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We describe an approach for integrating distance restraints from Double Electron-Electron Resonance (DEER) spectroscopy into Rosetta with the purpose of modeling alternative protein conformations from an initial experimental structure. Fundamental to this approach is a multilateration algorithm that harnesses sets of interconnected spin label pairs to identify optimal rotamer ensembles at each residue that fit the DEER decay in the time domain. Benchmarked relative to data analysis packages, the algorithm yields comparable distance distributions with the advantage that fitting the DEER decay and rotamer ensemble optimization are coupled. We demonstrate this approach by modeling the protonation-dependent transition of the multidrug transporter PfMATE to an inward facing conformation with a deviation to the experimental structure of less than 2Å Cα RMSD. By decreasing spin label rotamer entropy, this approach engenders more accurate Rosetta models that are also more closely clustered, thus setting the stage for more robust modeling of protein conformational changes., Author summary Proteins transition between different conformations during function. Double Electron-Electron Resonance (DEER) spectroscopy enables the direct observation of structural rearrangements that underpin these transitions. Typically, histograms of distances between spin labels, called distance distributions, are measured under different conditions. Structural rearrangements that underlie conformational transitions are manifested by changes in the averages and widths of the distance distributions. To transform these distance distributions into restraints for modeling alternate protein conformations, we developed an algorithm in the modeling suite Rosetta for direct analysis of DEER primary data that yield the optimum ensemble of spin label positions in space, referred to as rotamers, that account for the data. We benchmarked the effectiveness of this algorithm using experimental data collected in two proteins, the model system T4 Lysozyme and the multidrug transporter PfMATE in an outward-facing conformation. We then used optimized spin label rotamers to model the inward-facing conformation of PfMATE from the starting outward-facing conformation. Our results demonstrate substantial improvements in both precision and accuracy among the resulting models. Further improvement of this strategy will enable modeling of protein conformational changes involving complex modes of movements.

Among coupled exchangers, CLCs uniquely catalyze the exchange of oppositely charged ions (Cl– for H+). Transport-cycle models to describe and explain this unusual mechanism have been proposed based on known CLC structures. While the proposed models harmonize with many experimental findings, gaps and inconsistencies in our understanding have remained. One limitation has been that global conformational change – which occurs in all conventional transporter mechanisms – has not been observed in any high-resolution structure. Here, we describe the 2.6 Å structure of a CLC mutant designed to mimic the fully H+-loaded transporter. This structure reveals a global conformational change to improve accessibility for the Cl– substrate from the extracellular side and new conformations for two key glutamate residues. Together with DEER measurements, MD simulations, and functional studies, this new structure provides evidence for a unified model of H+/Cl– transport that reconciles existing data on all CLC-type proteins., eLife digest Cells are shielded from harmful molecules and other threats by a thin, flexible layer called the membrane. However, this barrier also prevents chloride, sodium, protons and other ions from moving in or out of the cell. Channels and transporters are two types of membrane proteins that form passageways for these charged particles. Channels let ions flow freely from one side of the membrane to the other. To do so, these proteins change their three-dimensional shape to open or close as needed. On the other hand, transporters actively pump ions across the membrane to allow the charged particles to accumulate on one side. The shape changes needed for that type of movement are different: the transporters have to open a passageway on one side of the membrane while closing it on the other side, alternating openings to one side or the other. In general, channels and transporters are not related to each other, but one exception is a group called CLCs proteins. Present in many organisms, this family contains a mixture of channels and transporters. For example, humans have nine CLC proteins: four are channels that allow chloride ions in and out, and five are ‘exchange transporters’ that make protons and chloride ions cross the membrane in opposite directions. These proteins let one type of charged particle move freely across the membrane, which generates energy that the transporter then uses to actively pump the other ion in the direction needed by the cell. Yet, the exact three-dimensional changes required for CLC transporters and channels to perform their roles are still unknown. To investigate this question, Chavan, Cheng et al. harnessed a technique called X-ray crystallography, which allows scientists to look at biological molecules at the level of the atom. This was paired with other methods to examine a CLC mutant that adopts the shape of a normal CLC transporter when it is loaded with a proton. The experiments revealed how various elements in the transporter move relative to each other to adopt a structure that allows protons and chloride ions to enter the protein from opposite sides of the membrane, using separate pathways. While obtained on a bacterial CLC, these results can be applied to other CLC channels and transporters (including those in humans), shedding light on how this family transports charged particles across membranes. From bone diseases to certain types of seizures, many human conditions are associated with poorly functioning CLCs. Understanding the way these structures change their shapes to perform their roles could help to design new therapies for these health problems.

Methodological and technological advances in EPR spectroscopy have enabled novel insight into the structural and dynamic aspects of integral membrane proteins. In addition to an extensive toolkit of EPR methods, multiple spin labels have been developed and utilized, among them Gd(III)-chelates which offer high sensitivity at high magnetic fields. Here, we applied a dual labeling approach, employing nitroxide and Gd(III) spin labels, in conjunction with Q-band and W-band double electron-electron resonance (DEER) measurements to characterize the solution structure of the detergent-solubilized multidrug transporter MdfA from E. coli. Our results identify highly flexible regions of MdfA, which may play an important role in its functional dynamics. Comparison of distance distribution of spin label pairs on the periplasm with those calculated using inward- and outward-facing crystal structures of MdfA, show that in detergent micelles, the protein adopts a predominantly outward-facing conformation, although more closed than the crystal structure. The cytoplasmic pairs suggest a small preference to the outward-facing crystal structure, with a somewhat more open conformation than the crystal structure. Parallel DEER measurements with the two types of labels led to similar distance distributions, demonstrating the feasibility of using W-band spectroscopy with a Gd(III) label for investigation of the structural dynamics of membrane proteins.

Summary Escherichia coli 5′-nucleotidase is a two-domain enzyme exhibiting a unique 96° domain motion that is required for catalysis. Here we present an integrated structural biology study that combines DEER distance distributions with structural information from X-ray crystallography and computational biology to describe the population of presumably almost isoenergetic open and closed states in solution. Ensembles of models that best represent the experimental distance distributions are determined by a Monte Carlo search algorithm. As a result, predominantly open conformations are observed in the unliganded state indicating that the majority of enzyme molecules await substrate binding for the catalytic cycle. The addition of a substrate analog yields ensembles with an almost equal mixture of open and closed states. Thus, in the presence of substrate, efficient catalysis is provided by the simultaneous appearance of open conformers (binding substrate or releasing product) and closed conformers (enabling the turnover of the substrate).

SummaryIonotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory signaling in the nervous system. Despite the profound importance of iGluRs to neurotransmission, little is known about the structures and dynamics of intact receptors in distinct functional states. Here, we elucidate the structures of the intact GluA2 AMPA receptor in an apo resting/closed state, in an activated/pre-open state bound with partial agonists and a positive allosteric modulator, and in a desensitized/closed state in complex with fluorowilliardiine. To probe the conformational properties of these states, we carried out double electron-electron resonance experiments on cysteine mutants and cryoelectron microscopy studies. We show how agonist binding modulates the conformation of the ligand-binding domain “layer” of the intact receptors and how, upon desensitization, the receptor undergoes large conformational rearrangements of the amino-terminal and ligand-binding domains. We define mechanistic principles by which to understand antagonism, activation, and desensitization in AMPA iGluRs.

DEER (Double Electron Electron Resonance) is a powerful pulsed ESR (electron spin resonance) technique allowing the determination of distance histograms between pairs of nitroxide spin-labels linked to a protein in a native-like solution environment. However, exploiting the huge amount of information provided by ESR/DEER histograms to refine structural models is extremely challenging. In this study, a restrained ensemble (RE) molecular dynamics (MD) simulation methodology is developed to address this issue. In RE simulation, the spin-spin distance distribution histograms calculated from a multiple-copy MD simulation are enforced, via a global ensemble-based energy restraint, to match those obtained from ESR/DEER experiments. The RE simulation is applied to 51 ESR/DEER distance histogram data from spin-labels inserted at 37 different positions in T4 lysozyme (T4L). The rotamer population distribution along the five dihedral angles connecting the nitroxide ring to the protein backbone is determined and shown to be consistent with available information from X-ray crystallography. For the purpose of structural refinement, the concept of a simplified nitroxide dummy spin-label is designed and parameterized on the basis of these all-atom RE simulations with explicit solvent. It is demonstrated that RE simulations with the dummy nitroxide spin-labels imposing the ESR/DEER experimental distance distribution data are able to systematically correct and refine a series of distorted T4L structures, while simple harmonic distance restraints are unsuccessful. This computationally efficient approach allows experimental restraints from DEER experiments to be incorporated into RE simulations for efficient structural refinement.

Small heat shock proteins (sHSPs) are ubiquitous chaperones that bind and sequester non-native proteins preventing their aggregation. Despite extensive studies of sHSPs chaperone activity, the location of the bound substrate within the sHSP oligomer has not been determined. In this paper, we used cryoelectron microscopy (cryoEM) to visualize destabilized mutants of T4 lysozyme (T4L) bound to engineered variants of the small heat shock protein Hsp16.5. In contrast to wild type Hsp16.5, binding of T4L to these variants does not induce oligomer heterogeneity enabling cryoEM analysis of the complexes. CryoEM image reconstruction reveals the sequestration of T4L in the interior of the Hsp16.5 oligomer primarily interacting with the buried N-terminal domain but also tethered by contacts with the α-crystallin domain shell. Analysis of Hsp16.5-WT/T4L complexes uncovers oligomer expansion as a requirement for high affinity binding. In contrast, a low affinity mode of binding is found to involve T4L binding on the outer surface of the oligomer bridging the formation of large complexes of Hsp16.5. These mechanistic principles were validated by cryoEM analysis of an expanded variant of Hsp16.5 in complex with T4L and Hsp16.5-R107G, which is equivalent to a mutant of human αB-crystallin linked to cardiomyopathy. In both cases, high affinity binding is found to involve conformational changes in the N-terminal region consistent with a central role of this region in substrate recognition.

As secondary active transporters, CLCs harness energy stored in one ion concentration gradient (Cl- or H+) to pump the other ion against its electrochemical gradient. This occurs through tight coupling of protein conformational changes to ion binding, unbinding, and translocation events. Crystal structures suggest that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Here, we combine spectroscopy, cross-linking studies, crystallography, and computation to evaluate this simple model. Using 19F NMR and double electron-electron resonance (DEER) spectroscopy, we show that as [H+] is increased to enrich the outward-facing state, residues distant from Gluex move. Consistent with the functional relevance of this motion, a cross-link designed to inhibit the motions reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. Together, the results indicate the existence of a previously uncharacterized “outward-facing open” conformational state. A computational model based on the asymmetry exchange hypothesis for transporters with inverted-topology repeats (Forrest et al. 2008, PNAS) predicts such a state. To test the computational model, we monitored H+-induced structural changes using DEER spectroscopy on >20 sets of doubly labeled ClC-ec1 protein samples. Experimental distance distributions are compared with those derived from the ClC-ec1 X-ray structure and the model using restrained-ensemble molecular dynamics simulations (Roux et al. 2013, J Phys Chem B). Preliminary results show H+-dependent structural changes consistent with the computational model that can also be used to refine it. These results highlight the relevance of global structural changes in CLC function and provide the necessary foundation for understanding the Cl-/H+ coupling mechanism.

How does the sequence of a single Small Heat Shock Protein (sHSP) assemble into oligomers of different sizes? To gain insight into the underlying structural mechanism, we determined the crystal structure of an engineered variant of Methanocaldococcus jannaschii Hsp16.5 wherein a 14 amino acid peptide from human heat shock protein 27 (Hsp27) was inserted at the junction of the N-terminal region and the α-crystallin domain. In response to this insertion, the oligomer shell expands from 24 to 48 subunits while maintaining octahedral symmetry. Oligomer rearrangement does not alter the fold of the conserved α-crystallin domain nor does it disturb the interface holding the dimeric building block together. Rather, the flexible C-terminal tail of Hsp16.5 changes its orientation relative to the α-crystallin domain which enables alternative packing of dimers. This change in orientation preserves a peptide-in-groove interaction of the C-terminal tail with an adjacent β-sandwich thereby holding the assembly together. The interior of the expanded oligomer, where substrates presumably bind, retains its predominantly non-polar character relative to the outside surface. New large windows in the outer shell provide increased access to these substrate-binding regions, thus accounting for the higher affinity of this variant to substrates. Oligomer polydispersity regulates sHSPs chaperone activity in vitro and has been implicated in their physiological roles. The structural mechanism of Hsp16.5 oligomer flexibility revealed here, which is likely to be highly conserved across the sHSP superfamily, explains the relationship between oligomer expansion observed in disease-linked mutants and changes in chaperone activity.

Trapping membrane proteins in the confines of a crystal lattice obscures dynamic modes essential for interconversion between multiple conformations in the functional cycle. Moreover, lattice forces could conspire with detergent solubilization to stabilize a minor conformer in an ensemble thus confounding mechanistic interpretation. Spin labeling in conjunction with electron paramagnetic resonance (EPR) spectroscopy offers an exquisite window into membrane protein dynamics in the native-like environment of a lipid bilayer. Systematic application of spin labeling and EPR identifies sequence-specific secondary structures, defines their topology and their packing in the tertiary fold. Long range distance measurements (60 Å-80 Å) between pairs of spin labels enable quantitative analysis of equilibrium dynamics and triggered conformational changes. This review highlights the contribution of spin labeling to bridging structure and mechanism. Efforts to develop methods for determining structures from EPR restraints and to increase sensitivity and throughput promise to expand spin labeling applications in membrane protein structural biology.

