong coupling regime and that precise distances can only be obtained if both the spin density delocalization and the pseudo-secular part of the dipolar coupling are included in the analysis.
Co-reporter:D. Abdullin, G. Hagelueken and O. Schiemann
Physical Chemistry Chemical Physics 2016 - vol. 18(Issue 15) pp:NaN10437-10437
Publication Date(Web):2016/03/14
DOI:10.1039/C6CP01307D
Pulsed electron–electron double resonance (PELDOR or DEER) in combination with site-directed spin labelling has emerged as an important method for measuring nanometer distance constraints that are used to obtain coarse-grained structures of biomolecules or to follow their conformational changes. Translating measured spin–spin distances between spin labels into structural information requires taking the conformational flexibility of spin label side chains into account. Here, we present an analysis of orientation selective PELDOR data recorded on six singly MTSSL-labelled azurin mutants. The analysis yielded conformational MTSSL ensembles, which are considerably narrower than those predicted using in silico spin labeling methods but match well with spin label conformations found in the corresponding crystal structures. The possible reasons and consequences for predicting spin label conformers in the fold of biomolecules are discussed.
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