Pulsed Electron-electron Double Resonance Research Articles

Structural Changes of a Doubly Spin-Labeled Chemically Driven Molecular Shuttle Probed by PELDOR Spectroscopy.

Gaining detailed information on the structural rearrangements associated with stimuli-induced molecular movements is of utmost importance for understanding the operation of molecular machines. Pulsed electron-electron double resonance (PELDOR) was employed to monitor the geometrical changes arising upon chemical switching of a [2]rotaxane that behaves as an acid-base-controlled molecular shuttle. To this aim, the rotaxane was endowed with stable nitroxide radical units in both the ring and axle components. The combination of PELDOR data and molecular dynamic calculations indicates that in the investigated rotaxane, the ring displacement along the axle, caused by the addition of a base, does not alter significantly the distance between the nitroxide labels, but it is accompanied by a profound change in the geometry adopted by the macrocycle.

Separation of intra- and intermolecular contributions to the PELDOR signal

Pulsed Electron–electron Double Resonance (PELDOR) is commonly used to measure distances between native paramagnetic centers or spin labels attached to complex biological macromolecules. In PELDOR the energies of electron magnetic dipolar interactions are measured by analyzing the oscillation frequencies of the recorded time resolved signal. Since PELDOR is an ensemble method, the detected signal contains contributions from intramolecular, as well as intermolecular electron spin interactions. The intramolecular part of the signal contains the information about the structure of the studied molecules, thus it is very important to accurately separate intra- and intermolecular contributions to the total signal. This separation can become ambiguous, when the length of the PELDOR signal is not much longer than twice the oscillation period of the signal. In this work we suggest a modulation depth scaling method, which can use short PELDOR signals in order to extract the intermolecular contribution. Using synthetic data we demonstrate the advantages of the new approach and analyze its stability with regard to signal noise. The method was also successfully tested on experimental data of three systems measured at Q-Band frequencies, two model compounds in deuterated and protonated solvents and one biological sample, namely BetP. The application of the new method with an assigned value of the signal modulation depth enables us to determine the interspin distances in all cases. This is especially interesting for the model compound with an interspin distance of 5.2nm in the protonated solvent and the biological sample, since an accurate separation of the intra- and intermolecular PELDOR signal contributions would be difficult with the standard approach in those cases.

Relative Orientation of POTRA Domains from Cyanobacterial Omp85 Studied by Pulsed EPR Spectroscopy

Many proteins of the outer membrane of Gram-negative bacteria and of the outer envelope of the endosymbiotically derived organelles mitochondria and plastids have a β-barrel fold. Their insertion is assisted by membrane proteins of the Omp85-TpsB superfamily. These proteins are composed of a C-terminal β-barrel and a different number of N-terminal POTRA domains, three in the case of cyanobacterial Omp85. Based on structural studies of Omp85 proteins, including the five POTRA-domain-containing BamA protein of Escherichia coli, it is predicted that anaP2 and anaP3 bear a fixed orientation, whereas anaP1 and anaP2 are connected via a flexible hinge. We challenged this proposal by investigating the conformational space of the N-terminal POTRA domains of Omp85 from the cyanobacterium Anabaena sp. PCC 7120 using pulsed electron-electron double resonance (PELDOR, or DEER) spectroscopy. The pronounced dipolar oscillations observed for most of the double spin-labeled positions indicate a rather rigid orientation of the POTRA domains in frozen liquid solution. Based on the PELDOR distance data, structure refinement of the POTRA domains was performed taking two different approaches: 1) treating the individual POTRA domains as rigid bodies; and 2) using an all-atom refinement of the structure. Both refinement approaches yielded ensembles of model structures that are more restricted compared to the conformational ensemble obtained by molecular dynamics simulations, with only a slightly different orientation of N-terminal POTRA domains anaP1 and anaP2 compared with the x-ray structure. The results are discussed in the context of the native environment of the POTRA domains in the periplasm.