EmrE, a member of the small multidrug transporters superfamily, extrudes positively charged hydrophobic compounds out of Escherichia coli cytoplasm in exchange for inward movement of protons down their electrochemical gradient. Although its transport mechanism has been thoroughly characterized, the structural basis of energy coupling and the conformational cycle mediating transport have yet to be elucidated. In this study, EmrE structure in liposomes and the substrate-induced conformational changes were investigated by systematic spin labeling and EPR analysis. Spin label mobilities and accessibilities describe a highly dynamic ligand-free (apo) conformation. Dipolar coupling between spin labels across the dimer reveals at least two spin label populations arising from different packing interfaces of the EmrE dimer. One population is consistent with antiparallel arrangement of the monomers, although the EPR parameters suggest deviations from the crystal structure of substrate-bound EmrE. Resolving these discrepancies requires an unusual disposition of TM3 relative to the membrane-water interface and a kink in its backbone that enables bending of its C-terminal part. Binding of the substrate tetraphenylphosphonium changes the environment of spin labels and their proximity in three transmembrane helices. The underlying conformational transition involves repacking of TM1, tilting of TM2, and changes in the backbone configurations of TM3 and the adjacent loop connecting it to TM4. A dynamic apo conformation is necessary for the polyspecificity of EmrE allowing the binding of structurally diverse substrates. The flexibility of TM3 may play a critical role in movement of substrates across the membrane.

SummaryAs many key proteins evade crystallization and remain too large for nuclear magnetic resonance spectroscopy, electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labeling offers an alternative approach for obtaining structural information. Such information must be translated into geometric restraints to be used in computer simulations. Here, distances between spin labels are converted into distance ranges between β carbons by using a “motion-on-a-cone” model, and a linear-correlation model links spin-label accessibility to the number of neighboring residues. This approach was tested on T4-lysozyme and αA-crystallin with the de novo structure prediction algorithm Rosetta. The results demonstrate the feasibility of obtaining highly accurate, atomic-detail models from EPR data by yielding 1.0 Å and 2.6 Å full-atom models, respectively. Distance restraints between amino acids far apart in sequence but close in space are most valuable for structure determination. The approach can be extended to other experimental techniques such as fluorescence spectroscopy, substituted cysteine accessibility method, or mutational studies.

We have identified sequence and structural determinants of oligomer size, symmetry, and polydispersity in the small heat shock protein super family. Using an insertion mutagenesis strategy that mimics evolutionary sequence divergence, we induced the ordered oligomer of Methanococcus jannaschii Hsp16.5 to transition to either expanded symmetric or polydisperse assemblies. A hybrid approach combining spin labeling EPR and cryoelectron microscopy imaging at 10A resolution reveals that the underlying plasticity is mediated by a packing interface with minimal contacts and a flexible C-terminal tether between dimers. Twenty-four dimeric building blocks related by octahedral symmetry assemble into the expanded symmetric oligomer. In contrast, the polydisperse variant has an ordered dimeric building block that heterogeneously packs to yield oligomers of various sizes. Increased exposure of the N-terminal region in the Hsp16.5 variants correlates with enhanced binding to destabilized mutants of T4 lysozyme, whereas deletion of this region reduces binding. Transition to larger intermediates with enhanced substrate binding capacity has been observed in other small heat shock proteins including lens alpha-crystallin mutants linked to congenital cataract. Together, these results provide a mechanistic perspective on substrate recognition and binding by the small heat shock protein superfamily.

Summary We report an approach for determining the structure of macromolecular assemblies by the combined application of cryo-electron microscopy (cryo-EM) and site-directed spin labeling electron paramagnetic resonance spectroscopy (EPR). This approach is illustrated for Hsp16.5, a small heat shock protein that prevents the aggregation of nonnative proteins. The structure of Hsp16.5 has been previously studied by both cryo-EM and X-ray crystallography. The crystal structure revealed a roughly spherical protein shell with dodecameric symmetry; however, residues 1–32 were found to be disordered. The cryo-EM reconstruction at 13 A resolution appeared similar to the crystal structure but with additional internal density corresponding to the N-terminal regions of the 24 subunits. In this study, a systematic application of site-directed spin labeling and EPR spectroscopy was carried out. By combining the EPR constraints from spin label accessibilities and proximities with the cryo-EM density, we obtained an atomic model for a portion of the Hsp16.5 N-terminal region in the context of the oligomeric complex.

Mammalian small heat shock proteins (sHSP) form polydisperse and dynamic oligomers that undergo equilibrium subunit exchange. Current models of their chaperone activity hypothesize that recognition and binding of protein non-native states involve changes in the oligomeric state. The equivalent thermodynamic representation is a set of three coupled equilibria that includes the sHSP oligomeric equilibrium, the substrate folding equilibrium, and the equilibrium binding between the sHSP and the substrate non-native states. To test this hypothesis and define the binding-competent oligomeric state of human Hsp27, we have perturbed the two former equilibria and quantitatively determined the consequences on binding. The substrate is a set of T4 lysozyme (T4L) mutants that bind under conditions that favor the folded state over the unfolded state by 10(2)-10(4)-fold. The concentration-dependent oligomer equilibrium of Hsp27 was perturbed by mutations that alter the relative stability of two major oligomeric states including phosphorylation-mimicking mutations that result in the dissociation to a small multimer over a wide range of concentrations. Correlation of binding isotherms with size exclusion chromatography analysis of the Hsp27 oligomer equilibrium demonstrates that the multimer is the binding-competent state. Binding occurs through two modes, each characterized by different affinity and number of binding sites, and results in T4L.Hsp27 complexes of different hydrodynamic properties. Mutants of the Hsp27 phosphorylation mimic that reverse the reduction in oligomer size also reduce the extent of T4L binding. Taken together, these results suggest a central role for the oligomeric equilibrium in regulating the chaperone activity of sHSP. The mutants identify sequence features important for modulating this equilibrium.

