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

Prediction and Selection of Spin Label Rotamers for High-Resolution Insight into Protein Structures by DEER Spectroscopy

Double electron-electron resonance (DEER) combined with site-directed spin labeling can provide distance distributions between selected protein residues to investigate protein structure and conformational heterogeneity. The utilization of the full quantitative information contained in DEER data requires effective protein and spin label modeling methods. Here, we review the application of DEER data to protein modeling. First, we discuss the significance of spin label modeling for accurate extraction of protein structural information and review the most popular label modeling methods. Next, we review several important aspects of protein modeling with DEER, including site selection, how DEER restraints are applied, common artifacts, and the unique potential of DEER data for modeling structural ensembles and conformational landscapes. Finally, we discuss common applications of protein modeling with DEER data and provide an outlook.

Analytical distance distributions in systems of spherical symmetry with applications to double electron–electron resonance

Modular assembly of bio-inspired supramolecular polymers is a powerful technique to develop new soft nanomaterials, and protein folding is a versatile basis for preparing such materials. Previous work demonstrated a significant difference in the physical properties of closely related supramolecular polymers composed of building blocks in which identical coiled-coil-forming peptides are cross-linked by one of two subtly different organic linkers (one flexible and the other rigid). Herein, we investigate the molecular basis for this observation by isolating a single subunit of the supramolecular polymer chain and probing its structure and conformational flexibility by double electron–electron resonance (DEER) spectroscopy. Experimental spin–spin distance distributions for two different labeling sites coupled with molecular dynamics simulations provide insights into how the linker structure impacts chain dynamics in the coiled-coil supramolecular polymer.

Structural Dynamics of Calmodulin using Bifunctional Spin Labels and Double Electron-Electron Resonance

Optimal Tikhonov regularization for DEER spectroscopy

Spectroscopy experiments are crucial to study membrane proteins for which traditional structure determination methods still prove challenging. Double electron-electron resonance (DEER) spectroscopy experiments provide protein residue-pair distance distributions that are indicative of their conformational heterogeneity. Atomistic molecular dynamics (MD) simulations are another tool that have been proven to be vital to study the structural dynamics of membrane proteins such as to identify inward-open, occluded, and outward-open conformations of transporter membrane proteins, among other partially open or closed states of the protein. Yet, studies have reported that there is no direct consensus between the distributional data from DEER experiments and MD simulations, which has challenged validation of structures obtained from long-timescale simulations and using simulations to design experiments. Current coping strategies for comparisons rely on heuristics, such as mapping the nearest matching peaks between two ensembles or biased simulations. Here we examine the differences in residue-pair distance distributions arising due to the choice of membranes around the protein and covalent modification of a pair of residues to nitroxide spin labels in DEER experiments. Through comparing MD simulations of two proteins, PepTSo and LeuT-both of which have been characterized using DEER experiments previously-we show that the proteins' dynamics are similar despite the choice of the detergent micelle as a membrane mimetic in DEER experiments. On the other hand, covalently modified residues show slight local differences in their dynamics and a huge divergence when the oxygen atom pair distances between spin labeled residues are measured rather than protein backbone distances. Given the computational expense associated with pairwise MTSSL labeled MD simulations, we examine the use of biased simulations to explore the conformational dynamics of the spin labels only to reveal that such simulations alter the underlying protein dynamics. Our study identifies the main cause for the mismatch between DEER experiments and MD simulations and will accelerate the development of potential mitigation strategies to improve the match.

<strong class="journal-contentHeaderColor">Abstract.</strong> Double electron–electron resonance (DEER) spectroscopy measures the distribution of distances between two electron spins in the nanometer range, often on doubly spin-labeled proteins, via the modulation of a refocused spin echo by the dipolar interaction between the spins. DEER is commonly conducted under conditions where the polarization of the spins is small. Here, we examine the DEER signal under conditions of high spin polarization, thermally obtainable at low temperatures and high magnetic fields, and show that the signal acquires a polarization-dependent out-of-phase component both for the intramolecular and intermolecular contributions. For the latter, this corresponds to a phase shift of the spin echo that is linear in the pump pulse position. We derive a compact analytical form of this phase shift and show experimental measurements using monoradical and biradical nitroxides at several fields and temperatures. The effect highlights a novel aspect of the fundamental spin physics underlying DEER spectroscopy.