A Bis‐Manganese(II)–DOTA Complex for Pulsed Dipolar Spectroscopy

High-spin gadolinium(III) and manganese(II) complexes have emerged as alternatives to standard nitroxide radical spin labels for measuring nanometric distances by using pulsed electron-electron double resonance (PELDOR or DEER) at high fields/frequencies. For certain complexes, particularly those with relatively small zero-field splitting (ZFS) and short distances between the two metal centers, the pseudosecular term of the dipolar coupling Hamiltonian is non-negligible. However, in general, the contribution from this term during conventional data analysis is masked by the flexibility of the molecule of interest and/or the long tethers connecting them to the spin labels. The efficient synthesis of a model system consisting of two [Mn(dota)](2-) (MnDOTA; DOTA(4-) =1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate) directly connected to the ends of a central rodlike oligo(phenylene-ethynylene) (OPE) spacer is reported. The rigidity of the OPE is confirmed by Q-band PELDOR measurements on a bis-nitroxide analogue. The Mn(II) -Mn(II) distance distribution profile determined by W-band PELDOR is in reasonable agreement with one simulated by using a simple rotamer analysis. The small degree of flexibility arising from the linking MnDOTA arm appears to outweigh the contribution from the pseudosecular term at this interspin distance. This study illustrates the potential of MnDOTA-based spin labels for measuring fairly short nanometer distances, and also presents an interesting candidate for in-depth studies of pulsed dipolar spectroscopy methods on Mn(II) -Mn(II) systems.

The histone chaperone Vps75 forms multiple oligomeric assemblies capable of mediating exchange between histone H3-H4 tetramers and Asf1-H3-H4 complexes.

Vps75 is a histone chaperone that has been historically characterized as homodimer by X-ray crystallography. In this study, we present a crystal structure containing two related tetrameric forms of Vps75 within the crystal lattice. We show Vps75 associates with histones in multiple oligomers. In the presence of equimolar H3–H4 and Vps75, the major species is a reconfigured Vps75 tetramer bound to a histone H3–H4 tetramer. However, in the presence of excess histones, a Vps75 dimer bound to a histone H3–H4 tetramer predominates. We show the Vps75–H3–H4 interaction is compatible with the histone chaperone Asf1 and deduce a structural model of the Vps75–Asf1-H3–H4 (VAH) co-chaperone complex using the Pulsed Electron-electron Double Resonance (PELDOR) technique and cross-linking MS/MS distance restraints. The model provides a molecular basis for the involvement of both Vps75 and Asf1 in Rtt109 catalysed histone H3 K9 acetylation. In the absence of Asf1 this model can be used to generate a complex consisting of a reconfigured Vps75 tetramer bound to a H3–H4 tetramer. This provides a structural explanation for many of the complexes detected biochemically and illustrates the ability of Vps75 to interact with dimeric or tetrameric H3–H4 using the same interaction surface.

Accurate Extraction of Nanometer Distances in Multimers by Pulse EPR.

Pulse electron paramagnetic resonance (EPR) is gaining increasing importance in structural biology. The PELDOR (pulsed electron–electron double resonance) method allows extracting distance information on the nanometer scale. Here, we demonstrate the efficient extraction of distances from multimeric systems such as membrane‐embedded ion channels where data analysis is commonly hindered by multi‐spin effects.

Ligand Induced Conformational Changes of a Membrane Transporter in E. coli Cells Observed with DEER/PELDOR.

An unrealized goal in structural biology is the determination of structure and conformational change at high resolution for membrane proteins within the cellular environment. Pulsed electron-electron double resonance (PELDOR) is a well-established technique to follow conformational changes in purified membrane protein complexes. Here we demonstrate the first proof of concept for the use of PELDOR to observe conformational changes in a membrane protein in intact cells. We exploit the fact that outer membrane proteins usually lack reactive cysteines and that paramagnetic spin labels entering the periplasm are selectively reduced to achieve specific labeling of the cobalamin transporter BtuB in Escherichia coli. We characterize conformational changes in the second extracellular loop of BtuB upon ligand binding and compare the PELDOR data with high-resolution crystal structures. Our approach avoids detergent extraction, purification, and reconstitution usually required for these systems. With this approach, structure, function, conformational changes, and molecular interactions of outer membrane proteins can be studied at high resolution in the cellular environment.