Age-related changes in protein-protein interactions in the lens play a critical role in the temporal evolution of its optical properties. In the relatively non-regenerating environment of the fiber cells, a critical determinant of these interactions is partial or global unfolding as a consequence of post-translational modifications or chemical damage to individual crystallins. One type of attractive force involves the recognition by α-crystallins of modified proteins prone to unfolding and aggregation. In this paper, we explore the energetic threshold and the structural determinants for the formation of a stable complex between α-crystallin and βB2-crystallin as a consequence of destabilizing mutations in the latter. The mutations were designed in the framework of a folding model that proposes the equilibrium population of a monomeric intermediate. Binding to α-crystallin is detected through changes in the emission properties of a bimane fluorescent probe site-specifically introduced at a solvent exposed site in βB2-crystallin. α-Crystallin binds the various βB2-crystallin mutants, although with a significantly lower affinity relative to destabilized T4 lysozyme mutants. The extent of binding, while reflective of the overall destabilization, is determined by the dynamic population of a folding intermediate. The existence of the intermediate is inferred from the biphasic bimane emission unfolding curve of a mutant designed to disrupt interactions at the dimer interface. The results of this paper are consistent with a model in which the interaction of α-crystallins with substrates is not solely triggered by an energetic threshold but also by the population of excited states even under favorable folding conditions. The ability of α-crystallin to detect subtle changes in the population of βB2-crystallin excited states supports a central role for this chaperone in delaying aggregation and scattering in the lens.

To elucidate the mechanism of alphaA-crystallin chaperone function, a detailed thermodynamic analysis of its binding to destabilized, site-directed mutants of T4 lysozyme was carried out. The selected mutants form a ladder of stabilities spanning the 5-10 kcal/mol range of free energy of unfolding. The crystal structures of the majority of the mutants have been previously determined and found to be similar to that of the wild type with no evidence of static local unfolding. Complex formation between alphaA-crystallin and T4 lysozyme was observed directly via the changes in the electron paramagnetic resonance lineshape of a nitroxide introduced at a non-destabilizing, solvent exposed site in T4 lysozyme. AlphaA-crystallin differentially interacts with the mutants, binding the more destabilized ones to a larger extent despite the similar structure of their native states. Our results suggest that the states recognized by alphaA-crystallin are non-native excited states distinct from the unfolded state. Stable complexes are formed when the free energy of binding to alphaA-crystallin is on the order of the free energy associated with the transition from the excited state to the native state. Biphasic binding isotherms reveal two modes of interactions with distinct affinities and stoichiometries. Highly destabilized mutants preferentially bind to the high capacity mode, suggesting conformational preference in the use of each mode. Furthermore, binding can be enhanced by increased temperature and pH, which may be reflecting conformational changes in alphaA-crystallin oligomeric structure.

We present a novel method for mapping proximity within proteins. The method exploits the quenching of the fluorescent label bimane by nearby Trp residues. In studies of T4 lysozyme we show that this effect appears to be distance dependent and orientation specific. Specifically, we show that a proximal Trp residue can reduce bimane fluorescence intensity by up to 500% and induce complicated fluorescence decay kinetics. Replacing the neighboring Trp residue with phenylalanine removes these spectral perturbations. The advantages of using the Trp quenching of bimane fluorescence for protein structural studies include the low amount of protein required and the substantial simplification of labeling strategies. We anticipate this method will prove suitable for a wide array of high-throughput protein studies such as protein folding, the detection of protein-protein interactions, and, most importantly, the dynamic monitoring of conformational changes.

ClC-ec1, a prokaryotic Cl-/H+ antiporter in the CLC family, has been crystallized under many conditions. Yet, aside from local structural differences at the chloride binding site, only one major conformation is observed. This failure of X-ray crystallography to reveal the unknown conformational states in the transport cycle motivates the use of alternative approaches. Using 19F-NMR, a previous study demonstrated that a tyrosine residue > 20 A from the chloride-transport pathway undergoes antiport-specific structural changes (Elvington et al. 2009. EMBO J). We combine computational and experimental approaches to further investigate possible protein movements during the antiport cycle. A computational model of ClC-ec1 based on the inverted topology repeat hypothesis (Forrest et al. 2008. PNAS) predicts structural changes that alter the accessibility of pathways to the central Cl- and H+ binding sites. To test the model, we introduced nitroxide paramagnetic spin labels to the ClC-ec1 homodimer via site-directed spin labeling and monitored structural changes using double electron-electron resonance (DEER) spectroscopy. Preliminary results are consistent with the inverted-topology repeat model and suggest that the antiport cycle involves structural changes beyond local movement at the chloride-binding site.

Active transporters couple the energetically uphill transport of substrate against their concentration gradients to direct use of ATP energy or the discharge of electrochemical ion gradients. Mapping the conformational motion that transduces the energy input into the work of substrate translocation is central to understanding transport mechanism. Spin labeling and electron paramagnetic resonance (EPR) spectroscopy offers a unique window into membrane protein conformational dynamics in the native-like environment of a lipid bilayer. Specifically, Double Electron-Electron Resonance (DEER) spectroscopy enables long range distance measurements between two spin labels yielding a distance distribution. The amplitude of movements can be determined from changes in the average distance while the width of the distance distribution can be interpreted in terms of underlying transporter conformational equilibria. We have applied spin labeling and DEER to define the proton-activated conformational switch of the multidrug antiporter LmrP, to uncover asymmetric conformations of the ABC heterodimer BrmCD, and to test models of the Na-symporter LeuT. These examples illustrate the importance of spectroscopic approaches for bridging structure and mechanism for membrane proteins.View Large Image | View Hi-Res Image | Download PowerPoint Slide

αA-Crystallin, a member of the small heat shock protein (sHsp) family, is a large multimeric protein composed of 30–40 identical subunits. Its quaternary structure is highly dynamic, with subunits capable of freely and rapidly exchanging between oligomers. We report here the development of a fluorescence resonance energy transfer method for measuring structural compatibility between αA-crystallin and other proteins. We found that Hsp27 and αB-crystallin readily exchanged with fluorescence-labeled αA-crystallin, but not with other proteins structurally unrelated to sHsps. Truncation of 19 residues from the N terminus or 10 residues from the C terminus of αA-crystallin did not significantly change its subunit organization or exchange rate constant. In contrast, removal of the first 56 or more residues converts αA-crystallin into a predominantly small multimeric form consisting of three or four subunits, with a concomitant loss of exchange activity. These findings suggest residues 20–56 are essential for the formation of large oligomers and the exchange of subunits. Similar results were obtained with truncated Hsp27 lacking the first 87 residues. We further showed that the exchange rate is independent of αA-crystallin concentration, suggesting subunit dissociation may be the rate-limiting step in the exchange reaction. Our findings reveal a quarternary structure of αA-crystallin, consisting of small multimers of αA-crystallin subunits in a dynamic equilibrium with the oligomeric complex.

