Molekulare Struktur mit Abstand - quantitative Interpretation von Puls Elektron-Elektron Doppelresonanzdaten

Pulsed electron-electron double resonance (PELDOR) is a well established method concerning nanometer distance measurements involving two nitroxide spin-labels. In this thesis the applicability of this method to count the number of spins is tested. Furthermore, this work explored the limits, up to which PELDOR data obtained on copper(II)-nitroxide complexes can be quantitatively interpreted. Spin counting provides access to oligomerization studies – monitoring the assembly of homo- or hetero-oligomers from singly labeled compounds. The experimental calibration was performed using model systems, which contain one to four nitroxide radicals. The results show that monomers, dimers, trimers, and tetramers can be distinguished within an error of 5% in the number of spins. Moreover, a detailed analysis of the distance distributions in model complexes revealed that more than one distance can be extracted from complexes bearing several spins, as for example three different distances were resolved in a model tetramer – the other three possible distances being symmetry related. Furthermore, systems exhibiting mixtures of oligomeric states complicate the analysis of the data, because the average number of spin centers contributes nonlinearly to the signal and different relaxation behavior of the oligomers has to be treated explicitly. Experiments solving these problems are proposed in the thesis. Thus, for the first time spin counting has been experimentally calibrated using fully characterized test systems bearing up to four spins. Moreover, the behavior of mixtures was quantitatively interpreted. In addition, it has been shown that several spin-spin distances within a molecule can be extracted from a single dataset. In the second part of the thesis PELDOR experiments on a spin-labeled copper(II)-porphyrin have been quantitatively analyzed. Metal-nitroxide distance measurements are a valuable tool for the triangulation of paramagnetic metal ions. Therefore, X-band PELDOR experiments at different frequencies have been performed. The data exhibits only weak orientation selection, but a fast damping of the oscillation. The experimental data has been interpreted based upon quantitative simulations. The influence of orientation selection, conformational flexibility, spin-density distribution, exchange interaction J, as well as anisotropy and strains of the g-tensor has been examined. An estimate of the spin-density delocalization has been obtained by density functional theory calculations. The dipolar interaction tensor was calculated from the point-charge model, the extension of the point-dipole approximation to several spin bearing centers. Even assuming asymmetric spin distributions induced by an ensemble of asymmetrically distorted porphyrins the effect of delocalization on the PELDOR time trace is weak. The observed damping of dipolar oscillations has been only reproduced by simulations, if a small distribution in J was assumed. It has been shown that the experimental damping of dipolar modulations is not solely due to conformational heterogeneity. In conclusion the quantitative interpretation of PELDOR data is extended to copper-nitroxide- and multi-spin-systems. The influence of the mean distance, of the number of coupled spins, of the conformational flexibility, of spin-density distribution and of the electronic structure of the spin centers has been analyzed using model systems. The insights on model compounds mimicking spin-labeled biomacromolecules – in oligomeric or metal bound states – calibrate the method with respect to the information that can be deduced from the experimental data. The resulting in-depth understanding allows correlating experimental results (from for example biological systems) with models of structure and dynamics. It also opens new fields for PELDOR as for example triangulation of metal centers and oligomerization studies. In general, this thesis has demonstrated that modern pulsed electron paramagnetic resonance techniques in combination with quantitative data analysis can contribute to a detailed insight into molecular structure and dynamics

Spectroscopically orthogonal labelling to disentangle site-specific nitroxide label distributions