Bayesian Statistical Methods in the Analysis of DEER Data

Double Electron-Electron Resonance (DEER), also known as Pulsed Electron Double Resonance (PELDOR), is an increasingly important pulsed Electron Paramagnetic Resonance (EPR) technique for long distance (20-80 A) measurements in biomacromolecules. Notably, DEER reports a distribution of distances, P(r), corresponding to protein conformational ensembles in frozen glassy solutions. Due to the ill-conditioned, inverse nature of DEER data analysis, numerical regularization is required to obtain P(r). Further, a robust method for quantifying uncertainty in DEER distance distributions does not currently exist, complicating interpretation. Here, we compare methods of regularization parameter selection and demonstrate Bayesian statistical methods to quantify uncertainty in DEER distance distributions arising from measurement noise. We generate corresponding Bayesian credible intervals for visualization and interpretation.

Determination of nitroxide spin label conformations via PELDOR and X-ray crystallography.

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.

Conformation, self-aggregation, and membrane interaction of peptaibols as studied by pulsed electron double resonance spectroscopy.

Pulsed EPR methods, in particular pulsed electron double resonance (PELDOR) [or double electron-electron resonance (DEER)], are very sensitive to the dipole ··· dipole interaction between electron spins in a pair of free radicals. Using PELDOR, the conformations of a number of double radical-containing biomolecules have been determined. In this review article, we focused our attention on the application of this spectroscopy to nitroxide-labeled peptaibols. This is an emerging class of naturally occurring, relatively short, linear, helical peptide molecules endowed with hydrophobic character, capability to interact with and to alter the structure of membranes, and antibiotic activity. We extracted detailed information on the secondary structures of specifically site-directed, double nitroxide-labeled peptaibols under a variety of experimental conditions, including biologically relevant environments. Moreover, we examined in-depth peptaibol clustering, related to the marked propensity of these molecules to undergo self-association in model and whole-cell membrane systems, using mainly mono-nitroxide-containing synthetic analogs. Finally, based on the PELDOR data accumulated, we proposed models of supramolecular (quaternary) structures of peptaibols and their binding modes to membranes.

Peptides on the Surface: Spin-Label EPR and PELDOR Study of Adsorption of the Antimicrobial Peptides Trichogin GA IV and Ampullosporin A on the Silica Nanoparticles

The properties of antimicrobial peptides adsorbed on inorganic or organic surfaces are of interest for their potential applications in intracellular drug delivery. In this work, continuous-wave (CW) electron paramagnetic resonance (EPR) and pulsed electron-electron double resonance (PELDOR) techniques were applied to study adsorption of the short-sequence trichogin GA IV and the medium-length sequence ampullosporin A antimicrobial peptides on the monodisperse colloidal silica nanospheres of 20 nm diameter. The results obtained by CW EPR support the view that the adsorbed peptides form close-packed clusters. PELDOR data show that both trichogin and ampullosporin adsorbed on the silica surface possess a more disordered conformation as compared to that in solution. For ampullosporin, disordering is much more pronounced than for trichogin. After desorption, the peptides restored their conformations; upon adsorption the peptides in some cases may lose partly their biradical character.

Carr-Purcell Pulsed Electron Double Resonance with Shaped Inversion Pulses.

Pulsed electron paramagnetic resonance (EPR) spectroscopy allows the determination of distances, in the range of 1.5-8 nm, between two spin-labels attached to macromolecules containing protons. Unfortunately, for hydrophobic lipid-bound or detergent-solubilized membrane proteins, the maximum distance accessible is much lower, because of a strongly reduced coherence time of the electron spins. Here we introduce a pulse sequence, based on a Carr-Purcell decoupling scheme on the observer spin, where each π-pulse is accompanied by a shaped sech/tanh inversion pulse applied to the second spin, to overcome this limitation. This pump/probe excitation scheme efficiently recouples the dipolar interaction, allowing a substantially longer observation time window to be achieved. This increases the upper limit and accuracy of distances that can be determined in membrane protein complexes. We validated the method on a bis-nitroxide model compound and applied this technique to the trimeric betaine transporter BetP. Interprotomer distances as long as 6 nm could be reliably determined, which is impossible with the existing methods.

Location of the High-Affinity Mn(2+) Site in Photosystem II Detected by PELDOR.

