Intermediate steps in the formation of neuronal SNARE complexes.

Neuronal exocytosis requires the assembly of three SNARE proteins, syntaxin and SNAP25 on the plasma membrane and synaptobrevin on the vesicle membrane. However, the precise steps in this process and the points at which assembly and fusion are controlled by regulatory proteins are unclear. In the present work, we examine the kinetics and intermediate states during SNARE assembly in vitro using a combination of time resolved fluorescence and EPR spectroscopy. We show that syntaxin rapidly forms a dimer prior to forming the kinetically stable 2:1 syntaxin:SNAP25 complex, and that the 2:1 complex is not diminished by the presence of excess SNAP25. Moreover, the 2:1 complex is temperature dependent with a reduced concentration at 37°C. The two segments of SNAP25 behave differently. The N-terminal SN1 segment of SNAP25 exhibits a pronounced increase in backbone ordering from the N- to the C-terminus that is not seen in the C-terminal SNAP25 segment SN2. Both the SN1 and SN2 segments of SNAP25 will assemble with syntaxin; however, while the association of the SN1 segment with syntaxin produces a stable 2:2 (SN1:syntaxin) complex, the complex formed between SN2 and syntaxin is largely disordered. Synaptobrevin fails to bind syntaxin alone, but will associate with syntaxin in the presence of either the SN1 or SN2 segments; however, the synaptobrevin:syntaxin:SN2 complex remains disordered. Taken together, these data suggest that synaptobrevin and syntaxin do not assemble in the absence of SNAP25, and that the SN2 segment of SNAP25 is the last to enter the SNARE complex.

A guideline for the distance measurement plans of site-directed spin labels for structural prediction of nucleic acids.

CONTEXT AND RESULTS: Site-directed spin labeling (SDSL) combined with electron paramagnetic resonance spectroscopy methods has been successfully used to predict the structures of nucleic acids. These methods measure the distances between spin labels yielding distance equations that are solved using numerical algorithms to provide one or several structural predictions. In this work, the minimum number of SDSL distance measurements and distance measurement types required to predict a unique nucleic acid structure were investigated. Our results indicate that at least six distance measurements should be obtained given that the distance measurements do not connect one SDSL on one arm with more than three SDSLs on the other arm. Moreover, there may be a preference for 1-to-1 SLs distance measurements rather than 1-to-many SLs as the latter was linked to undefined structures discussed in this study. METHODS: Pairs of double-helical arms of nucleic acid were simulated using the finite element software Pro/ENGINEER (PTC Inc., Boston, MA). In each simulation, a specific SDSL distance measurement plan was adopted and the resulting structure was tested for movability. Immovable structures indicate that this plan will potentially result in a unique structural prediction of the nucleic acid. All the possible plans for SDSL distance measurements were investigated either by direct measurement or by extrapolation.

Manipulating solid-state spin concentration through charge transport.

Solid-state defects are attractive platforms for quantum sensing and simulation, e.g., in exploring many-body physics and quantum hydrodynamics. However, many interesting properties can be revealed only upon changes in the density of defects, which instead is usually fixed in material systems. Increasing the interaction strength by creating denser defect ensembles also brings more decoherence. Ideally one would like to control the spin concentration at will while keeping fixed decoherence effects. Here, we show that by exploiting charge transport, we can take some steps in this direction, while at the same time characterizing charge transport and its capture by defects. By exploiting the cycling process of ionization and recombination of NV centers in diamond, we pump electrons from the valence band to the conduction band. These charges are then transported to modulate the spin concentration by changing the charge state of material defects. By developing a wide-field imaging setup integrated with a fast single photon detector array, we achieve a direct and efficient characterization of the charge redistribution process by measuring the complete spectrum of the spin bath with micrometer-scale spatial resolution. We demonstrate a two-fold concentration increase of the dominant spin defects while keeping the T2 of the NV center relatively unchanged, which also provides a potential experimental demonstration of the suppression of spin flip-flops via hyperfine interactions. Our work paves the way to studying many-body dynamics with temporally and spatially tunable interaction strengths in hybrid charge-spin systems.

