Double Electron-electron Resonance Spectroscopy Research Articles

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

We have used site-directed spin labeling and double electron-electron resonance spectroscopy (DEER) to probe the structural dynamics of calmodulin in the context of regulating the skeletal muscle Ca release channel, the ryanodine receptor (RyR1). Conventional spin labels that attach using a single flexible di-sulfide linkage allow local spin label motion that limits the resolution and accuracy of distance or orientation measurements. To avoid this problem, we use a bifunctional spin label (BSL) that provides stereospecific attachment at Cys sites four residues apart on an α helix. A mutant calmodulin construct containing bifunctional labeling sites on helices in the N-lobe (T34CS38C) and in the C-lobe (R106CT110C) was expressed and labeled with BSL. DEER was used to measure the spin-spin distance distribution in the presence and absence of Ca. Our results show that in the absence of Ca, CaM is predominantly in one structure with a center distance of 4.22 nm, which is in in good agreement with the predicted structure based on the published NMR solution structure (4.30 nm). In the presence of Ca, CaM takes on two structures with center distances at 2.73 (34%) and 4.11 nm (66%). Neither of these distances agrees with that predicted by the x-ray crystal structure of Ca-CaM (5.6 nm). This discrepancy may be due to artifacts from the crystalline environment in x-ray, whereas our DEER measurement was made in frozen solution. In conclusion, these results indicate the potential of BSL for accurate distance measurement and suggest new structures of Ca-bound CaM in solution. Ongoing studies are expanding these experiments to CaM bound to a synthetic peptide that is part of the CaM-binding domain on RyR1. This work was supported by NIH grants to DDT (R37 AG26160, T32 AR007612).

Detecting Structural Changes in Myosin using Bifunctional Spin Labels

Actomyosin interaction is responsible for driving a vast number of cellular processes, including force generation during muscle contraction. In this context, myosin is the molecular “motor” that produces muscle contraction. The myosin motor domain is comprised from three major subdomains: the force-generating domain (relay helix, SH-1, and converter), the ATP-binding pocket and the actin-binding cleft. During the cycle of force generation, myosin undergoes a series of structural rearrangements including “bending” of the relay helix, which communicates the biochemical state of the ATP binding pocket to the lever arm. The relay helix has previously been shown to reflect changes in distance, dynamics and orientation by electron paramagnetic resonance (EPR) with nucleotide state of the myosin catalytic domain. We now observe relay helix conformational changes using two rigidly attached bifunctional spin labels (BSL) on the relay helix (492.496) and in the lower 50 kDa domain (639.643) of the Dictyostelium myosin II motor domain by double electron electron resonance spectroscopy (DEER). We observe changes in relay helix conformation with various allosteric effectors - including potent myosin II inhibitors and physiological inhibitors such as site-directed oxidation. This approach allows for characterization of perturbations in chemo-mechanical coupling within the actomyosin complex under inhibitory conditions, as well as showing the reliability of relay helix spin probe sensitivity to the changes in myosin structural dynamics.

Structural-Function Study of Membrane Proteins with Restrained-Ensemble and Dummy Spin-Label Molecular Dynamics Simulations

Membrane proteins perform numerous physiological functions that are critical for human health. DEER (Double Electron Electron Resonance) spectroscopy is playing an increasingly important role in characterizing the conformational states of these proteins. DEER is a powerful-pulsed EPR technique allowing the determination of spin-spin distance histograms between site-directed nitroxide label sites on a protein. Combining the DEER structural data with molecular dynamics (MD) simulation methods can be a powerful strategy to study membrane protein structure and function.

Radial Tilting of the Extracellular Domain of GLIC Revealed by EPR Spectroscopy

Signaling in the brain depends on rapid opening and closing of pentameric ligand-gated ion channels (pLGICs). Defects in these proteins are linked to a variety of diseases including myasthenia, epilepsy and sleep-disorders. In addition, these proteins are therapeutic targets for various drugs including anti-convulsants, anxiolytics, anesthetics and sedatives. While recent crystal structures of prokaryotic and eukaryotic pLGICs solved in detergent micelles have shed light on the molecular architecture of these proteins, describing their conformational dynamics in physiological lipids is essential for understanding their function. Crystal structures of GLIC and the glutamate-activated chloride channel predict that agonist-mediated channel opening is associated with a quaternary twisting and a radial un-blooming (inward tilting) of the extracellular binding domain (ECD). Using site directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy and functional GLIC channels reconstituted in liposomes, we are testing these gating predictions. Residues located at the top of β-strands (β1, 2, 5, 6, 8) were mutated to cysteine, labeled with the spin probe MTSL and proton-induced gating motions were measured using continuous wave (CW) and double electron-electron resonance (DEER) spectroscopy. CW EPR spectroscopy data showed the spin label attached to I92C, S93C, V94C (β5), T99C and Q101C (β6) moved into a slightly more constrained/immobile environment at pH 3.0 as compared to pH 7.6. DEER distance distributions for K48 (β2), S93 (β5), T99 and Q101 (β6) revealed the probes moved closer together at pH 3.0, suggesting that the inner β-sheet of the extracellular domain of GLIC tilt inwards upon agonist activation.

