Double Electron-electron Resonance Data Research Articles

Bayesian Probabilistic Inference of Nonparametric Distance Distributions in DEER Spectroscopy.

Double electron-electron resonance (DEER) spectroscopy measures distance distributions between spin labels in proteins, yielding important structural and energetic information about conformational landscapes. Analysis of an experimental DEER signal in terms of a distance distribution is a nontrivial task due to the ill-posed nature of the underlying mathematical inversion problem. This work introduces a Bayesian probabilistic inference approach to analyze DEER data, assuming a nonparametric distance distribution with a Tikhonov smoothness prior. The method uses Markov Chain Monte Carlo sampling with a compositional Gibbs sampler to determine a posterior probability distribution over the entire parameter space, including the distance distribution, given an experimental data set. This posterior contains all of the information available from the data, including a full quantification of the uncertainty about the model parameters. The corresponding uncertainty about the distance distribution is visually captured via an ensemble of posterior predictive distributions. Several examples are presented to illustrate the method. Compared with bootstrapping, it performs faster and provides slightly larger uncertainty intervals.

Conformations of influenza A M2 protein in DOPC/DOPS and E. coli native lipids and proteins

We compared the conformations of the transmembrane domain (TMD) of influenza A M2 (IM2) protein reconstituted in 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPC/DOPS) bilayers to those in isolated Escherichia coli (E. coli) membranes, having preserved its native proteins and lipids. IM2 is a single-pass transmembrane protein known to assemble into a homo-tetrameric proton channel. To represent this channel, we made a construct containing the IM2’s TMD region flanked by the juxtamembrane residues. The single cysteine substitution, L43C, of leucine located in the bilayer polar region was paramagnetically tagged with a methanethiosulfonate nitroxide label for the electron spin resonance (ESR) study. For this particular residue, we probed the conformations of the spin-labeled IM2 reconstituted in DOPC/DOPS and isolated E. coli membranes using continuous-wave ESR and double electron-electron resonance (DEER) spectroscopy. The total protein-to-lipid molar ratio spanned the range from 1:230 to 1:10,400. The continuous-wave ESR spectra corresponded to very slow spin-label motion in both environments. In all cases, the DEER data were reconstructed into distance distributions with well-resolved peaks at 1.68 and 2.37 nm in distance and amplitude ratios of 1.41 ± 0.2 and 2:1, respectively. This suggests four nitroxide spin labels located at the corners of a square, indicative of an axially symmetric tetramer. The distance modeling of DEER data with molecular modeling software applied to the NMR molecular structures (PDB: 2L0J) confirmed the symmetry and closed state of the C-terminal exit pore of the IM2 TMD tetramer in agreement with the model. Thus, we can conclude that, under conditions of pH 7.4 used in this study, IM2 TMD has similar conformations in model lipid bilayers and membranes made of native E. coli lipids and proteins of comparable thickness and fluidity, notwithstanding the complexity of the E. coli membranes caused by their lipid diversity and the abundance of integral and peripheral membrane proteins.

Quantitative analysis of sterol-modulated monomer–dimer equilibrium of the β1-adrenergic receptor by DEER spectroscopy

G protein-coupled receptors (GPCR) activate numerous intracellular signaling pathways. The oligomerization properties of GPCRs, and hence their cellular functions, may be modulated by various components within the cell membrane (such as the presence of cholesterol). Modulation may occur directly via specific interaction with the GPCR or indirectly by affecting the physical properties of the membrane. Here, we use pulsed Q-band double electron-electron resonance (DEER) spectroscopy to probe distances between R1 nitroxide spin labels attached to Cys163 and Cys344 of the β1-adrenergic receptor (β1AR) in n-dodecyl-β-D-maltoside micelles upon titration with two soluble cholesterol analogs, cholesteryl hemisuccinate (CHS) and sodium cholate. The former, like cholesterol, inserts itself into the lipid membrane, parallel to the phospholipid chains; the latter is aligned parallel to the surface of membranes. Global quantitative analysis of DEER echo curves upon titration of spin-labeled β1AR with CHS and sodium cholate reveal the following: CHS binds specifically to the β1AR monomer at a site close to the Cys163-R1 spin label with an equilibrium dissociation constant [Formula: see text]~1.4± 0.4mM. While no direct binding of sodium cholate to the β1AR receptor was observed by DEER, sodium cholate induces specific β1AR dimerization ([Formula: see text]~35± 6mM and a Hill coefficient n~ 2.5±0.4) with intersubunit contacts between transmembrane helices 1 and 2 and helix 8. Analysis of the DEER data obtained upon the addition of CHS to the β1AR dimer in the presence of excess cholate results in dimer dissociation with species occupancies as predicted from the individual KD values.