Double electron-electron resonance (DEER) spectroscopy measures the distribution of distances between two electron spins in the nanometer range, often on doubly spin-labeled proteins, via the modulation of a refocused spin echo by the dipolar interaction between the spins. DEER is commonly conducted under conditions where the polarization of the spins is small. Here, we examine the DEER signal under conditions of high spin polarization, thermally obtainable at low temperatures and high magnetic fields, and show that the signal acquires a polarization-dependent out-of-phase component, both for the intra- and the inter-molecular contribution. For the latter, this corresponds to a phase shift of the spin echo that is linear in the pump pulse position. We derive a compact analytical form of this phase shift and show experimental measurements using monoradical and biradical nitroxides at several fields and temperatures. The effect highlights a novel aspect of the fundamental spin physics underlying DEER spectroscopy.

Spectroscopy and imaging are widely used to characterise systems structurally and functionally, and this information allows for the rational development of novel drugs and theranostics in many diverse areas of medicine. This thesis focuses on applying Electron Paramagnetic Resonance (EPR) techniques to determine the structure and function of protein molecules. A feasibility study to develop paramagnetic probes for EPR imaging is also presented.In EPR the probe is a paramagnetic centre and information is obtained by measuring interactions of this probe with its environment. In this thesis continuous wave (CW) EPR and Double Electron Electron Resonance (DEER) in conjunction with a site-directed spin labelling (SDSL) were employed to study structure and function of spin-labelled protein molecules. The techniques allow examination of molecular systems that cannot be crystallised or are too big for efficient NMR investigation. DEER provides information on distance distributions between two spin labels located on Cys-mutated residues of the protein molecule and can access longer distances in comparison with NMR. CW EPR provides information about the mobility of a single spin label which can be used to characterise protein dynamics. Structural modelling was used to combine the crystal structures and Molecular Dynamics (MD) simulations with DEER distance constraints to present a DEER-based structural model of the conformational variability of the protein molecule.Chapters 2 and 3 focus on characterising two metal ion substrate binding proteins (SBP), Zn2+-binding AdcA and Mn2+-binding PsaA, using DEER and CW EPR. The ATP-Binding Cassette (ABC) permeases, with which the SBPs are associated, are a primary importer used by bacteria to scavenge the essential first-row metal ions (e.g. iron, zinc, manganese) from a host environment. The process is essential for bacterial survival and propagation. Understanding the metal ion acquisition mechanisms by these SBPs can provide new opportunities for targeted drug development. Collectively, the CW EPR and DEER data along with crystal structures, differential scanning fluorimetry (DSF), Molecular Dynamics simulation, and smFRET microscopy, were able to determine a structural model for Zn2+-binding in the AdcA protein referred to as “trap-door” mechanism. Our data show that the “trap-door” mechanism employed by AdcA is different from the “spring-hammer” mechanism employed by PsaA.Non-Ribosomal Peptide Synthetase (NRPS) are the topic of chapter 4. NRPS’s are a family of mega enzymes that produce a diverse range of pharmaceuticals, yet how these molecular machines operate to produce such complex chemicals is very poorly understood. This project will investigate module 7 of the teicoplanin NRPS, a 200 kDa protein, which is the last step in the production of the glycopeptide antibiotics (GPA). A comprehensive structural and functional understanding of the teicoplanin NRPS machinery will provide a paradigm for this enzyme family, enabling the tailored re-engineering of the in vivo NRPS biosynthesis for the development of new antibiotics and pharmaceuticals. This thesis concentrates on a di-domain complex PCP-X from module 7 (Tcp12) of teicoplanin NRPS which contains the peptidyl carrier protein (PCP) domain and the X-domain. These two domains are joined by a long linker which is thought to be flexible. The goal is to investigate conformational dynamics of the PCP-X construct in different states. This includes the PCP domain in loaded and unloaded states, and how the PCP-X construct interacts with an OxyB (P450) protein. Using DEER data from a large number of double spin-labelled PCP-X mutants, rigid-body structural models based on the domain’s crystal structures were investigated to characterise the mobility of the PCP-X di-domain and compare it to very limited crystal structure data of the larger domain construct.Chapter 5 describes the application of the Maximum Entropy (MaxEnt) approach to 2D HYSCORE image reconstruction and its comparison with the conventionally utilised Discrete Fourier Transformation (DFT). HYSCORE is a widely used electron spin echo envelope (ESEEM) experiment used for measuring the nuclear quadrupole and hyperfine couplings. While EPR experiments are generally very sensitive, the time required for two and higher dimensional techniques are a significant limitation. The results show that the MaxEnt algorithm allows sampling of data non-uniformity, provides better sensitivity and an increase in resolution compared to DFT.Lastly, chapter 6 describes the development of PEG-based hyperbranched polymers and pPEGMA-coated gold nanoparticles as EPR imaging agents and sensors. Both nanoparticles have been shown as promising platforms that can be functionalized with targeting ligands and various imaging probes (i.e. a paramagnetic spin label) for the purpose of using multiple molecular imaging modalities for in vivo theranostics. The choice of a spin label dictates the possible application of the final agent, either for EPR imaging or EPR spectroscopy. The preliminary study assessed the sensitivity of commercially available EPR equipment and showed that nitroxides are more suitable for use as a sensor rather than an imaging probe. Future work based on the results in the thesis are discussed at the end of this chapter.