Funding: The authors acknowledge support by the Wellcome Trust (204821/Z/16/Z), the Leverhulme Trust (RPG-2018-397), and the EPSRC (EP/X016455/1). BEB acknowledges equipment funding by BBSRC (BB/R013780/1 and BB/T017740/1).Biomolecular applications of pulse dipolar electron paramagnetic resonance spectroscopy (PDS) are becoming increasingly valuable in structural biology. Site-directed spin labelling of proteins is routinely performed using nitroxides, with paramagnetic metal ions and other organic radicals gaining popularity as alternative spin centres. Spectroscopically orthogonal spin labelling using different types of labels potentially increases the information content available from a single sample. When analysing experimental distance distributions between two nitroxide spin labels, the site-specific rotamer information has been projected into the distance and is not readily available, and the contributions of individual labelling sites to the width of the distance distribution are not obvious from the PDS data. Here, we exploit the exquisite precision of labelling double-histidine (dHis) motifs with CuII chelate complexes. The contribution of this label to the distance distribution widths in model protein GB1 has been shown to be negligible. By combining a dHis CuII labelling site with cysteine-specific nitroxide labelling, we gather insights on the label rotamers at two distinct sites, comparing their contributions to distance distributions based on different in silico modelling approaches and structural models. From this study, it seems advisable to consider discrepancies between different in silico modelling approaches when selecting labelling sites for PDS studies.Publisher PDFPeer reviewe

Pulse dipolar electron paramagnetic resonance spectroscopy distance measurements at low nanomolar concentrations : the CuII-trityl case

Funding: To meet institutional and research funder open access requirements, any accepted manuscript arising shall be open access under a Creative Commons Attribution (CC BY) reuse licence with zero embargo. The authors acknowledge support by a University of St Andrews-University of Bonn Collaborative Research Grant, by the Wellcome Trust (204821/Z/16/Z), and by the EPSRC (EP/X016455/1). B.E.B. acknowledges equipment funding by BBSRC (BB/R013780/1 and BB/T017740/1). O.S. thanks the DFG for funding (420322655). C.A.H. thanks the DAAD for a travel and research scholarship. The authors thank the StAnD (St Andrews and Dundee) EPR grouping for long-standing support and the St Andrews mass spectrometry and proteomics facility for equipment access.Recent sensitivity enhancements in pulse dipolar EPR spectroscopy (PDS) have afforded distance measurements at submicromolar spin concentrations. This development opens the path for new science, as more biomolecular systems can be investigated at their respective physiological concentrations. Here, we demonstrate that the combination of orthogonal spin labelling using CuII ions and trityl yields a more than 3-fold sensitivity increase compared to the established CuII-nitroxide labelling strategy. Application of the recently developed variable-time RIDME method yields a further approximately 2.5-fold increase compared to the commonly used constant-time RIDME. This overall increase in sensitivity of almost an order of magnitude makes distance measurements in the range of 3 nm with protein concentrations as low as 10 nM feasible, more than two times lower than previously reported. We expect that experiments at single digit nanomolar concentrations are imminent, which has the potential to transform biological PDS applications.Publisher PDFPeer reviewe

In-lipid structure of pressure sensitive domains hints mechanosensitive channel functional diversity

This project was supported by a BBSRC grant (BB/S018069/1) to C.P., who was supported by the Royal Society of Edinburgh, Tenovus (T15/41) and Carnegie Trust (OS000256), at the initial stages of this project. Further C.P. acknowledges support from the University of St Andrews for the C.K. studentship and the University of Leeds and the Chinese Scholarship Council for the Y.M. studentship. B.E.B. and C.P. acknowledge support by the Leverhulme Trust (RPG-2018–397). This work was also supported by previous Wellcome Trust [099149/Z/12/Z] and BBSRC equipment grants (BB/R013780/1).The mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis has been used as structural model for rationalizing functional observations in multiple MscL orthologues. Although these orthologues adopt similar structural architectures, they reportedly present significant functional differences. Subtle structural discrepancies on mechanosensitive channel nano-pockets are known to affect mechanical gating and may be linked to large variability in tension sensitivity among these membrane channels. Here we modify the nano-pocket regions of MscL from Escherichia coli and Mycobacterium tuberculosis and employ PELDOR/DEER distance and 3pESEEM deuterium accessibility measurements to interrogate channel structure within lipids, in which both channels adopt a closed conformation. Significant in-lipid structural differences between the two constructs suggest a more compact EcMscL at the membrane inner-leaflet, as a consequence of a rotated TM2 helix. Observed differences within lipids could explain EcMscL’s higher tension sensitivity and should be taken into account in extrapolated models used for MscL gating rationalization.Publisher PDFPeer reviewe