An improvement to capture and visualize the protein conformational ensembles in double electron-electron resonance measurements.

Over the past decades, pulsed ESR double electron-electron resonance (DEER) has emerged as an effective biophysical tool for revealing protein conformational ensembles. With a collection of numerous interspin distances by DEER, they can be utilized to reveal the dynamic structural ensembles as opposed to static structures from other techniques. In a continuing effort to improve the technique, we present a combined approach of experimental and simulation advances that allows one to capture and visualize protein conformational ensembles in the absence of cryoprotectant additives. First, we present a rapid freezing method using a flexible fused silica microcapillary, enabling DEER measurement without the use of cryoprotectants. Second, we present a Monte Carlo based algorithm that allows one to efficiently utilize the sparse DEER data for deriving ensemble structures of a target protein. The Bid protein, a highly glycerol-sensitive protein, is used as a model. We show that our rapid-freezing DEER method can capture the Bid ensembles in the absence of glycerol, which was not possible with the conventional DEER setup. With the newly developed algorithm, we can efficiently visualize the Bid conformational ensembles, saving a lot for performing advanced structural modeling. Our combined method advances DEER for capturing and visualizing protein conformational ensembles.

Integrated AlphaFold2 and DEER investigation of the conformational dynamics of a pH-dependent APC antiporter

The Amino Acid-Polyamine-Organocation (APC) transporter GadC contributes to the survival of pathogenic bacteria under extreme acid stress by exchanging extracellular glutamate for intracellular γ-aminobutyric acid (GABA). Its structure, determined in an inward-facing conformation at alkaline pH, consists of the canonical LeuT-fold with a conserved five-helix inverted repeat, thereby resembling functionally divergent transporters such as the serotonin transporter SERT and the glucose-sodium symporter SGLT1. However, despite this structural similarity, it is unclear if the conformational dynamics of antiporters such as GadC follow the blueprint of these or other LeuT-fold transporters. Here, we used double electron-electron resonance (DEER) spectroscopy to monitor the conformational dynamics of GadC in lipid bilayers in response to acidification and substrate binding. To guide experimental design and facilitate the interpretation of the DEER data, we generated an ensemble of structural models in multiple conformations using a recently introduced modification of AlphaFold2 . Our experimental results reveal acid-induced conformational changes that dislodge the Cterminus from the permeation pathway coupled with rearrangement of helices that enables isomerization between inward- and outward-facing states. The substrate glutamate, but not GABA, modulates the dynamics of an extracellular thin gate without shifting the equilibrium between inward- and outward-facing conformations. In addition to introducing an integrated methodology for probing transporter conformational dynamics, the congruence of the DEER data with patterns of structural rearrangements deduced from ensembles of AlphaFold2 models illuminates the conformational cycle of GadC underpinning transport and exposes yet another example of the divergence between the dynamics of different families in the LeuT-fold.

Comparative evaluation of spin-label modeling methods for protein structural studies

Site-directed spin-labeling electron paramagnetic resonance spectroscopy is a powerful technique for the investigation of protein structure and dynamics. Accurate spin-label modeling methods are essential to make full quantitative use of site-directed spin-labeling electron paramagnetic resonance data for protein modeling and model validation. Using a set of double electron-electron resonance data from seven different site pairs on maltodextrin/maltose-binding protein under two different conditions using five different spin labels, we compare the ability of two widely used spin-label modeling methods, based on accessible volume sampling and rotamer libraries, to predict experimental distance distributions. We present a spin-label modeling approach inspired by canonical side-chain modeling methods and compare modeling accuracy with the established methods.