Confidence Analysis of DEER Data and its Structural Interpretation with Ensemble-Biased Metadynamics

Ionoids: Macrocyclic, organic and small inorganic building blocks electrostatically self-assemble in solution. The formed nano-objects feature a mobile interior. The molecules inside the aggregates seem to rotate freely on certain spatial positions. The hydrodynamic radius of the nano-assemblies, however, is precisely defined. Many societal challenges at the beginning of the 21st century lead to an apparent and growing need for functional materials and novel ways of materials synthesis and assembly. Rising to the challenge, the utilization of small, self-assembling building blocks for the bottom-up construction of new types of polymers and nanostructures has enjoyed increasing popularity among materials researchers in the recent past. Supramolecular materials like foldamers, surface films, nanoparticles, etc. are created by exploiting noncovalent forces1 leading to an ordered arrangement of nanoscale building blocks.2 In the search for new polymers based on noncovalent molecular forces, we are motivated by the idea of supramolecular or even polymer-like structures by self-assembly of small ionic monomers, merely formed from electrostatic and solvent interactions. We, in particular, focus on applying intrinsically nondirected electrostatic interactions to create nonetheless well- organized supramolecular structures.3 To achieve this aim, we here make use of a recently highlighted, multicationic molecular box (14+; Figure 1 a), developed by Sessler and co-workers, which has already been applied in self-assembly applications of metal–organic rotaxane frameworks.4 The molecular box 14+ seems to facilitate structure generation through self- assembly, indicated by, for example, the surprising ability to build supramolecular necklaces through incorporation of electron-rich aromatic guest molecules.5 However, not focused on short-ranged interactions but on new applications of long-ranged electrostatic forces at the nanoscale,6 we combine 14+ with small di-anionic salts, (KSO3)2CH2 (22−) and (KSO3)2NO (32−), to generate a defined assembly of cations and anions in solution (Figure 1 a). However, self-assembly due to electrostatic attraction is complex to describe qualitatively as well as quantitatively, because not only Coulomb forces but also entropy changes due to counterion release, solvation of ions, depletion forces, etc., contribute to the total free energy of a system. A central question in any application of solution-based ionic self-assembly therefore is: does any kind of ordered arrangement of the ionic building blocks take place?7 Intuitively one would assume the distribution of ions in solution to be of random, rather homogeneous nature. a) Molecular structure of 14+ (left) and 22−/32− (top right). b) A typical monoexponential fit of an autocorrelation function gained from DLS on 22−/14+ ionoids. c) Hydrodynamic radii of the 22−/14+ ionoids plotted against time, as derived from DLS measurements. t=0 days refers to the date of initial preparation. The upper and lower lines indicate error estimates derived from the fits of the autocorrelation functions. In the following, we show dynamic light scattering (DLS) data, which clearly illustrate that low-concentrated mixtures of anionic 22− and cationic 14+ self-assemble in solution to highly defined, monodisperse ionic clusters. In this special case, the distribution of ions in solution is therefore not at all random but well-ordered due to long-range electrostatic correlations. Performing molecular dynamics (MD) and Monte Carlo (MC) simulations, we further elucidate the internal constitution of the ionic clusters and show that a long-ranged order may be induced by electrostatic correlations of the cationic macrocycles. The simulations indicate that the monodisperse objects are constituted of relatively loosely bound ions that, however, occupy certain fixed coordinates in the clusters for at least orders of nanoseconds. The reliability of the computational data is additionally supported by double electron–electron resonance (DEER)8 spectroscopy data. Taking long-ranged (DLS, MC) and molecular (MD, DEER) insights together, we demonstrate, to the best of our knowledge for the first time, that size-controlled self-assembly through electrostatic interaction between small ions in solution is possible. The so formed objects can be regarded as loosely bound ion-based colloids, thus we propose the name ionoids. These ionoids are peculiar as they result from ion–ion correlations of moderately charged constituents at low concentrations, while long-range electrostatic correlations have so far only been discussed in the context