Interaction of barley ß-glucan with food dye molecules - An insight from pulse dipolar EPR spectroscopy.

The interactions between dietary fibers (DFs) and small molecules are of great interest to food chemistry and nutrition science. However, the corresponding interaction mechanisms and structural rearrangements of DFs at the molecular level are still opaque due to the usually weak binding and the lack of appropriate techniques to determine details of conformational distributions in such weakly organized systems. By combining our previously established methodology on stochastic spin-labelling of DFs with the appropriately revised set of pulse electron paramagnetic resonance techniques, we present here a toolkit to determine the interactions between DFs and small molecules, using barley ß-glucan as an example for neutral DF and a selection of food dye molecules as examples for small molecules. The proposed here methodology allowed us to observe subtle conformational changes of ß-glucan by detecting multiple details of the local environment of the spin labels. Substantial variations of binding propensities were detected for different food dyes.

Biophysical investigations of class A GPCRs.

G protein-coupled receptors (GPCRs) play a central role in cellular communication, converting external stimuli into intracellular responses. GPCRs bind a very broad panel of ligands, such as hormones, neurotransmitters, peptides and lipids. Ligand binding triggers a series of receptor conformational rearrangements, enabling the coupling to intracellular partners and the activation of signaling cascades. The major breakthrough in GPCRs structural biology of the past decade has considerably advanced our understanding of GPCR activation. However, structural information cannot fully explain the molecular details of GPCRs pharmacology. Biophysical investigations reveal that GPCRs are very dynamic proteins, capable of exploring a wide range of conformational states. Binding to ligands of various pharmacological classes, as well as intracellular effectors and allosteric modulators, can shift the equilibrium between these states and the kinetic of interconversions among the different conformers. Investigation of GPCR dynamic interplay is therefore important to better understand the complex pharmacology and signaling profile of these receptors.

Mapping Phase Diagram of Tau-RNA LLPS Under Live Cell Coculturing Conditions.

Protein liquid-liquid phase separation (LLPS) has been associated with biological functions and pathological aggregation. Mapping the phase separation conditions is the first step to identify and quantify the driving forces of LLPS. Here, we describe the protocols to draw the phase diagram of tau-RNA LLPS and use the mapped diagram to guide experimental conditions for LLPS-cell coculturing, electron resonance spectroscopy in particular double electron-electron resonance spectroscopy, crosslinking immunoprecipitation, and isothermal titration calorimetry.

Integration of Nanometer-Range Label-to-Label Distances and Their Distributions into Modelling Approaches.

Labelling techniques such as electron paramagnetic resonance spectroscopy and single-molecule fluorescence resonance energy transfer, allow access to distances in the range of tens of angstroms, corresponding to the size of proteins and small to medium-sized protein complexes. Such measurements do not require long-range ordering and are therefore applicable to systems with partial disorder. Data from spin-label-based measurements can be processed into distance distributions that provide information about the extent of such disorder. Using such information in modelling presents several challenges, including a small number of restraints, the influence of the label itself on the measured distance and distribution width, and balancing the fitting quality of the long-range restraints with the fitting quality of other restraint subsets. Starting with general considerations about integrative and hybrid structural modelling, this review provides an overview of recent approaches to these problems and identifies where further progress is needed.

PP2A is activated by cytochrome c upon formation of a diffuse encounter complex with SET/TAF-Iß.