Quantitative Agreement between Conformational Substates of Holo Calcium-Loaded Calmodulin Detected by Double Electron–Electron Resonance EPR and Predicted by Molecular Dynamics Simulations

Calcium-loaded calmodulin (CaM/4Ca2+) comprises two domains that undergo rigid body reorientation from a predominantly extended conformation to a compact one upon binding target peptides. A recent replica-exchange molecular dynamics (MD) simulation on holo CaM/4Ca2+ suggested the existence of distinct structural clusters (substates) along the path from extended to compact conformers in the absence of substrates. Here, we experimentally demonstrate the existence of CaM/4Ca2+ substates trapped in local minima by three freezing/annealing regimes (slow, 40 s; intermediate, 1.5 s; fast, 0.5 ms) using pulsed Q-band double electron-electron resonance (DEER) EPR spectroscopy to measure interdomain distances between nitroxide spin-labels positioned at A17C and A128C in the N- and C-terminal domains, respectively. The DEER echo curves were directly fit to population-optimized P(r) pairwise distance distributions calculated from the coordinates of the MD clusters and compact crystal structure. DEER data on fully deuterated CaM/4Ca2+ were acquired at multiple values of the second echo period (10-35 μs) and analyzed globally to eliminate instrumental and overfitting artifacts and ensure accurate populations, peak positions, and widths. The DEER data for all three freezing regimes are quantitatively accounted for within experimental error by 5-6 distinct conformers comprising a predominantly populated extended form (60-75%) and progressively more compact states whose populations decrease as the degree of compactness increases. The shortest interdomain separation is found in the compact crystal structure, which has an occupancy of 4-6%. Thus, CaM/4Ca2+ samples high energy local minima comprising a few discrete substates of increasing compactness in a rugged energy landscape.

DEER experiments reveal fundamental differences between calmodulin complexes with IQ and MARCKS peptides in solution

Calmodulin (CaM) is a calcium-binding protein that regulates the function of many proteins by indirectly conferring Ca2+ sensitivity, and it undergoes a large conformational change on partners' binding. We compared the solution binding mode of the target peptides MARCKS and IQ by double electron-electron resonance (DEER) distance measurements and paramagnetic NMR. We combined nitroxide and Gd(III) spin labels, including specific substitution of one of the Ca2+ ions in the CaM mutant N60D by a Gd(III) ion. The binding of MARCKS to holo-CaM resulted neither in a closed conformation nor in a unique relative orientation between the two CaM domains, in contrast with the crystal structure. Binding of IQ to holo-CaM did generate a closed conformation. Using elastic network modeling and 12 distance restraints obtained from multiple holo-CaM/IQ DEER data, we derived a model of the solution structure, which is in reasonable agreement with the crystal structure.

Protein functional dynamics from the rigorous global analysis of DEER data: Conditions, components, and conformations.

The potential of spin labeling to reveal the dynamic dimension of macromolecules has been recognized since the dawn of the methodology in the 1960s. However, it was the development of pulsed electron paramagnetic resonance spectroscopy to detect dipolar coupling between spin labels and the availability of turnkey instrumentation in the 21st century that realized the full promise of spin labeling. Double electron-electron resonance (DEER) spectroscopy has seen widespread applications to channels, transporters, and receptors. In these studies, distance distributions between pairs of spin labels obtained under different biochemical conditions report the conformational states of macromolecules, illuminating the key movements underlying biological function. These experimental studies have spurred the development of methods for the rigorous analysis of DEER spectroscopic data along with methods for integrating these distributions into structural models. In this tutorial, we describe a model-based approach to obtaining a minimum set of components of the distance distribution that correspond to functionally relevant protein conformations with a set of fractional amplitudes that define the equilibrium between these conformations. Importantly, we review and elaborate on the error analysis reflecting the uncertainty in the various parameters, a critical step in rigorous structural interpretation of the spectroscopic data.

Pinholin S21 mutations induce structural topology and conformational changes

The bacteriophage infection cycle is terminated at a predefined time to release the progeny virions via a robust lytic system composed of holin, endolysin, and spanin proteins. Holin is the timekeeper of this process. Pinholin S21 is a prototype holin of phage Φ21, which determines the timing of host cell lysis through the coordinated efforts of pinholin and antipinholin. However, mutations in pinholin and antipinholin play a significant role in modulating the timing of lysis depending on adverse or favorable growth conditions. Earlier studies have shown that single point mutations of pinholin S21 alter the cell lysis timing, a proxy for pinholin function as lysis is also dependent on other lytic proteins. In this study, continuous wave electron paramagnetic resonance (CW-EPR) power saturation and double electron-electron resonance (DEER) spectroscopic techniques were used to directly probe the effects of mutations on the structure and conformational changes of pinholin S21 that correlate with pinholin function. DEER and CW-EPR power saturation data clearly demonstrate that increased hydrophilicity induced by residue mutations accelerate the externalization of antipinholin transmembrane domain 1 (TMD1), while increased hydrophobicity prevents the externalization of TMD1. This altered hydrophobicity is potentially accelerating or delaying the activation of pinholin S21. It was also found that mutations can influence intra- or intermolecular interactions in this system, which contribute to the activation of pinholin and modulate the cell lysis timing. This could be a novel approach to analyze the mutational effects on other holin systems, as well as any other membrane protein in which mutation directly leads to structural and conformational changes.