Detecting Ligand-Induced Motions in Pentameric Ligand-Gated Ion Channels

Chemical signaling in the brain and periphery relies on rapid opening and closing of pentameric ligand-gated ion channels (pLGICs). While recent high resolution crystal structures of pLGICs in apparently closed, open and desensitized channel conformations are providing invaluable insights into possible activation mechanisms, whether these static protein structures, solved in detergent micelles, accurately capture the conformational gating transitions that a functional pLGIC undergoes when embedded in a lipid bilayer is unknown. Using site-directed spin labeling of a prototypical pentameric ligand-gated ion channel, GLIC, and double electron-electron resonance (DEER) spectroscopy, we are mapping motions underlying ligand-driven gating transitions in functional channels reconstituted into liposomes. In regions at the boundary between the ligand binding domain (loops 2 and 9) and the channel domain (M2-M3 loop), we found that proton-driven gating transitions induced a large movement of loop 9 and an immobilization of loop 2. In the extracellular β-sandwich domain, spin probes attached to the inner beta-sheet moved closer together at pH 3.0, suggesting that the extracellular binding domain of GLIC tilts radially inward when the channel transitions from a closed to open/desensitized states. These data, combined with recently solved structures, expand our understanding of signaling mechanisms underlying pLGIC gating transitions.

Distance measurements between paramagnetic ligands bound to parallel stranded guanine quadruplexes.

Aside from a double helix, deoxyribonucleic acid (DNA) folds into non-canonical structures, one of which is the guanine quadruplex. Cationic porphyrins bind guanine quadruplexes, but the effects of ligand binding on the structure of guanine quadruplexes with more than four contiguous guanine quartets remains to be fully elucidated. Double electron-electron resonance (DEER) spectroscopy conducted at 9.5 GHz (X-band) using broadband, shaped inversion pulses was used to measure the distances between cationic copper porphyrins bound to model parallel-stranded guanine quadruplexes with increasing numbers of guanine quartets. A single Gaussian component was found to best model the time domain datasets, characteristic of a 2 : 1 binding stoichiometry between the porphyrins and each quadruplex. The measured Cu(2+)-Cu(2+) distances were found to be linearly proportional with the number of guanines. Rather unexpectedly, the ligand end-stacking distance was found to monotonically decreases the overall quadruplex length was extended, suggesting a conformational change in the quadruplex secondary structure dependent upon the number of successive guanine quartets.

Interaction of triarylmethyl radicals with DNA termini revealed by orientation-selective W-band double electron-electron resonance spectroscopy.

Spin labels selectively attached to biomolecules allow high-accuracy nanoscale distance measurements using pulsed electron paramagnetic resonance (EPR), in many cases providing the only access to the structure of complex biosystems. Triarylmethyl (TAM) radicals have recently emerged as a new class of spin labels expanding the applicability of the method to physiological temperatures. Along with other factors, the accuracy of the obtained distances crucially relies on the understanding of interactions between biomolecules and spin labels. In this work, we consider such crucial interactions and their impact on pulsed EPR distance measurements in TAM-labeled DNAs. Using orientation-selective high-frequency (94 GHz) double electron-electron resonance (DEER) we demonstrate strong specific interactions between DNA termini and TAM labels, leading to a significant restriction of their conformational mobility. An understanding of such interactions guides the way to select optimum TAM-labeling strategies, thus refining nanoscale EPR distance measurements in nucleic acids and their complexes under physiological conditions.

Conformations of Human Telomeric G-Quadruplex Studied Using a Nucleotide-Independent Nitroxide Label.