The folding pattern of the alpha-crystallin domain, a conserved protein module encoding the molecular determinants of structure and function in the small heat-shock protein superfamily, was determined in the context of the lens protein alphaA-crystallin by systematic application of site-directed spin labeling. The sequence-specific secondary structure was assigned primarily from nitroxide scanning experiments in which the solvent accessibility and mobility of a nitroxide probe were measured as a function of residue number. Seven beta-strands were identified and their orientation relative to the aqueous solvent determined, thus defining the residues lining the hydrophobic core. The pairwise packing of adjacent strands in the primary structure was deduced from patterns of proximities in nitroxide pairs with one member on the exposed surface of each strand. In addition to identifying supersecondary structures, these proximities revealed that the seven strands are arranged in two beta-sheets. The overall packing of the two sheets was determined by application of the general rules of protein structure and from proximities in nitroxide pairs designed to distinguish between known all beta-sheet folds. Our data are consistent with an immunoglobulin-like fold consisting of two aligned beta-sheets. Comparison of this folding pattern to that of the evolutionary distant alpha-crystallin domain in Methanococcus jannaschii heat-shock protein 16.5 reveals a conserved core structure with the differences sequestered at one edge of the beta-sandwich. A beta-strand deletion in alphaA-crystallin disrupts a subunit interface and allows for a different dimerization motif. Putative substrate binding regions appear to include a buried loop and a buried turn, suggesting that the chaperone function involves a disassembly of the oligomer.

Site-directed spin labeling was used to investigate quaternary interactions along a conserved sequence in the α-crystallin domain of αA-crystallin, heat-shock protein 27 (HSP 27), and Mycobacterium tuberculosis heat-shock protein (HSP 16.3). In previous work, it was demonstrated that this sequence in αA-crystallin and HSP 27 forms a β-strand involved in subunit contacts. In this study, the symmetry and geometry of the resulting interface were investigated. For this purpose, the pattern of spin-spin interactions was analyzed, and the number of interacting spins was determined in αA-crystallin and HSP 27. The results reveal a 2-fold symmetric interface consisting of two β-strands interacting near their N termini in an antiparallel fashion. Remarkably, subunit interactions along this interface persist when the α-crystallin domains are expressed in isolation. Because this domain in αA-crystallin forms dimers and tetramers, it is inferred that interactions along this interface mediate the formation of a basic dimeric unit. In contrast, in HSP 16.3, spin-spin interactions are observed at only one site near the C terminus of the sequence. Furthermore, cysteine substitutions at residues flanking the N terminus resulted in the dissociation of the oligomeric structure. Analysis of the spin-spin interactions and size exclusion chromatography indicates a 3-fold symmetric interface. Taken together, our results demonstrate that subunit interactions in the α-crystallin domain of mammalian small heat-shock proteins assemble a basic building block of the oligomeric structure. Sequence divergence in this domain results in variations in the size and symmetry of the quaternary structure between distant members of the small heat-shock protein family.

The LeuT Fold is characterized by a five transmembrane helix, inverted repeat structure that has been observed in an increasing number of physiologically important transporter families. The recurrence of this structural scaffold among the LeuT Fold transporters has stimulated conjectures of a unified mechanism of alternating access despite significant diversity in sequence, function, and ion coupling stoichiometries. Mhp1, a Na+-coupled symporter of the Nucleobase:Cation Symporter 1 (NCS1) family, was the first LeuT Fold member to be structurally characterized by a full complement of canonical states including outward-facing, inward-facing, and occluded conformations, which implied a rocking bundle mechanism. Here we report an investigation of Mhp1 dynamics by electron paramagnetic resonance (EPR) spectroscopy. In this analysis, double electron-electron resonance (DEER) distance distributions between pairs of spin labels monitored conformational changes in response to ligand-binding. These experimental distance distributions were directly compared with the Mhp1 crystal structures by applying MD simulations of spin label rotamers at the double mutant sites and simulating distance histograms. The results of this investigation support the assertion that the crystallographically-captured conformations of Mhp1 are reflected in solution as major conformations and that Mhp1 operates primarily through a rocking bundle-like mechanism. However, unlike LeuT, Na+ binding does not induce transitions between inward- and outward-facing conformations, rather it appears that Na+ powers transport exclusively through modulation of substrate binding affinity. The model of Mhp1 transport proposed here depends critically on the description of conformational equilibria, as the Mhp1 transport cycle progresses through states using low probability transitions. A comparative analysis of the LeuT and Mhp1 mechanisms highlight significant mechanistic divergence that we speculate may in part be explained by the differential mechanisms of coupling to the Na+ gradient.

The folding pattern of the segment of alphaA-crystallin encoded by exon 2 and containing putative substrate binding sites was explored using site-directed spin labeling (SDSL). For this purpose, a nitroxide scan was carried out between residues 60 and 108. At each site, structural constraints describing the local environment and topography were obtained from analysis of the nitroxide mobility and its solvent accessibility. Periodic patterns in the sequence-specific variation of these parameters were used to assign the secondary structure along the sequence. Geometric constraints describing the packing of secondary structure were deduced from patterns of proximities in 20 nitroxide pairs, specifically designed to differentiate between supersecondary structural motifs. Our data, in conjunction with those of Berengian et al. [Berengian, A. R., Bova, M. P., and Mchaourab, H. S. (1997) Biochemistry 36, 9951-9957], reveal that the fold of the segment between residues 84 and 120 consists of an antiparallel beta-sheet of three strands arranged in consecutive beta-hairpins. The boundaries of the sheet are defined at one end by a surface of isologous association and on the other end by an unstructured, charged interdomain segment. One of the putative substrate binding segments overlaps a buried loop, suggesting that the structural origin of the thermal activation of binding is the transient exposure of this site. This paper describes and implements a general strategy for experimental fold recognition using SDSL. The results of its application to alphaA-crystallin provide the first experimental insight into the folding pattern of the subunit and establish the structural context necessary to understand molecular recognition and substrate binding.

Twelve sequential single cysteine mutants of alphaA-crystallin extending between amino acids Y109 and L120 were prepared and reacted with a sulfhydryl specific spin label in order to investigate the role of this sequence in the assembly of the alphaA-crystallin quaternary structure and its chaperone-like function. The sequence is located in the region of highest homology in the alpha-crystallin domain, a stretch of 100 amino acids conserved among lens alpha-crystallins and small heat-shock proteins (sHSPs). Analysis of the solvent accessibility and mobility of the attached nitroxides reveals that the sequence, as a whole, is relatively sequestered from the aqueous solvent. Furthermore, as teh nitroxide is scanned across the sequence, both mobility and accessibility vary with a periodicity of 2, demonstrating that the backbone conformation is that of a beta-strand. Once face of the strand, containing the highly conserved residues R112 and R116, is buried with virtually no accessibility to the aqueous solvent. Equivalent strands from different subunits are in close spatial proximity, as inferred from spin-spin interactions between identical residues along the strand. Taken together, our results are consistent with the hypothesis that the alpha-crystallin domain is a building block of the alpha-crystallins quaternary structure and suggest that the charge conservation observed in the alpha-crystallins evolution might be important for the assembly of the oligomer. This work reports the first use of SDSL to identify a beta-strand in an unknown structure and demonstrates the feasibility of using this technique to investigate the oligomeric structure of the alpha-crystallins and sHSPs.