Pulse EPR distance measurements to study multimers and multimerisation

This work was supported by funding from the European Union (Marie Curie Actions REA 334496), the Carnegie Trust (70098), the EPSRC (EP/M024660/1) and a Wellcome Trust multi-user equipment grant (099149/Z/12/Z).Pulse dipolar electron paramagnetic resonance (PD-EPR) has become a powerful tool for structural biology determining distances on the nanometre scale. Recent advances in hardware, methodology, and data analysis have widened the scope to complex biological systems. PD-EPR can be applied to systems containing lowly populated conformers or displaying large intrinsic flexibility, making them all but intractable for cryo-electron microscopy and crystallography. Membrane protein applications are of particular interest due to the intrinsic difficulties for obtaining high-resolution structures of all relevant conformations. Many drug targets involved in critical cell functions are multimeric channels or transporters. Here, common approaches for introducing spin labels for PD-EPR cause the presence of more than two electron spins per multimeric complex. This requires careful experimental design to overcome detrimental multi-spin effects and to secure sufficient distance resolution in presence of multiple distances. In addition to obtaining mere distances, PD-EPR can also provide information on multimerisation degrees allowing to study binding equilibria and to determine dissociation constants.PostprintPeer reviewe

A general model to optimise CuII labelling efficiency of double-histidine motifs for pulse dipolar EPR applications

JLW is supported by the BBSRC DTP Eastbio. We thank the Leverhulme Trust for support (RPG-2018-397). This work was supported by equipment funding through the Wellcome Trust (099149/Z/12/Z) and BBSRC (BB/R013780/1). We gratefully acknowledge ISSF support to the University of St Andrews from the Wellcome Trust.Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to studies of biomolecules underpinning health and disease by providing highly accurate and precise geometric constraints. Combining double-histidine (dH) motifs with CuII spin labels shows promise for further increasing the precision of distance measurements, and for investigating subtle conformational changes. However, non-covalent coordination-based spin labelling is vulnerable to low binding affinity. Dissociation constants of dH motifs for CuII-nitrilotriacetic acid were previously investigated via relaxation induced dipolar modulation enhancement (RIDME), and demonstrated the feasibility of exploiting the double histidine motif for EPR applications at sub-μM protein concentrations. Herein, the feasibility of using modulation depth quantitation in CuII-CuII RIDME to simultaneously estimate a pair of non-identical independent KD values in such a tetra-histidine model protein is addressed. Furthermore, we develop a general speciation model to optimise CuII labelling efficiency, in dependence of pairs of identical or disparate KD values and total CuII label concentration. We find the dissociation constant estimates are in excellent agreement with previously determined values, and empirical modulation depths support the proposed model.Publisher PDFPeer reviewe

Isolation of EPR spectra and estimation of spin-states in two-component mixtures of paramagnets

This work was supported by Sasol Ltd. SC is grateful for a St Leonard’s postgraduate fellowship by the University of St Andrews.The presence of multiple paramagnetic species can lead to overlapping electron paramagnetic resonance (EPR) signals. This complication can be a critical obstacle for the use of EPR to unravel mechanisms and aid the understanding of earth abundant metal catalysis. Furthermore, redox or spin-crossover processes can result in the simultaneous presence of metal centres in different oxidation or spin states. In this contribution, pulse EPR experiments on model systems containing discrete mixtures of Cr(I) and Cr(III) or Cu(II) and Mn(II) complexes demonstrate the feasibility of the separation of the EPR spectra of these species by inversion recovery filters and the identification of the relevant spin states by transient nutation experiments. We demonstrate the isolation of component spectra and identification of spin states in a mixture of catalyst precursors. The usefulness of the approach is emphasised by monitoring the fate of the chromium species upon activation of an industrially used precatalyst system.PostprintPeer reviewe