Intrinsic protein flexibility is of overwhelming relevance for intermolecular recognition and adaptability of highly dynamic ensemble of complexes, and the phenomenon is essential for the understanding of numerous biological processes. These conformational ensembles-encounter complexes-lack a unique organization, which prevents the determination of well-defined high resolution structures. This is the case for complexes involving the oncoprotein SET/template-activating factor-Iß (SET/TAF-Iß), a histone chaperone whose functions and interactions are significantly affected by its intrinsic structural plasticity. Besides its role in chromatin remodeling, SET/TAF-Iß is an inhibitor of protein phosphatase 2A (PP2A), which is a key phosphatase counteracting transcription and signaling events controlling the activity of DNA damage response (DDR) mediators. During DDR, SET/TAF-Iß is sequestered by cytochrome c (Cc) upon migration of the hemeprotein from mitochondria to the cell nucleus. Here, we report that the nuclear SET/TAF-Iß:Cc polyconformational ensemble is able to activate PP2A. In particular, the N-end folded, globular region of SET/TAF-Iß (a.k.a. SET/TAF-Iß ΔC)-which exhibits an unexpected, intrinsically highly dynamic behavior-is sufficient to be recognized by Cc in a diffuse encounter manner. Cc-mediated blocking of PP2A inhibition is deciphered using an integrated structural and computational approach, combining small-angle X-ray scattering, electron paramagnetic resonance, nuclear magnetic resonance, calorimetry and molecular dynamics simulations.

Light-induced pulsed dipolar EPR spectroscopy for distance and orientation analysis.

Measuring distances in biology at the molecular level is of great importance for understanding the structure and function of proteins, nucleic acids and other biological molecules and their complexes. Pulsed Dipolar Spectroscopy (PDS) offers advantages with respect to other methods as it is uniquely sensitive and specific to electronic spin centers and allows measurements in near-native conditions, comprising the in-cell environment. PDS methods measure the electron spin-spin dipolar interaction, therefore they require the presence of at least two paramagnetic centers, which are often stable radicals. Recent developments have introduced transient triplet states, photo-activated by a laser pulse, as spin labels and probes, thereby establishing a new family of techniques-Light-induced PDS (LiPDS). In this chapter, an overview of these methods is provided, looking at the chromophores that can be used for LiPDS and some of the technical aspects of the experiments. A guide to the choice of technique that can yield the best results, depending on the type of system studied and the information required, is provided. Examples of previous LiPDS studies of model systems and proteins are given. Characterization data for the chromophores used in these studies is tabulated to help selection of appropriate triplet state probes in future studies.

PELDOR Measurements on Nitroxide-Labeled Oligonucleotides.

In the past decades, pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) has emerged as a powerful tool in biophysical chemistry to study the structure, dynamics, and function of biomolecules like oligonucleotides and proteins. Structural information is obtained from PDS methods in form of a distribution of distances between spin centers. Such spin centers can either be intrinsically present paramagnetic metal ions and organic radicals or may be attached to the biomolecule by means of site-directed spin labeling. The most common PDS experiment for probing interspin distances in the nanometer range is pulsed electron-electron double resonance (PELDOR or DEER). In the protocol presented here, we provide a step-by-step workflow on how to set up a PELDOR experiment on a commercially available pulsed EPR spectrometer, outline the data analysis, and highlight potential pitfalls. We suggest PELDOR measurements on nitroxide-labeled oligonucleotides to study the structure of either RNA-cleaving DNAzymes in complex with their RNA targets or modified DNAzymes with different functions and targets, in which deoxynucleotides are substituted by nitroxide-labeled nucleotides.

Assessing the Role of Lipids in the Molecular Mechanism of Membrane Proteins.

Membrane proteins have evolved to work optimally within the complex environment of the biological membrane. Consequently, interactions with surrounding lipids are part of their molecular mechanism. Yet, the identification of lipid-protein interactions and the assessment of their molecular role is an experimental challenge. Recently, biophysical approaches have emerged that are compatible with the study of membrane proteins in an environment closer to the biological membrane. These novel approaches revealed specific mechanisms of regulation of membrane protein function. Lipids have been shown to play a role in oligomerization, conformational transitions or allosteric coupling. In this review, we summarize the recent biophysical approaches, or combination thereof, that allow to decipher the role of lipid-protein interactions in the mechanism of membrane proteins.