T4 lysozyme and mutants thereof crystallize in different conformations that are related to each other by a bend about a hinge in the molecule. This observation suggests that the wild type protein may undergo a hinge-bending motion in solution to allow substrate access to an otherwise closed active site cleft [Faber, H.R., & Matthews, B.W. (1990) Nature 348, 263-266]. To test this hypothesis, either single or pairs of nitroxide side chains were introduced into the protein to monitor tertiary contact interactions and inter-residue distances, respectively, in solution. A set of constraints for these structural parameters was derived from a reference state, a covalent enzyme-substrate adduct where the enzyme is locked in the closed state. In the absence of substrate, differences in both inter-residue distances and tertiary contact interactions relative to this reference state are consistent with a hinge-bending motion that opens the active site cleft. Quantitative analysis of spin-spin interactions between nitroxide pairs reveals an 8 A relative domain movement upon substrate binding. In addition, it is demonstrated that the I3P mutation, which produces a large hinge-bending angle in the crystal, has no effect on the solution conformation. Thus, the hinge motion is not the result of the mutation but is an integral part of T4 lysozyme catalysis in solution, as suggested recently [Zhang, X.J., Wozniak, J.A., & Matthews, B.W. (1995) J. Mol. Biol. 250, 527-552]. The strategy employed here, based on site-directed spin labeling, should be generally applicable to the study of protein conformation and conformational changes in solution.

The small multidrug transporter from E. coli, EmrE, couples the energetically uphill extrusion of hydrophobic cations out of the cell to the transport of two protons down their electrochemical gradient (1-2). While the principal elements of the antiport mechanism have been revealed (3), the structural record is limited to the crystal structure of the substrate-bound state (4). Here we utilize spin labeling and Q-band Double Electron Electron Resonance (DEER; 5) to uncover the conformational changes that are subsequent to protonation of critical acidic residues, a central and missing link in the structure/mechanism relationship. We find that protonation of E14 leads to extensive helix rotation and tilt in conjunction with repacking of loops, conformational changes which alter the orientation of side chains that coordinate substrate binding and modulate substrate access from the lipid bilayer. The model that emerges from our data posits a proton-bound, but occluded, resting state. Substrate binding from the inner leaflet of the bilayer releases the protons and triggers isoenergetic alternating access between inward- and outward facing conformations of the substrate-loaded transporter (6), thus enabling antiport without dissipating the proton gradient.(1) Schuldiner S (2009) Biochim Biophys Acta 1794(5): 748-762.(2) Amadi ST, Koteiche HA, Mishra S, Mchaourab HS (2010) J Biol Chem 285(34): 26710-26718.(3) Yerushalmi H, Schuldiner S (2000) FEBS Lett 476(1-2): 93-97.(4) Chen YJ, et al. (2007) Proc Natl Acad Sci USA 104(48): 18999-19004.(5) Jeschke G (2012) Annu Rev Phys Chem 63: 419-446.(6) Morrison EA, et al. (2012) Nature 481(7379): 45-50.

Bacteriophage T4 Lysozyme (T4L) catalyzes the hydrolysis of the peptidoglycan layer of the bacterial cell wall late in the infection cycle. It has long been postulated that equilibrium dynamics enable substrate access to the active site located at the interface between the N- and C-terminal domains. Crystal structures of WT-T4L and point mutants captured a range of conformations that differ by the hinge-bending angle between the two domains. Evidence of equilibrium between open and closed conformations in solution was gleaned from distance measurements between the two domains but the nature of the equilibrium and the timescale of the underlying motion have not been investigated. Here, we used fluorescence fluctuation spectroscopy to directly detect T4L equilibrium conformational fluctuations in solution. For this purpose, Tetramethylrhodamine probes were introduced at pairs of cysteines in regions of the molecule that undergo relative displacement upon transition from open to closed conformations. Correlation analysis of Tetramethylrhodamine intensity fluctuations reveals hinge-bending motion that changes the relative distance and orientation of the N- and C-terminal domains with ≅15 μs relaxation time. That this motion involves interconversion between open and closed conformations was further confirmed by the dampening of its amplitude upon covalent substrate trapping. In contrast to the prevalent two-state model of T4L equilibrium, molecular brightness and number of particles obtained from cumulant analysis suggest that T4L populates multiple intermediate states, consistent with the wide range of hinge-bending angles trapped in the crystal structure of T4L mutants.

Human small heat shock protein 27 (Hsp27) undergoes concentration-dependent equilibrium dissociation from an ensemble of large oligomers to a dimer. This phenomenon plays a critical role in Hsp27 chaperone activity in vitro enabling high affinity binding to destabilized proteins. In vivo dissociation, which is regulated by phosphorylation, controls Hsp27 role in signaling pathways. In this study, we explore the sequence determinants of Hsp27 dissociation and define the structural basis underlying the increased affinity of Hsp27 dimers to client proteins. A systematic cysteine mutagenesis is carried out to identify residues in the N-terminal domain important for the equilibrium between Hsp27 oligomers and dimers. In addition, spin labels were attached to the cysteine mutants to enable electron paramagnetic resonance (EPR) analysis of residue environment and solvent accessibility in the context of the large oligomers, upon dissociation to the dimer, and following complex formation with the model substrate T4 Lysozyme (T4L). The mutagenic analysis identifies residues that modulate the equilibrium dissociation in favor of the dimer. EPR analysis reveals that oligomer dissociation disrupts subunit contacts leading to the exposure of Hsp27 N-terminal domain to the aqueous solvent. Moreover, regions of this domain are highly dynamic with no evidence of a packed core. Interaction between T4L and sequences in this domain is inferred from transition of spin labels to a buried environment in the substrate/Hsp27 complex. Together, the data provides the first structural analysis of sHSP dissociation and supports a model of chaperone activity wherein unstructured and highly flexible regions in the N-terminal domain are critical for substrate binding.

Major Facilitator Superfamily (MFS) transporters harness the free energy stored in ion or solute gradients for active transport of a variety of substrates. Although most of the 58 distinct families of MFS transporters are substrate-specific, six families contain multidrug transporters (MDR). EmrD is a MFS-MDR from E.coli whose crystal structure has been determined. Similar to the lac permease, the topology consists of 12 transmembrane helices arranged in two bundles of six helices. However, EmrD has a more compact conformation that occludes access to a hydrophobic cavity/chamber from both sides of the membranes. To define the conformational motion involved in substrate transport, we are using spin labeling and electron paramagnetic resonance (EPR) spectroscopy. Spin labels were introduced at selected sites along transmembrane helices 5, 6 and 8. EPR analysis of mobilities and accessibilities reveal distinct changes in spin label dynamics and exposure to NiEDDA upon protonotation of the transporter. The sites of these changes cluster at the periplasmic side of EmrD suggesting that this end of the transporter undergoes conformational rearrangements at low pH. Double electron electron resonance analysis is in progress to determine the nature and amplitude of the protein conformational motion.