Dipolar-coupled entangled molecular 4f qubits

S.P. thanks the Novo Nordisk Foundation for research grant NNF20OC0065610. EPR Equipment funding by BBSRC to B. E. B. (17 Alert grant BB/R013780/1) is also gratefully acknowledged.We demonstrate by use of continuous wave- and pulse-electron paramagnetic resonance spectroscopy on oriented single crystals of magnetically dilute YbIII ions in Yb0.01Lu0.99(trensal) that molecular entangled two-qubit systems can be constructed by exploiting dipolar interactions between neighboring YbIII centers. Furthermore, we show that the phase memory time and Rabi frequencies of these dipolar-interaction-coupled entangled two-qubit systems are comparable to the ones of the corresponding single qubits.Publisher PDFPeer reviewe

Historical Overview of the Human Population-Genetic Studies in Bosnia and Herzegovina: Small Country, Great Diversity

Modern Bosnia and Herzegovina is a multinational and multi-religious country, situated in the western part of the Balkan Peninsula in South-eastern Europe. According to recent archaeological findings, Bosnia and Herzegovina has been occupied by modern humans since the Palaeolithic period. The structure of Bosnia-Herzegovina’s human populations is very complex and specific, due to which it is interesting for various population-genetic surveys. The population of Bosnia and Herzegovina has been the focus of bio-anthropological and population genetics studies since the 19th century. The first known bio-anthropological analyses of Bosnia-Herzegovina population were primarily based on the observation of some phenotypic traits. Later examinations included cytogenetic and DNA based molecular markers. The results of all studies which have been done up to date showed no accented genetic difference among the populations (based on geographical regions) with quite high diversity within them. Human population of Bosnia and Herzegovina is closely related to other populations in the Balkans. However, there are still many interesting features hidden within the existing diversity of local human populations that are still waiting to be discovered and described

Unveiling the catalytic mechanism of a processive metalloaminopeptidase

Funding: C.M.C. is funded by the Wellcome Trust (210486/Z/18/Z and [204821/Z/16/Z] to the University of StAndrews). M.C.S.is funded by a PhD studentship from the University of St Andrews. B.E.B. acknowledges equipment funding by BBSRC (BB/R013780/1).Intracellular leucine aminopeptidases (PepA) are metalloproteases from the family M17. These enzymes catalyze peptide bond cleavage, removing N-terminal residues from peptide and protein substrates, with consequences for protein homeostasis and quality control. While general mechanistic studies using model substrates have been conducted on PepA enzymes from various organisms, specific information about their substrate preferences and promiscuity, choice of metal, activation mechanisms, and the steps that limit steady-state turnover remain unexplored. Here, we dissected the catalytic and chemical mechanisms of PaPepA: a leucine aminopeptidase from Pseudomonas aeruginosa. Cleavage assays using peptides and small-molecule substrate mimics allowed us to propose a mechanism for catalysis. Steady-state and pre-steady-state kinetics, pH rate profiles, solvent kinetic isotope effects, and biophysical techniques were used to evaluate metal binding and activation. This revealed that metal binding to a tight affinity site is insufficient for enzyme activity; binding to a weaker affinity site is essential for catalysis. Progress curves for peptide hydrolysis and crystal structures of free and inhibitor-bound PaPepA revealed that PaPepA cleaves peptide substrates in a processive manner. We propose three distinct modes for activity regulation: tight packing of PaPepA in a hexameric assembly controls substrate length and reaction processivity; the product leucine acts as an inhibitor, and the high concentration of metal ions required for activation limits catalytic turnover. Our work uncovers catalysis by a metalloaminopeptidase, revealing the intricacies of metal activation and substrate selection. This will pave the way for a deeper understanding of metalloenzymes and processive peptidases/proteases.Publisher PDFPeer reviewe