Structural intermediates observed only in intact Escherichia coli indicate a mechanism for TonB-dependent transport.

Outer membrane TonB-dependent transporters facilitate the uptake of trace nutrients and carbohydrates in Gram-negative bacteria and are essential for pathogenic bacteria and the health of the microbiome. Despite this, their mechanism of transport is still unknown. Here, pulse electron paramagnetic resonance (EPR) measurements were made in intact cells on the Escherichia coli vitamin B12 transporter, BtuB. Substrate binding was found to alter the C-terminal region of the core and shift an extracellular substrate binding loop 2 nm toward the periplasm; moreover, this structural transition is regulated by an ionic lock that is broken upon binding of the inner membrane protein TonB. Significantly, this structural transition is not observed when BtuB is reconstituted into phospholipid bilayers. These measurements suggest an alternative to existing models of transport, and they demonstrate the importance of studying outer membrane proteins in their native environment.

Bacteria must obtain nutrients from their surrounding environment in order to survive. In Gram-negative bacteria, proteins in the outer membrane surrounding the cell actively transport carbohydrates and trace nutrients like iron into the cell's interior. Although the structures of many of these transport proteins have been determined, the mechanism they use to move molecules across the membrane is poorly understood. To better understand this process, Nilaweera, Nyenhuis and Cafiso examined the structure of BtuB, a transport protein found in the outer membrane of Escherichia coli that is responsible for absorbing vitamin B₁₂. Previous experiments analyzing the structure of BtuB, and other similar transporters, have been carried out on purified proteins that were extracted from the outer membrane. However, these isolated proteins fail to replicate the transport activity observed in bacterial cells. Nilaweera, Nyenhuis and Cafiso therefore wanted to see how the structure of BtuB changes when it is still enclosed in the membrane of E. coli. This revealed that BtuB undergoes large structural changes when it binds to vitamin B₁₂, suggesting that this is an important part of the transport process. However, when purified BtuB was placed into an artificial membrane, these structural changes did not occur. This indicates that the cellular environment in the bacteria is needed for BtuB to carry out its transport role, and explains why previous experiments using purified proteins struggled to see this structural shift. This work highlights the importance of studying bacterial membrane proteins in their native cell environment. BtuB and similar transporters represent a large family of proteins unique to Gram-negative bacteria that have an impact on human health. Since these proteins are structurally alike, the results of this study may help resolve the transport mechanisms of other proteins, ultimately leading to new ways to control bacterial growth.

Conserved binding site in the N-lobe of prokaryotic MATE transporters suggests a role for Na+ in ion-coupled drug efflux.

In both prokaryotes and eukaryotes, multidrug and toxic-compound extrusion (MATE) transporters catalyze the efflux of a broad range of cytotoxic compounds, including human-made antibiotics and anticancer drugs. MATEs are secondary-active antiporters, i.e., their drug-efflux activity is coupled to, and powered by, the uptake of ions down a preexisting transmembrane electrochemical gradient. Key aspects of this mechanism, however, remain to be delineated, such as its ion specificity and stoichiometry. We previously revealed the existence of a Na+-binding site in a MATE transporter from Pyroccocus furiosus (PfMATE) and hypothesized that this site might be broadly conserved among prokaryotic MATEs. Here, we evaluate this hypothesis by analyzing VcmN and ClbM, which along with PfMATE are the only three prokaryotic MATEs whose molecular structures have been determined at atomic resolution, i.e. better than 3 Å. Reinterpretation of existing crystallographic data and molecular dynamics simulations indeed reveal an occupied Na+-binding site in the N-terminal lobe of both structures, analogous to that identified in PfMATE. We likewise find this site to be strongly selective against K+, suggesting it is mechanistically significant. Consistent with these computational results, DEER spectroscopy measurements for multiple doubly-spin-labeled VcmN constructs demonstrate Na+-dependent changes in protein conformation. The existence of this binding site in three MATE orthologs implicates Na+ in the ion-coupled drug-efflux mechanisms of this class of transporters. These results also imply that observations of H+-dependent activity likely stem either from a site elsewhere in the structure, or from H+ displacing Na+ under certain laboratory conditions, as has been noted for other Na+-driven transport systems.