This work demonstrates the use of multiquantum EPR to study the magnetic properties of copper complexes and copper proteins. Pure absorption spectra are obtained because of the absence of field modulation. The signal intensity of 3-quantum spectra is proportional to the spin lattice relaxation time T1, while its linewidth in a frequency difference sweep is T1(-1). A change in lineshape for the EPR detectable mixed value [Cu(1.5) . . . Cu(1.5)] site in nitrous oxide reductase is attributed to suppression of the forbidden transitions. The data confirm the unusually fast relaxation time for this site, which requires temperatures of less than 100 K to resolve hyperfine structure. The T1's for the mixed valence [Cu(1.5) . . . Cu(1.5)] site in nitrous oxide reductase are very similar to T1's for the Cua site in cytochrome c oxidase. The similar relaxation properties, together with previous multifrequency EPR results, support the hypothesis that the EPR detectable sites in cytochrome c oxidase and nitrous oxide reductase are mixed valence [Cu(1.5) . . . Cu(1.5)] configurations.

Crystallographic, computational and functional analyses of LeuT have revealed details of the molecular architecture of Na(+)-coupled transporters and the mechanistic nature of ion/substrate coupling, but the conformational changes that support a functional transport cycle have yet to be described fully. We have used site-directed spin labeling and electron paramagnetic resonance (EPR) analysis to capture the dynamics of LeuT in the region of the extracellular vestibule associated with the binding of Na(+) and leucine. The results outline the Na(+)-dependent formation of a dynamic outward-facing intermediate that exposes the primary substrate binding site and the conformational changes that occlude this binding site upon subsequent binding of the leucine substrate. Furthermore, the binding of the transport inhibitors tryptophan, clomipramine and octyl-glucoside is shown to induce structural changes that distinguish the resulting inhibited conformation from the Na(+)/leucine-bound state.

Fluorescence correlation spectroscopy (FCS) and cumulant analysis were applied to study the conformational dynamics of T4 Lysozyme (T4L) in solution. Previous EPR studies (Mchaourab et al., 1997)∗ have shown that T4L undergoes a hinge-bending motion in its native state - an oscillatory motion between an open and a closed conformation. To observe this motion on a single molecule level, we took advantage of the self quenching of the probe tetramethyl rhodamine (TAMRA) at short distances. Pairs of fluorescent probes were placed at specific residues predicted to undergo relative movement. FCS autocorrelation showed two components consistent with two conformational states of T4L in solution. Fits to a diffusion/kinetic model yielded a relaxation time in the range of 5 to 25 microseconds. Molecular brightness values obtained in the two conformations correlate with expected proximity in the structure and with distances between pairs of spin labels introduced at the same sites. We further found that the structural fluctuations as revealed by the autocorrelation curve are diminished when a substrate is bound to T4L. A novel finding is that the hinge motion modulates the dynamics of the long inter-domain helix. We are currently extending these studies to membrane transporters to detect and characterize functionally-relevant fluctuations in their structures.∗Mchaourab HS, Oh KJ, Fang CJ and Hubbell WL. 1997. Conformation of T4 Lysozyme in solution. Hinge-bending motion and the substrate-induced conformational transition studied by site-directed spin labeling. Biochemistry36:307-316.

Driven by the energy of ATP binding and hydrolysis, ATP-binding cassette transporters alternate between inward- and outward-facing conformations, allowing vectorial movement of substrates. Conflicting models have been proposed to describe the conformational motion underlying this switch in access of the transport pathway. One model, based on three crystal structures of the lipid flippase MsbA, envisions a large-amplitude motion that disengages the nucleotide-binding domains and repacks the transmembrane helices. To test this model and place the crystal structures in a mechanistic context, we use spin labeling and double electron-electron resonance spectroscopy to define the nature and amplitude of MsbA conformational change during ATP hydrolysis cycle. For this purpose, spin labels were introduced at sites selected to provide a distinctive pattern of distance changes unique to the crystallographic transformation. Distance changes in liposomes, induced by the transition from nucleotide-free MsbA to the highest energy intermediate, fit a simple pattern whereby residues on the cytoplasmic side undergo 20-30 A closing motion while a 7- to 10-A opening motion is observed on the extracellular side. The transmembrane helices undergo relative movement to create the outward opening consistent with that implied by the crystal structures. Double electron-electron resonance distance distributions reveal asymmetric backbone flexibility on the two sides of the transporter that correlates with asymmetric opening of the substrate-binding chamber. Together with extensive accessibility analysis, our results suggest that these structures capture features of the motion that couples ATP energy expenditure to work, providing a framework for the mechanism of substrate transport.

A central step in understanding lens aging is to characterize the thermodynamic stability of its proteins and determine the consequences of changes in the primary sequence on their folding equilibria. For this purpose, destabilized mutations were introduced in betaB1-crystallin targeting the domain interface within the fold of a subunit. Global unfolding was monitored by tryptophan fluorescence while concomitant structural changes at the dimer interface were monitored by fluorescence and spin labels. Both spectral probes report explicit evidence of multi-state unfolding equilibrium. The biphasic nature of the unfolding curves was more pronounced at higher protein concentration. Distinct shifts in the midpoint of the second transition reflect the population of a dimeric intermediate. This intermediate may be a critical determinant for the life-long stability of the beta-crystallins and has important consequences on interactions with alpha-crystallin.

The determinants of the oligomeric assembly of Hsp16.5, a small heat-shock protein (sHSP) from Methanococcus jannaschii, were explored via site-directed truncation and site-directed spin labeling. For this purpose, subunit contacts around the two-, three- and four-fold symmetry axes were fingerprinted using patterns of proximities between nitroxide spin labels introduced at selected sites. The lack of change in this fingerprint in an N-terminal truncation of the protein demonstrates that the interactions are encoded in the α-crystallin domain. In contrast, the truncation of the N-terminal domain of Mycobacterium tuberculosis Hsp16.3, a bacterial sHSP with an equally short N-terminal region, results in the dissociation of the oligomer to a trimer. These results, in conjunction with those from previous truncation studies in mammalian sHSP, suggest that as the α-crystallin domain evolved to encode a smaller basic unit than the overall oligomer, the control of the assembly and dynamics of the oligomeric structure became encoded in the N-terminal domain.

The near completion of a large number of genome sequencing projects is ushering in a new perspective on how research in biological and biomedical sciences will be conducted in the future, both at the conceptual and experimental levels. The rush is on to transform the molecular blueprints of life into global views of the biochemical and physiological circuitry that interconnect to form entire organisms. The evolutionary histories, now available as multiple sequence alignments, will be complemented with pictures of evolving structures. In this post genomic era, the process of scientific discovery will be the result of large-scale parallel measurements to answer questions formulated from a genomic perspective.