Guanine-rich oligonucleotides can form a unique G-quadruplex (GQ) structure with stacking units of four guanine bases organized in a plane through Hoogsteen bonding. GQ structures have been detected in vivo and shown to exert their roles in maintaining genome integrity and regulating gene expression. Understanding GQ conformation is important for understanding its inherent biological role and for devising strategies to control and manipulate functions based on targeting GQ. Although a number of biophysical methods have been used to investigate structure and dynamics of GQs, our understanding is far from complete. As such, this work explores the use of the site-directed spin labeling technique, complemented by molecular dynamics simulations, for investigating GQ conformations. A nucleotide-independent nitroxide label (R5), which has been previously applied for probing conformations of noncoding RNA and DNA duplexes, is attached to multiple sites in a 22-nucleotide DNA strand derived from the human telomeric sequence (hTel-22) that is known to form GQ. The R5 labels are shown to minimally impact GQ folding, and inter-R5 distances measured using double electron-electron resonance spectroscopy are shown to adequately distinguish the different topological conformations of hTel-22 and report variations in their occupancies in response to changes of the environment variables such as salt, crowding agent, and small molecule ligand. The work demonstrates that the R5 label is able to probe GQ conformation and establishes the base for using R5 to study more complex sequences, such as those that may potentially form multimeric GQs in long telomeric repeats.

Doubly Spin-Labeled RNA as an EPR Reporter for Studying Multicomponent Supramolecular Assemblies

mRNAs are involved in complicated supramolecular complexes with human 40S and 80S ribosomes responsible for the protein synthesis. In this work, a derivative of nonaribonucleotide pUUCGUAAAA with nitroxide spin labels attached to the 5′-phosphate and to the C8 atom of the adenosine in sixth position (mRNA analog) was used for studying such complexes using double electron-electron resonance/pulsed electron-electron double resonance spectroscopy. The complexes were assembled with participation of tRNAPhe, which targeted triplet UUC of the derivative to the ribosomal peptidyl site and predetermined location of the adjacent GUA triplet coding for Val at the aminoacyl (A) site. The interspin distances were measured between the two labels of mRNA analog attached to the first nucleotide of the peptidyl site bound codon and to the third nucleotide of the A site bound codon, in the absence/presence of second tRNA bound at the A site. The values of the obtained interspin distances agree with those calculated for available near-atomic structures of similar complexes of 40S and 80S ribosomes, showing that neither 60S subunit nor tRNA at the A site have a noticeable effect on arrangement of mRNA at the codon-anticodon interaction area. In addition, the shapes of distance distributions in four studied ribosomal complexes allowed conclusions on conformational flexibility of mRNA in these complexes. Overall, the results of this study are the first, to our knowledge, demonstration of double electron-electron resonance/pulsed electron-electron double resonance application for measurements of intramolecular distances in multicomponent supramolecular complexes involving intricate cellular machineries and for evaluating dynamic properties of ligands bound to these machineries.

A chimeric prokaryotic pentameric ligand-gated channel reveals distinct pathways of activation.

Recent high resolution structures of several pentameric ligand-gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron-electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand-gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand-gated ion channel, which is activated by protons. We found that the chimera was independently gated by primary amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators.

Mechanism of influenza A M2 transmembrane domain assembly in lipid membranes

M2 from influenza A virus functions as an oligomeric proton channel essential for the viral cycle, hence it is a high-priority pharmacological target whose structure and functions require better understanding. We studied the mechanism of M2 transmembrane domain (M2TMD) assembly in lipid membranes by the powerful biophysical technique of double electron-electron resonance (DEER) spectroscopy. By varying the M2TMD-to-lipid molar ratio over a wide range from 1:18,800 to 1:160, we found that M2TMD exists as monomers, dimers, and tetramers whose relative populations shift to tetramers with the increase of peptide-to-lipid (P/L) molar ratio. Our results strongly support the tandem mechanism of M2 assembly that is monomers-to-dimer then dimers-to-tetramer, since tight dimers are abundant at small P/L’s, and thereafter they assemble as dimers of dimers in weaker tetramers. The stepwise mechanism found for a single-pass membrane protein oligomeric assembly should contribute to the knowledge of the association steps in membrane protein folding.

SIGNAL TRANSDUCTION. Structural basis for nucleotide exchange in heterotrimeric G proteins.