of macroions.9 DLS experiments were performed on a system containing a mixture of 22−/14+ at an approximate ratio of 3 mM:1 mM in DMSO/88 % aqueous glycerol (1:1). Note that the exact concentration of 22− within the light-scattering sample is difficult to determine, as the solubility of the pure component 22−, in contrast to 32−, is only 3 mM in our solvent mixture. The measurements revealed monodisperse small objects in the solution, after an initial period of several days where no correlation could be detected. The fit of one correlation function measured at a scattering angle of 90 ° is depicted in Figure 1 b. Note the monoexponential nature of this fit, which indicates monodispersity of the aggregates in the solution. The intercept of the correlation function is unusually low, due to the small signal-to-noise ratio of <3, that is, the scattered intensity from the pure solvent mixture is nearly 40 % of the overall scattered intensity. As, in the absence of 22−, we found no correlation at all but only the DLS signature of the pure solvent mixture, it is at hand to deduce that these small objects stem from self- assembly of 22−–14+ clusters. The hydrodynamic radius of the self-assembled 22−–14+ cluster is quite small, that is, only 6.2 nm±0.2 nm can be observed 10 days after sample preparation, thus, growing very slow (+2 nm after 140 days; see Figure 1 c) and fostering an analogy with charge-stabilized colloids. It is important to note that all correlation functions measured over time can be fitted by a single exponential decay, and the resulting diffusion coefficient is independent of the scattering angle, which underlines the monodispersity in aggregate size. Thus, one can conclude that molecules 22− are necessary to overcome like-charge repulsion and to bring together 3–4 molecules 14+ (estimated from the size of approx. 6–7 nm) in an extraordinarily uniform manner. To support the reliability of the DLS data and to elucidate the internal constitution of the ionic clusters, we conducted all-atom MD simulations corresponding to a 32−/14+ system at a molar ratio of 6:2. In order to check the reliability of the MD simulation, we supplement it with DEER data and therefore replaced 22− by paramagnetic 32−. (This change may have an influence on special interactions in the ionoids, yet long-range electrostatic interactions remain unchanged. Note that the concentration in the simulations was three times higher than in the DEER measurements, to reduce the computation time). Starting from a random distribution of all molecules, we let the system equilibrate for 0.2 ns, and subsequently we extracted the trajectories for 8 ns with the aim of calculating the radial distribution function, g(r), of 32−. Figure 2 a shows a snapshot of the simulation after 2 ns, illustrating the molecular arrangement of the ions in such an ionoid-type cluster. Figure 2 b depicts the radial distance distribution of 32− summed up over 8 ns of simulation and over different time intervals of 1 ns. Prominent features in the overall g(r) at 1.9 nm, 2.4 nm, 3.5 nm are observable. Thus, the distribution of 32− in the simulated system is far from being random. Also, the distributions summed up in each case over 1 ns of simulation indicate favored population of distinct distances in the 32−/14+ system. At first sight, it is astonishing that the maxima in g(r) occur with a spacing of approximately 0.5 nm, indicating a periodic arrangement of the spin probes (Figure 2 b) . a) Snapshot after 2 ns of MD of the system 32−/14+ at 6:2 molar ratio. b) Pair correlation function of 32− molecules gained from MD simulation of the system 32−/14+ (6:2) summed up over different time intervals. The overall distance distribution is shown in orange (—). Note that over time, certain maxima in the distributions appear frequently, as indicated by the dashed lines. The black distribution depicts the distances gained from DEER on the same system (—). c) The Monte Carlo-derived radial distribution function for 32− calculated for two 14+ and six 32− molecules (—). The box size was 263 nm3. As can be seen, there are no distinct maxima in the distance distribution. Hence, the aggregation of 32− and 14+ is not based on Coulomb interaction and excluded volume effects alone. Solvent entropy may possibly contribute significantly to the generation of the ionoids, as the MD-derived distributions lead to reasonable agreements with the experimental data. The 14+–14+ distribution (—) reveals a long-range correlation at approximately 15 nm. For more details, see the Supporting Information. Although the inner constitution of the 32−–14+ aggregates could be assessed with MD, the global size of the so formed aggregates cannot be deduced