Experimental restraints are critical to the expansion of de novo proteinfolding as restraints limit the conformational search space and increase the proportion of quality models. Our laboratory has shown that only a small percentage of randomly selected distance restraints, obtained from EPR analysis of spin labeled protein, are responsible for the improvements in model quality associated with restraint-based folding (1). Furthermore, we demonstrated that information content, a ratio of geometric distance between residues and their separation in the sequence, is a critical feature of successful distance restraints selection. Sequence coverage, a measure of the representation of the entire sequence, was identified as another important feature. For the use of experimental restraints to be practical, restraint patterns must be optimized to limit the number of measurements required to produce quality models and increase the throughput of the technique. The focus of this work is the creation of an algorithm that predicts from primary sequence a set of distance measurements that would most effectively guide tertiary structure prediction. For our prediction algorithm, we have developed four individual terms that approximate these features: sequence separation, label density, secondary structure placement, and secondary structure connections. We used these terms to generate patterns of distance measurements in T4 Lysozyme, simulated these distance measurements from the crystal structure (2LZM), and ran restraint-based folding with Rosetta. We observed a significant improvement in RMSD distribution when using the optimized restraint patterns when compared to both randomized patterns and folding without restraints.1.Alexander, N., Bortolus, M., Al-Mestarihi, A., Mchaourab, H., Meiler, J. Structure. De novo high-resolution protein structure determination from sparse spin-labeling EPR data. 16, 181-195 (2008).

To explore the spatial organization and functional dynamics of the citrate transport protein (CTP), a nitroxide scan was carried out along 22 consecutive residues within the fourth transmembrane domain (TMDIV). This domain has been implicated as being of unique importance to the CTP mechanism due to (i) the presence of two intramembranous positive charges that are essential for CTP function and (ii) the existence of a transmembrane aqueous surface within this domain which likely corresponds to a portion of the citrate translocation pathway. The sequence-specific variation in the mobilities of the introduced nitroxides and their accessibilities to molecular O(2) reveal an alpha-helical conformation along the sequence. The accessibilities to NiEDDA are out of phase with accessibilites to O(2), indicating that one face of the helix is solvated by the lipid bilayer while the other is solvated by an aqueous environment. A gradient of NiEDDA accessibility is observed along the helix surface facing the aqueous phase, and the EPR spectral line shapes at these sites indicate considerable motional restriction. In the context of the model where TMDIV lines the translocation pathway, these data suggest a barrier to passive diffusion through the pathway. This paper reports the first use of site-directed spin labeling to study mitochondrial transporter structure.

We report an investigation of how much protein structural information could be obtained using a site-directed fluorescence labeling (SDFL) strategy. In our experiments, we used 21 consecutive single-cysteine substitution mutants in T4 lysozyme (residues T115-K135), located in a helix-turn-helix motif. The mutants were labeled with the fluorescent probe monobromobimane and subjected to an array of fluorescence measurements. Thermal stability measurements show that introduction of the label is substantially perturbing only when it is located at buried residue sites. At buried sites (solvent surface accessibility of

A spin-labeled derivative of the ion channel peptide cecropin AD (Fink et al., 1988) was synthesized and used to investigate its aggregation state in water and in the presence of a helix-promoting solvent. A cysteine was introduced at position 33 and spin-labeled using the methanethiosulfonate spin label. In low ionic strength aqueous solution, the peptide is monomeric, and the ESR spectrum indicates a high degree of segmental flexibility at the nitroxide attachment point, consistent with a predominantly random coil conformation. Upon addition of 5-10% (v/v) hexafluoro-2-propanol (HFP), the peptide is induced to aggregate as evidenced by significant motional restriction of the spin label and spin-spin broadening of the ESR lines. At higher concentrations of HFP, the peptide reverts to a monomeric state but retains its folded conformation. Our data suggest that between 5 and 10% HFP the peptide undergoes two structural transitions. The first transition starts at 5% and is very cooperative. Its dependence on ionic strength, temperature, and pH indicates that it involves the interconversion between a random coil and an ordered state stabilized by interpeptide electrostatic and hydrophobic interactions. The second transition, which occurs at 11% v/v HFP, is between the self-associated form and an ordered monomeric form. The analysis of our experimental results demonstrates aggregate formation at 5-10% HFP. This may be relevant to the mechanism of channel formation by cecropins in membranes.

Site-directed spin labeling and electron paramagnetic resonance were used to investigate the structure and conformational dynamics of the small multidrug resistance transporter EmrE from Escherichia coli. The data set consists of the mobilities, solvent accessibilities of 110 spin labeled residues as well as selected pairwise distances. These parameters were interpreted as constraints on the local steric environment, the orientation of helices in the lipid phase and packing of the transmembrane helices in each monomer as well as across the dimer interface. The data collectively suggest that in liposomes, ligand-free EmrE average structure is not compatible with the crystal structure and an EM-based model. Spin label pairs at the dimer interface located at opposite sides of helix 3 reveal dipolar interaction between the spins separated by less than 15A which contradicts their inverted relative orientation in the crystal structure. Furthermore, distances between spin labels along helix 4 are not consistent with its tight packing at the dimer interface. The spectroscopic data is supported by an expanded topological analysis of EmrE-GFP chimeras which reveals that the previous interpretation of Rapp et al is not unequivocal. Binding of tetraphenyl phosphonium (TPP+) to EmrE increases the structural order as manifested by a narrowing of spin label distance distributions but does not lead to major conformational rearrangements. The EPR-based constraints are being used to generate a model of EmrE in liposomes.

Site-Directed Spin-Labeling Electron Paramagnetic Resonance (SDSL-EPR), in combination with the Rosetta protein folding algorithm (Rohl et al 2004), could serve as an alternative method in structure elucidation of proteins that continue to evade traditional techniques, such as x-ray crystallography and NMR. A spin-label "motion-on-a-cone" model was used during de novo folding of T4-lysozyme and αA-crystallin, which resulted in full-atom models at 1.0A and 2.6A to the crystal structures, respectively (Alexander et al 2008). This spin-label model and already-existing EPR distance data have been used to generate EPR distance and accessibility knowledge-based potentials, which can be implemented as folding constraints into Rosetta. In addition, we have introduced a rotamer library of the methanethiosulfonate spin-label (MTSSL). Spin-label rotamers have been derived from conformations observed in crystal structures of spin-labeled T4-lysozyme. The method was benchmarked using a set of proteins where the spin-label was positioned at various levels of exposure. The results indicate that the method is able to recover important aspects of spin-label orientation with up to 0.4A RMSD. In particular, experimental distances and distance distributions observed for T4-lysozyme were reproduced with relatively high accuracy.