The location of the high-affinity Mn(2+) site in apo-photosystem (PS) II was investigated by pulsed EPR. The electron-electron magnetic dipole interaction of 1.7 MHz between the YD(•) radical and Mn(2+) ion was observed using the pulsed electron-electron double resonance (PELDOR) technique, and the Mn(2+) ion was bound to one apo-PS II in the absence and presence of Ca(2+). PELDOR signals were calculated using the previously determined spin distribution on the YD(•) radical and its known position in the crystal structure, assuming that the specific Mn(2+) site was located in the oxygen evolving complex. The results show that the high-affinity Mn(2+) site is located at the position denoted by Mn4(A) in the native crystal structure. The Mn(2+) is coordinated with axial ligands Asp170 and Glu333 in the D1 polypeptide.

Pulse Electron Double Resonance Detected Multinuclear NMR Spectra of Distant and Low Sensitivity Nuclei and Its Application to the Structure of Mn(II) Centers in Organisms.

The ability to characterize the structure of metal centers beyond their primary ligands is important to understanding their chemistry. High-magnetic-field pulsed electron double resonance detected NMR (ELDOR-NMR) is shown to be a very sensitive approach to measuring the multinuclear NMR spectra of the nuclei surrounding Mn(II) ions. Resolved spectra of intact organisms with resonances arising from (55)Mn, (31)P, (1)H, (39)K, (35)Cl, (23)Na, and (14)N nuclei surrounding Mn(2+) centers were obtained. Naturally abundant cellular (13)C could be routinely measured as well. The amplitudes of the (14)N and (2)H ELDOR-NMR spectra were found to be linearly dependent on the number of nuclei in the ligand sphere. The evolution of the Mn(II) ELDOR-NMR spectra as a function of excitation time was found to be best described by a saturation phenomenon rather than a coherently driven process. Mn(II) ELDOR-NMR revealed details about not only the immediate ligands to the Mn(II) ions but also more distant nuclei, providing a view of their extended structures. This will be important for understanding the speciation and chemistry of the manganese complexes as well as other metals found in organisms.

Nanometric distance measurements between Mn(ii)DOTA centers.

Pulse electron-electron double resonance (PELDOR) is a versatile technique for probing the structures and functions of complex biological systems. Despite the recent interest in high-spin metal-ions for high field/frequency applications, PELDOR measurements of Mn(ii) remain relatively underexplored. Here we present Mn(ii)-Mn(ii) PELDOR distance measurements at 94 GHz on polyproline II (PPII) helices doubly spin-labeled with Mn(ii)DOTA, which are distinguished by their small zero-field interaction. The measured Mn-Mn distances and distribution profiles were in good agreement with the expected values from molecular models. Additional features in the frequency-domain spectra became apparent at certain combinations of detect and pump frequencies. Spin-Hamiltonian calculations showed that they likely arose from contributions from the pseudo-secular component of the dipolar interaction that were found to be non-negligible for Mn(ii)DOTA. However, the influence of the pseudo-secular component on the distance distribution profiles apparently was limited. The results show the potential of Mn(ii)DOTA spin labels for high-field PELDOR distance measurements in proteins and other biological systems.

Spin labeling and Double Electron-Electron Resonance (DEER) to Deconstruct Conformational Ensembles of HIV Protease.

An understanding of macromolecular conformational equilibrium in biological systems is oftentimes essential to understand function, dysfunction, and disease. For the past few years, our lab has been utilizing site-directed spin labeling (SDSL), coupled with electron paramagnetic resonance (EPR) spectroscopy, to characterize the conformational ensemble and ligand-induced conformational shifts of HIV-1 protease (HIV-1PR). The biomedical importance of characterizing the fractional occupancy of states within the conformational ensemble critically impacts our hypothesis of a conformational selection mechanism of drug-resistance evolution in HIV-1PR. The purpose of the following chapter is to give a timeline perspective of our SDSL EPR approach to characterizing conformational sampling of HIV-1PR. We provide detailed instructions for the procedure utilized in analyzing distance profiles for HIV-1PR obtained from pulsed electron-electron double resonance (PELDOR). Specifically, we employ a version of PELDOR known as double electron-electron resonance (DEER). Data are processed with the software package "DeerAnalysis" (http://www.epr.ethz.ch/software), which implements Tikhonov regularization (TKR), to generate a distance profile from electron spin-echo amplitude modulations. We assign meaning to resultant distance profiles based upon a conformational sampling model, which is described herein. The TKR distance profiles are reconstructed with a linear combination of Gaussian functions, which is then statistically analyzed. In general, DEER has proven powerful for observing structural ensembles in proteins and, more recently, nucleic acids. Our goal is to present our advances in order to aid readers in similar applications.