from the simulation and compared with RH because of a box size of only 83 nm3. Consequently, we performed Monte Carlo (MC) simulations with a box size of 263 nm3 to elucidate the individual cluster size. These simulations included electrostatic potentials as well as excluded volume effects. However, the MC simulation showed that, due to mere electrostatic interactions and excluded volume, no distinct short-range association takes place (Figure 2 c). Yet, the MC simulations do reveal a surprising long-range correlation of 14+ at about 15 nm, suggesting a defined size of the observed ionic clusters, electrostatic and excluded volume effects to be essential for the formation of the observed ionic clusters (Figure 2 c and the Supporting Information), because the experimentally determined radius (6.2 nm–8.2 nm) and the size suggested from MC simulations (R≈7.5 nm) are in good agreement. Yet the distinct 32−–32− distances, as gained from MD, seem to be a consequence of additional entropic or solvent effects next to mere electrostatic effects. To further support the reliability of the computational data, we performed electron paramagnetic resonance (EPR)-based nanoscale distance measurement (DEER) experiments on a freeze-quenched 32−/14+ system to extract the experimental distance distribution of 32−. The resulting distance distribution is depicted in Figure 2 b (black curve). It shows the population of all 32−–32− distances of 6 mM molecules 32− around 2 mM molecules 14+ and can be compared directly with the g(r) gained from MD. Taking into account that the simulation features significantly higher concentrations than the experimentally measured system (the 14+ concentration was three times higher than in the experiment to increase the speed of calculation) and the restricted number of simulated 14+ and 32− molecules, the simulation-derived and the experimental distribution overall are in reasonable agreement. However, there are no distinct dipolar modulations observable in the DEER time traces (see the Supporting Information). Hence, the extracted distances (by Tikhonov regularization) have to be treated with caution. Yet, as they are clearly indicative of nonhomogeneous/nonrandom distributions of 32− and in addition are even in reasonably good agreement with the simulated distributions, one can assume that the distances extracted from the MD simulation resemble the situation inside the ionoids to a certain degree. Thus, judging from the simulation, the situation as shown in Figure 2 a can be taken as a representative snapshot of the distribution of ions in the solution. As it does not change significantly over time, it seems like the molecules 14+ are forced into a short-distance arrangement in order to minimize the potential electrostatic energy of the whole system. Divalent molecules 32−, thereby, could be acting as 'ionic glue' between macrocycles 14+. In doing so, the positions of local maxima in the distance distribution as derived from the MD simulation do not change significantly over time. This means that the spatial conformational ensemble covered seems to be relatively small. Once 32− is 'trapped' in an ensemble, it remains at a certain position for at least several nanoseconds. Continuous wave (CW)-EPR data at room temperature heavily support this hypothesis: the 32− probes still rotate relatively freely, although their rotational mobility is slightly restricted when small amounts of 14+ are added to a 32− solution (see the Supporting Information for the CW-EPR spectra and corresponding spectral simulations). At the same time, they still favor certain coordinates in the ionoids because of electrostatic attraction to 14+ and repulsion among themselves. It is likely that the combination of larger organic and small, yet doubly charged, inorganic building blocks is necessary for the emergence of ordered ionic assemblies, which persist in solution over time. Also, solvent effects could play a role, as DMSO is known to feature anomalous properties because of self-association.10 Taking all the experimental and computational data into account, one can conclude that the self-assembled objects feature a defined radius, although, on a molecular scale, their interior seems not to be strongly fixed: 'gluing' 32− molecules can still rotate relatively undisturbed. Yet, on the supramolecular scale, certain defined coordinates inside the aggregates are preferentially populated for at least some nanoseconds. Hence, the position of the 'glue′ is not arbitrary. From a physico-chemical point of view it is highly intriguing that an intrinsically nondirected force like electrostatic attraction/repulsion leads to the formation of highly defined nanoscopic objects. One can even