Viewing rare conformations of the ß2 adrenergic receptor with pressure-resolved DEER spectroscopy.

The ß2 adrenergic receptor (ß2AR) is an archetypal G protein coupled receptor (GPCR). One structural signature of GPCR activation is a large-scale movement (ca. 6 to 14 Å) of transmembrane helix 6 (TM6) to a conformation which binds and activates a cognate G protein. The ß2AR exhibits a low level of agonist-independent G protein activation. The structural origin of this basal activity and its suppression by inverse agonists is unknown but could involve a unique receptor conformation that promotes G protein activation. Alternatively, a conformational selection model proposes that a minor population of the canonical active receptor conformation exists in equilibrium with inactive forms, thus giving rise to basal activity of the ligand-free receptor. Previous spin-labeling and fluorescence resonance energy transfer experiments designed to monitor the positional distribution of TM6 did not detect the presence of the active conformation of ligand-free ß2AR. Here we employ spin-labeling and pressure-resolved double electron-electron resonance spectroscopy to reveal the presence of a minor population of unliganded receptor, with the signature outward TM6 displacement, in equilibrium with inactive conformations. Binding of inverse agonists suppresses this population. These results provide direct structural evidence in favor of a conformational selection model for basal activity in ß2AR and provide a mechanism for inverse agonism. In addition, they emphasize 1) the importance of minor populations in GPCR catalytic function; 2) the use of spin-labeling and variable-pressure electron paramagnetic resonance to reveal them in a membrane protein; and 3) the quantitative evaluation of their thermodynamic properties relative to the inactive forms, including free energy, partial molar volume, and compressibility.

A Two-Armed Probe for In-Cell DEER Measurements on Proteins*.

The application of double electron-electron resonance (DEER) with site-directed spin labeling (SDSL) to measure distances in proteins and protein complexes in living cells puts rigorous restraints on the spin-label. The linkage and paramagnetic centers need to resist the reducing conditions of the cell. Rigid attachment of the probe to the protein improves precision of the measured distances. Here, three two-armed GdIII complexes, GdIII -CLaNP13a/b/c were synthesized. Rather than the disulfide linkage of most other CLaNP molecules, a thioether linkage was used to avoid reductive dissociation of the linker. The doubly GdIII labeled N55C/V57C/K147C/T151C variants of T4Lysozyme were measured by 95 GHz DEER. The constructs were measured in vitro, in cell lysate and in Dictyostelium discoideum cells. Measured distances were 4.5 nm, consistent with results from paramagnetic NMR. A narrow distance distribution and typical modulation depth, also in cell, indicate complete and durable labeling and probe rigidity due to the dual attachment sites.

Electron Paramagnetic Resonance as a Tool for Studying Membrane Proteins.

Membrane proteins possess a variety of functions essential to the survival of organisms. However, due to their inherent hydrophobic nature, it is extremely difficult to probe the structure and dynamic properties of membrane proteins using traditional biophysical techniques, particularly in their native environments. Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) is a very powerful and rapidly growing biophysical technique to study pertinent structural and dynamic properties of membrane proteins with no size restrictions. In this review, we will briefly discuss the most commonly used EPR techniques and their recent applications for answering structure and conformational dynamics related questions of important membrane protein systems.

Cryo-EM structure of the native rhodopsin dimer in nanodiscs.