EPR Distance Measurements in Deuterated Proteins.

Pulsed electron double resonance technique, also known as double electron-electron resonance, jointly with site-directed spin labeling (SDSL) have been used extensively for studying structures and structural change. During the last decades, significant enhancements have been made by optimization of the experimental protocols, introducing new techniques for artifact suppression, and developing data analysis programs for extracting more reliable distance distributions. However, the distance determination by pulsed electron paramagnetic resonance is still facing some limitations, especially when studying spin-labeled proteins, due mainly to the fast relaxation time that imposes severe limitations on the maximum distances measurable and upon the sensitivity of such experiments. In the present work, we demonstrate the impact of the deuteration of the underlying protein, in addition to the solvent, on relaxation times, sensitivity, and on distance measurements.

A Conformational Sampling Model for Radical Catalysis in Pyridoxal Phosphate- and Cobalamin-dependent Enzymes

Cobalamin-dependent enzymes enhance the rate of C-Co bond cleavage by up to ∼10(12)-fold to generate cob(II)alamin and a transient adenosyl radical. In the case of the pyridoxal 5'-phosphate (PLP) and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5 aminomutase (OAM), it has been proposed that a large scale domain reorientation of the cobalamin-binding domain is linked to radical catalysis. Here, OAM variants were designed to perturb the interface between the cobalamin-binding domain and the PLP-binding TIM barrel domain. Steady-state and single turnover kinetic studies of these variants, combined with pulsed electron-electron double resonance measurements of spin-labeled OAM were used to provide direct evidence for a dynamic interface between the cobalamin and PLP-binding domains. Our data suggest that following ligand binding-induced cleavage of the Lys(629)-PLP covalent bond, dynamic motion of the cobalamin-binding domain leads to conformational sampling of the available space. This supports radical catalysis through transient formation of a catalytically competent active state. Crucially, it appears that the formation of the state containing both a substrate/product radical and Co(II) does not restrict cobalamin domain motion. A similar conformational sampling mechanism has been proposed to support rapid electron transfer in a number of dynamic redox systems.

Modelling multi-protein complexes using PELDOR distance measurements for rigid body minimisation experiments using XPLOR-NIH

Crystallographic and NMR approaches have provided a wealth of structural information about protein domains. However, often these domains are found as components of larger multi domain polypeptides or complexes. Orienting domains within such contexts can provide powerful new insight into their function. The combination of site specific spin labelling and Pulsed Electron Double Resonance (PELDOR) provide a means of obtaining structural measurements that can be used to generate models describing how such domains are oriented. Here we describe a pipeline for modelling the location of thio-reactive nitroxyl spin locations to engineered sties on the histone chaperone Vps75. We then use a combination of experimentally determined measurements and symmetry constraints to model the orientation in which homodimers of Vps75 associate to form homotetramers using the XPLOR-NIH platform. This provides a working example of how PELDOR measurements can be used to generate a structural model.

Conformationally restricted isoindoline-derived spin labels in duplex DNA: distances and rotational flexibility by pulsed electron-electron double resonance spectroscopy.

Three structurally related isoindoline-derived spin labels that have different mobilities were incorporated into duplex DNA to systematically study the effect of motion on orientation-dependent pulsed electron-electron double resonance (PELDOR) measurements. To that end, a new nitroxide spin label, (ExIm)U, was synthesized and incorporated into DNA oligonucleotides. (ExIm)U is the first example of a conformationally unambiguous spin label for nucleic acids, in which the nitroxide N-O bond lies on the same axis as the three single bonds used to attach the otherwise rigid isoindoline-based spin label to a uridine base. Continuous-wave (CW) EPR measurements of (ExIm)U confirm a very high rotational mobility of the spin label in duplex DNA relative to the structurally related spin label (Im)U, which has restricted mobility due to an intramolecular hydrogen bond. The X-band CW-EPR spectra of (ExIm)U can be used to identify mismatches in duplex DNA. PELDOR distance measurements between pairs of the spin labels (Im)U, (Ox)U, and (ExIm)U in duplex DNA showed a strong angular dependence for (Im)U, a medium dependence for (Ox)U, and no orientation effect for (ExIm)U. Thus, precise distances can be extracted from (ExIm)U without having to take orientational effects into account.