Conformational ensemble of a multidomain protein explored by Gd3+ electron paramagnetic resonance

Despite their importance in function, the conformational state of proteins and its changes are often poorly understood, mainly because of the lack of an efficient tool. MurD, a 47-kDa protein enzyme responsible for peptidoglycan biosynthesis, is one of those proteins whose conformational states and changes during their catalytic cycle are not well understood. Although it has been considered that MurD takes a single conformational state in solution as shown by a crystal structure, the solution nuclear magnetic resonance (NMR) study suggested the existence of multiple conformational state of apo MurD in solution. However, the conformational distribution has not been evaluated. In this work, we investigate the conformational states of MurD by the use of electron paramagnetic resonance (EPR), especially intergadolinium distance measurement using double electron-electron resonance (DEER) measurement. The gadolinium ions are fixed on specific positions on MurD via a rigid double-arm paramagnetic lanthanide tag that has been originally developed for paramagnetic NMR. The combined use of NMR and EPR enables accurate interpretation of the DEER distance information to the structural information of MurD. The DEER distance measurement for apo MurD shows a broad distance distribution, whereas the presence of the inhibitor narrows the distance distribution. The results suggest that MurD exists in a wide variety of conformational states in the absence of ligands, whereas binding of the inhibitor eliminates variation in conformational states. The multiple conformational states of MurD were previously implied by NMR experiments, but our DEER data provided structural characterization of the conformational variety of MurD.

Tylopeptin B peptide antibiotic in lipid membranes at low concentrations: Self-assembling, mutual repulsion and localization

The medium-length peptide Tylopeptin B possesses activity against Gram-positive bacteria. It binds to bacterial membranes altering their mechanical properties and increasing their permeability. This action is commonly related with peptide self-assembling, resulting in the formation of membrane channels. Here, pulsed double electron-electron resonance (DEER) data for spin-labeled Tylopeptin B in palmitoyl-oleoyl-glycero-phosphocholine (POPC) model membrane reveal that peptide self-assembling starts at concentration as low as 0.1 mol%; above 0.2 mol% it attains a saturation-like dependence with a mean number of peptides in the cluster <n> = 3.3. Using the electron spin echo envelope modulation (ESEEM) technique, Tylopeptin B molecules are found to possess a planar orientation in the membrane. In the peptide concentration range between 0.1 and 0.2 mol%, DEER data show that the peptide clusters have tendency of mutual repulsion, with a circle of inaccessibility of radius around 20 nm. It may be proposed that within this radius the peptides destabilize the membrane, providing so the peptide antimicrobial activity. Exploiting spin-labeled stearic acids as a model for free fatty acids (FFA), we found that at concentrations of 0.1–0.2 mol% the peptide promotes formation of lipid-mediated FFA clusters; further increase in peptide concentration results in dissipation of these clusters.

Bayesian Probabilistic Analysis of DEER Spectroscopy Data Using Parametric Distance Distribution Models.

Double electron-electron resonance (DEER) spectroscopy measures distance distributions between spin labels in proteins, yielding important structural and energetic information about conformational landscapes. Analysis of an experimental DEER signal in terms of a distance distribution is a nontrivial task due to the ill-posed nature of the underlying mathematical inversion problem. This work introduces a Bayesian probabilistic inference approach to analyze DEER data, using a multi-Gauss mixture model for the distance distribution. The method uses Markov chain Monte Carlo (MCMC) sampling to determine a posterior probability distribution over model parameter space. This distribution contains all the information available from the data, including a full quantification of the uncertainty about the parameters. The corresponding uncertainty about the distance distribution is captured via an ensemble of posterior predictive distributions. Several synthetic examples illustrate the method. An experimental example shows the importance of model checking and comparison using residual analysis and Bayes factors. Overall, the Bayesian approach allows for more robust inference about protein conformations from DEER spectroscopy.