control the initial size of the self-assembled ionoids precisely and keep them stable in solution for months. Thus, by choosing the right ions and counterions, their distribution in solution is not more or less homogeneous as one would expect intuitively for electrolyte solutions.10–11 Contrarily, they assemble into discrete arrangements as has been observed for macroions such as in DNA-condensation years ago .12 As the observed ionoids are based on noncovalent interaction among constituents, they probably are sensitive towards even minor changes in the solvent properties, ionic strength of the solution, and concentration of the constituents. These factors are likely to influence their composition and size. Furthermore, we expect that the observed growth of the ionoids will be fostered by a decrease in solvent viscosity, and that their occurrence is limited to certain solvents. Altogether, we have presented experimental and computational evidence for the formation of electrostatically self-assembled supramolecular structures in solution. The nano-assemblies have a well-defined size, and molecules in their interior populate, at least transiently, certain fixed positions in the cluster, while keeping a certain rotational freedom. The so formed ionoids can possibly open new routes towards the design and self-assembly of novel materials by exploiting electrostatic interactions between small molecules which have not attracted much attention in this field of research so far. In addition, these nano-objects are intriguing from a theoretical point of view, as long-range electrostatic correlations in solution have been postulated but, to the best of our knowledge, have not yet been observed directly for low-molecular electrolytes (unlike macroions).13 From the slow but steady size increase over the course of two months (Figure 1 c) one could even speculate that ionoids could be some sort of solvated precursor for precipitation or even crystallization nuclei. More experimental evidence would be needed to further substantiate such a picture, which currently is a mere speculation. Considering the small size of the ionoids, it is likely that X-ray and neutron scattering experiments will yield substantial insight into the specific properties of these solution structures. Such measurements are currently on our agenda. The responsiveness of ionoids towards stimuli such as concentration, solvent-mixture or charge ratio could also become interesting in the course of the search for new responsive materials. We thank Christian Bauer for technical support and Prof. Hans W. Spiess for continuing support. D.K. acknowledges support by the Gutenberg Academy of the University of Mainz. This work was financially supported by the Deutsche Forschungsgemeinschaft (DFG) under grant number HI 1094/2-1 and the Max Planck Graduate Center with the University of Mainz (MPGC). Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Hsp90 plays a central role in cell homeostasis by assisting folding and maturation of a large variety of clients. It is a homo-dimer, which functions via hydrolysis of ATP-coupled to conformational changes. Hsp90's conformational cycle in the absence of cochaperones is currently postulated as apo-Hsp90 being an ensemble of "open"/"closed" conformations. Upon ATP binding, Hsp90 adopts an active ATP-bound closed conformation where the N-terminal domains, which comprise the ATP binding site, are in close contact. However, there is no consensus regarding the conformation of the ADP-bound Hsp90, which is considered important for client release. In this work, we tracked the conformational states of yeast Hsp90 at various stages of ATP hydrolysis in frozen solutions employing electron paramagnetic resonance (EPR) techniques, particularly double electron-electron resonance (DEER) distance measurements. Using rigid Gd(III) spin labels, we found the C domains to be dimerized with same distance distribution at all hydrolysis states. Then, we substituted the ATPase Mg(II) cofactor with paramagnetic Mn(II) and followed the hydrolysis state using hyperfine spectroscopy and measured the inter-N-domain distance distributions via Mn(II)-Mn(II) DEER. The point character of the Mn(II) spin label allowed us resolve 2 different closed states: The ATP-bound (prehydrolysis) characterized by a distance distribution having a maximum of 4.3 nm, which broadened and shortened, shifting the mean to 3.8 nm at the ADP-bound state (posthydrolysis). This provides experimental evidence to a second closed conformational state of Hsp90 in solution, referred to as "compact." Finally, the so-called high-energy state, trapped by addition of vanadate, was found structurally similar to the posthydrolysis state.