Imaging of rod photoreceptor outer-segment disc membranes by atomic force microscopy and cryo-electron tomography has revealed that the visual pigment rhodopsin, a prototypical class A G protein-coupled receptor (GPCR), can organize as rows of dimers. GPCR dimerization and oligomerization offer possibilities for allosteric regulation of GPCR activity, but the detailed structures and mechanism remain elusive. In this investigation, we made use of the high rhodopsin density in the native disc membranes and of a bifunctional cross-linker that preserves the native rhodopsin arrangement by covalently tethering rhodopsins via Lys residue side chains. We purified cross-linked rhodopsin dimers and reconstituted them into nanodiscs for cryo-EM analysis. We present cryo-EM structures of the cross-linked rhodopsin dimer as well as a rhodopsin dimer reconstituted into nanodiscs from purified monomers. We demonstrate the presence of a preferential 2-fold symmetrical dimerization interface mediated by transmembrane helix 1 and the cytoplasmic helix 8 of rhodopsin. We confirmed this dimer interface by double electron-electron resonance measurements of spin-labeled rhodopsin. We propose that this interface and the arrangement of two protomers is a prerequisite for the formation of the observed rows of dimers. We anticipate that the approach outlined here could be extended to other GPCRs or membrane receptors to better understand specific receptor dimerization mechanisms.

EPR Techniques to Probe Insertion and Conformation of Spin-Labeled Proteins in Lipid Bilayers.

Electron paramagnetic resonance (EPR) spectroscopy of spin-labeled membrane proteins is a valuable biophysical technique to study structural details and conformational transitions of proteins close to their physiological environment, for example, in liposomes, membrane bilayers, and nanodiscs. Unlike in nuclear magnetic resonance (NMR) spectroscopy, having only one or few specific side chains labeled at a time with paramagnetic probes makes the size of the object under investigation irrelevant in terms of technique sensitivity. As a drawback, extensive site-directed mutagenesis is required in order to analyze the properties of the protein under investigation. EPR can provide detailed information on side chain dynamics of large membrane proteins or protein complexes embedded in membranes with an exquisite sensitivity for flexible regions and on water accessibility profiles across the membrane bilayer. Moreover, distances between the two spin-labeled side chains in membrane proteins can be detected with high precision at cryogenic temperatures. The application of EPR to membrane proteins still presents some challenges in terms of sample preparation, sensitivity and data interpretation, thus it is difficult to give ready-to-go methodological recipes. However, new technological developments (arbitrary waveform generators) and new spin labels spectroscopically orthogonal to nitroxides increased the range of applicability from in vitro toward in-cell EPR experiments. This chapter is an updated version of the one published in the first edition of the book and describes the state of the art in the application of nitroxide-based site-directed spin labeling EPR to membrane proteins, addressing new tools such as arbitrary waveform generators and spectroscopically orthogonal labels, such as Gd(III)-based labels. We will present challenges in sample preparation and data analysis for functional and structural membrane protein studies using site-directed spin labeling techniques and give experimental details on EPR techniques providing information on side chain dynamics and water accessibility using nitroxide probes. An updated optimal Q-band DEER setup for nitroxide probes will be described, and its extension to gadolinium-containing samples will be addressed.

Angiotensin Analogs with Divergent Bias Stabilize Distinct Receptor Conformations.

"Biased" G protein-coupled receptor (GPCR) agonists preferentially activate pathways mediated by G proteins or ß-arrestins. Here, we use double electron-electron resonance spectroscopy to probe the changes that ligands induce in the conformational distribution of the angiotensin II type I receptor. Monitoring distances between 10 pairs of nitroxide labels distributed across the intracellular regions enabled mapping of four underlying sets of conformations. Ligands from different functional classes have distinct, characteristic effects on the conformational heterogeneity of the receptor. Compared to angiotensin II, the endogenous agonist, agonists with enhanced Gq coupling more strongly stabilize an "open" conformation with an accessible transducer-binding site. ß-arrestin-biased agonists deficient in Gq coupling do not stabilize this open conformation but instead favor two more occluded conformations. These data suggest a structural mechanism for biased ligand action at the angiotensin receptor that can be exploited to rationally design GPCR-targeting drugs with greater specificity of action.

Structural Characterization of Biomolecules through Atomistic Simulations Guided by DEER Measurements.

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