Application of spherical harmonics for DEER data analysis in systems with a conformational distribution

Double electron–electron resonance (DEER) and other pulse electron paramagnetic resonance (EPR) techniques are valuable tools for determining distances between paramagnetic centres. DEER theory is well developed for a scenario where relative orientations of paramagnetic centres do not affect the DEER data. In particular, such theory enables a number of approaches for extracting distance distributions. However, in a more general case, when orientation selection effects become substantial, the analytical theory of DEER is less well developed, therefore quite commonly researchers rely on a comparison of some model-based simulations with experimental data. This work elaborates the theory of DEER with orientation selection effects, focusing on a scenario of a moderate conformational disorder, leading to an orientation distribution in a pair of paramagnetic centres. The analytical treatment based on expansions into spherical harmonics, provides important insights into the structure of DEER data. As follows from this treatment, DEER spectra with orientation selection can be represented as a linear combination of modified Pake pattern (MPP) components. The conformational disorder has a filtering effect on the weights of MPP components, specifically by significantly suppressing MPP components of higher degrees. The developed theory provides a pathway for model-based simulations of DEER data where orientation distribution is defined by analytical functions with parameters. The theory based on spherical harmonics expansions was also applied to develop an iterative processing algorithm based on Tikhonov regularization, which disentangles the distance and orientation information in a model-free manner. As an input, this procedure takes several DEER datasets measured at various positions of an EPR line, and outputs a distance distribution and orientation distribution information encoded in a set of coefficients related to the weights of MPP components. The model-based and model-free approaches based on the developed theory were validated for a nitroxide biradical and a spin-labelled protein.

Submillisecond Freezing Permits Cryoprotectant-Free EPR Double Electron-Electron Resonance Spectroscopy.

Double electron-electron resonance (DEER) EPR spectroscopy is a powerful method for obtaining distance distributions between pairs of engineered nitroxide spin-labels in proteins and other biological macromolecules. These measurements require the use of cryogenic temperatures (77 K or less) to prolong the phase memory relaxation time (Tm ) sufficiently to enable detection of a DEER echo curve. Generally, a cryoprotectant such as glycerol is added to protein samples to facilitate glass formation and avoid protein clustering (which can result in a large decrease in Tm ) during relatively slow flash freezing in liquid N2 . However, cryoprotectants are osmolytes and can influence protein folding/unfolding equilibria, as well as species populations in weak multimeric systems. Here we show that submillisecond rapid freezing, achieved by high velocity spraying of the sample onto a rapidly spinning, liquid nitrogen cooled copper disc obviates the requirement for cryoprotectants and permits high quality DEER data to be obtained in absence of glycerol. We demonstrate this approach on five different protein systems: protein A, the metastable drkN SH3 domain, urea-unfolded drkN SH3, HIV-1 reverse transcriptase, and the transmembrane domain of HIV-1 gp41 in lipid bicelles.

Conformational Response of N-Terminally Truncated Cytochrome P450 3A4 to Ligand Binding in Solution.

Human cytochrome P450 3A4 (CYP3A4) is a membrane-associated monooxygenase that is responsible for metabolizing >50% of the pharmaceuticals in the current market, so studying its chemical mechanism and structural changes upon ligand binding will help provide deeper insights into drug metabolism and further drug development. The best-characterized cytochrome P450 is a bacterial form, P450cam, which undergoes significant conformational changes upon binding substrate and its redox partner, putidaredoxin. In contrast, most crystal structures of CYP3A4 with or without ligands have shown few changes, although allosteric effects and multiple-substrate binding in solution are well-documented. In this study, we use double electron-electron resonance (DEER) to measure distances between spatially separated spin-labels on CYP3A4 and molecular dynamics to interpret the DEER data. These methods were applied to a soluble N-terminally truncated CYP3A4 form, and the results show that there are few changes in the average structure upon binding ketoconazole, ritonavir, or midazolam. However, binding of midazolam, but not ketoconazole or ritonavir, resulted in a significant change in the motion and/or disorder in the F/G helix region near the substrate binding pocket. These results suggest that soluble CYP3A4 behaves in a unique way in response to inhibitor and substrate binding.

CHARMM-GUI DEER facilitator for spin-pair distance distribution calculations and preparation of restrained-ensemble molecular dynamics simulations.