Double electron-electron resonance (DEER) EPR is a powerful tool in structural biology, providing distances between pairs of spin labels. When the sample consists of a mixture of oligomeric species (e.g., monomer and dimer), the question arises as to how to assign the peaks in the DEER-derived probability distance distribution to the individual species. Here, we propose incorporating an EPR longitudinal electron relaxation (T1) inversion recovery experiment within a DEER pulse sequence to resolve this problem. The apparent T1 between dipolar coupled electron spins measured from the inversion recovery time (τinv) dependence of the peak intensities in the T1-edited DEER-derived probability P(r) distance distribution will be affected by the number of nitroxide labels attached to the biomolecule of interest, for example, two for a monomer and four for a dimer. We show that global fitting of all the T1-edited DEER echo curves, recorded over a range of τinv values, permits the deconvolution of distances between spin labels originating from monomeric (longer T1) and dimeric (shorter T1) species. This is especially useful when the trapping of spin labels in different conformational states during freezing gives rise to complex P(r) distance distributions. The utility of this approach is demonstrated for two systems, the β1 adrenergic receptor and a construct of the huntingtin exon-1 protein fused to the immunoglobulin domain of protein G, both of which exist in a monomer-dimer equilibrium.

The signaling state of the photoactive yellow protein (PYP) photoreceptor is transiently developed via isomerization of its blue-light-absorbing chromophore. The associated structural rearrangements have large amplitude but, due to its transient nature and chemical exchange reactions that complicate NMR detection, its accurate three-dimensional structure in solution has been elusive. Here we report on direct structural observation of the transient signaling state by combining double electron electron resonance spectroscopy (DEER), NMR, and time-resolved pump-probe X-ray solution scattering (TR-SAXS/WAXS). Measurement of distance distributions for doubly spin-labeled photoreceptor constructs using DEER spectroscopy suggests that the signaling state is well ordered and shows that interspin-label distances change reversibly up to 19 Å upon illumination. The SAXS/WAXS difference signal for the signaling state relative to the ground state indicates the transient formation of an ordered and rearranged conformation, which has an increased radius of gyration, an increased maximum dimension, and a reduced excluded volume. Dynamical annealing calculations using the DEER derived long-range distance restraints in combination with short-range distance information from (1)H-(15)N HSQC perturbation spectroscopy give strong indication for a rearrangement that places part of the N-terminal domain in contact with the exposed chromophore binding cleft while the terminal residues extend away from the core. Time-resolved global structural information from pump-probe TR-SAXS/WAXS data supports this conformation and allows subsequent structural refinement that includes the combined energy terms from DEER, NMR, and SAXS/WAXS together. The resulting ensemble simultaneously satisfies all restraints, and the inclusion of TR-SAXS/WAXS effectively reduces the uncertainty arising from the possible spin-label orientations. The observations are essentially compatible with reduced folding of the I(2)' state (also referred to as the 'pB' state) that is widely reported, but indicates it to be relatively ordered and rearranged. Furthermore, there is direct evidence for the repositioning of the N-terminal region in the I(2)' state, which is structurally modeled by dynamical annealing and refinement calculations.