The double electron-electron resonance (DEER) is a powerful structural biology technique to obtain distance information in the range of 18 to 80 å by measuring the dipolar coupling between two unpaired electron spins. The distance distributions obtained from the experiment provide valuable structural information about the protein in its native environment that can be exploited using restrained ensemble molecular dynamics (reMD) simulations. We present a new tool DEER Facilitator in CHARMM-GUI that consists of two modules Spin-Pair Distributor and reMD Prepper to setup simulations that utilize information from DEER experiments. Spin-Pair Distributor provides a web-based interface to calculate the spin-pair distance distribution of labeled sites in a protein using MD simulations. The calculated distribution can be used to guide the selection of the labeling sites in experiments as well as validate different protein structure models. reMD Prepper facilities the setup of reMD simulations using different types of spin labels in four different environments including vacuum, solution, micelle, and bilayer. The applications of these two modules are demonstrated with several test cases. Spin-Pair Distributor and reMD Prepper are available at http://www.charmm-gui.org/input/deer and http://www.charmm-gui.org/input/deerre. DEER Facilitator is expected to facilitate advanced biomolecular modeling and simulation, thereby leading to an improved understanding of the structure and dynamics of complex biomolecular systems based on experimental DEER data. © 2019 Wiley Periodicals, Inc.

Hybrid Refinement of Heterogeneous Conformational Ensembles Using Spectroscopic Data.

Multistructured biomolecular systems play crucial roles in a wide variety of cellular processes but have resisted traditional methods of structure determination, which often resolve only a few low-energy states. High-resolution structure determination using experimental methods that yield distributional data remains extremely difficult, especially when the underlying conformational ensembles are quite heterogeneous. We have therefore developed a method to integrate sparse, multimultimodal spectroscopic data to obtain high-resolution estimates of conformational ensembles. We have tested our method by incorporating double electron-electron resonance data on the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein syntaxin-1a into biased molecular dynamics simulations. We find that our method substantially outperforms existing state-of-the-art methods in capturing syntaxin's open-closed conformational equilibrium and further yields new conformational states that are consistent with experimental data and may help in understanding syntaxin's function. Our improved methods for refining heterogeneous conformational ensembles from spectroscopic data will greatly accelerate the structural understanding of such systems.

Interpretation of spectroscopic data using molecular simulations for the secondary active transporter BetP.

Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and channels requires accurate depictions of conformational ensembles, and the manner in which they interchange as a function of environmental factors including substrates, lipids, and inhibitors. Spectroscopic techniques such as electron spin resonance (ESR) pulsed electron-electron double resonance (PELDOR), also known as double electron-electron resonance (DEER), provide a complement to atomistic structures obtained from x-ray crystallography or cryo-EM, since spectroscopic data reflect an ensemble and can be measured in more native solvents, unperturbed by a crystal lattice. However, attempts to interpret DEER data are frequently stymied by discrepancies with the structural data, which may arise due to differences in conditions, the dynamics of the protein, or the flexibility of the attached paramagnetic spin labels. Recently, molecular simulation techniques such as EBMetaD have been developed that create a conformational ensemble matching an experimental distance distribution while applying the minimal possible bias. Moreover, it has been proposed that the work required during an EBMetaD simulation to match an experimentally determined distribution could be used as a metric with which to assign conformational states to a given measurement. Here, we demonstrate the application of this concept for a sodium-coupled transport protein, BetP. Because the probe, protein, and lipid bilayer are all represented in atomic detail, the different contributions to the work, such as the extent of protein backbone movements, can be separated. This work therefore illustrates how ranking simulations based on EBMetaD can help to bridge the gap between structural and biophysical data and thereby enhance our understanding of membrane protein conformational mechanisms.

Structural Characterization of Biomolecules through Atomistic Simulations Guided by DEER Measurements

Double electron-electron resonance (DEER) is a popular technique that exploits attached spin labels to probe the collective dynamics of biomolecules in a native environment. Like most spectroscopic approaches, DEER detects an ensemble of states accounting for biomolecular dynamics as well as the labels' intrinsic flexibility. Hence, the DEER data alone do not provide high-resolution structural information. To disentangle this variability, we introduce a minimally biased simulation method to sample a structural ensemble that reproduces multiple experimental signals within the uncertainty. In contrast to previous approaches, our method targets the raw data themselves, and thereby it brings forth an unbiased molecular interpretation of the experiments. After validation on the T4 lysozyme, we apply this technique to interpret recent DEER experiments on a membrane transporter binding protein (VcSiaP). The results highlight the large-scale conformational movement that occurs upon substrate binding and reveal that the unbound VcSiaP is more open in solution than the X-ray structure.