G protein-coupled receptors (GPCRs) relay diverse extracellular signals into cells by catalyzing nucleotide release from heterotrimeric G proteins, but the mechanism underlying this quintessential molecular signaling event has remained unclear. Here we use atomic-level simulations to elucidate the nucleotide-release mechanism. We find that the G protein α subunit Ras and helical domains-previously observed to separate widely upon receptor binding to expose the nucleotide-binding site-separate spontaneously and frequently even in the absence of a receptor. Domain separation is necessary but not sufficient for rapid nucleotide release. Rather, receptors catalyze nucleotide release by favoring an internal structural rearrangement of the Ras domain that weakens its nucleotide affinity. We use double electron-electron resonance spectroscopy and protein engineering to confirm predictions of our computationally determined mechanism.

Structural Insights into the Dynamic Process of β2-Adrenergic Receptor Signaling

G-protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the β2-adrenergic receptor (β2AR) using (19)F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound β2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shiftthe equilibrium toward a conformation capable of engaging cytoplasmic G proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate, and active states. Complete transition to the active conformation requires subsequent interaction with a G protein or an intracellular G protein mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G-protein-coupling interface that may generally be responsible for the complex signaling behavior observed for many GPCRs.

Conformational dynamics of metallo-β-lactamase CcrA during catalysis investigated by using DEER spectroscopy.

Previous crystallographic and mutagenesis studies have implicated the role of a position-conserved hairpin loop in the metallo-β-lactamases in substrate binding and catalysis. In an effort to probe the motion of that loop during catalysis, rapid-freeze-quench double electron-electron resonance (RFQ-DEER) spectroscopy was used to interrogate metallo-β-lactamase CcrA, which had a spin label at position 49 on the loop and spin labels (at positions 82, 126, or 233) 20-35 Å away from residue 49, during catalysis. At 10 ms after mixing, the DEER spectra show distance increases of 7, 10, and 13 Å between the spin label at position 49 and the spin labels at positions 82, 126, and 233, respectively. In contrast to previous hypotheses, these data suggest that the loop moves nearly 10 Å away from the metal center during catalysis and that the loop does not clamp down on the substrate during catalysis. This study demonstrates that loop motion during catalysis can be interrogated on the millisecond time scale.

Gating Topology of the Proton-Coupled Oligopeptide Symporters

SummaryProton-coupled oligopeptide transporters belong to the major facilitator superfamily (MFS) of membrane transporters. Recent crystal structures suggest the MFS fold facilitates transport through rearrangement of their two six-helix bundles around a central ligand binding site; how this is achieved, however, is poorly understood. Using modeling, molecular dynamics, crystallography, functional assays, and site-directed spin labeling combined with double electron-electron resonance (DEER) spectroscopy, we present a detailed study of the transport dynamics of two bacterial oligopeptide transporters, PepTSo and PepTSt. Our results identify several salt bridges that stabilize outward-facing conformations and we show that, for all the current structures of MFS transporters, the first two helices of each of the four inverted-topology repeat units form half of either the periplasmic or cytoplasmic gate and that these function cooperatively in a scissor-like motion to control access to the peptide binding site during transport.

Determining Oligomeric Order of a Membrane Protein by Double Electron-Electron Resonance Spectroscopy

Many different classes of membrane proteins are known to form oligomers in cellular membranes in order to carry out specific cellular functions. Detection and detailed structural characterization of protein oligomers in lipid milieu is by no means a trivial task. Here we demonstrate the use of spin-labeling and Double Electron-Electron Resonance (DEER) spectroscopy to determine the oligomeric order of a membrane protein. Specifically, we investigate oligomerization of a seven-helical membrane photoreceptor Anabaena Sensory Rhodopsin (ASR) from Anabaena sp. PCC7120. Recently, ASR structure has been solved by both x-ray protein crystallography (Science 2004, 306, 1390) and solid-state NMR (Nat Methods 2013, 10, 1007). Here we show that the same spin-labeling sites we employed for paramagnetic relaxation enhancement (PRE) NMR can also be used for DEER experiments. The results demonstrate that DEER restraints can not only differentiate between the dimer (x-ray) and trimer (ssNMR) models that have very different interfaces, but further rule out hypothetical tetramer and other higher order polygon models. The crux of our DEER-based approach relies on taking advantage of the multi-spin effects and analyzing experimental DEER traces by direct fitting to the multispin models. Overall, the observed profound effect of higher order spin correlations on the DEER trace allows for a reliable differentiation between oligomer models. In the specific case of ASR, the DEER trace modeling allowed us to unambiguously discard all but the trimer model. Furthermore, the addition of DEER electron-electron distances to the NMR restraints in the structure calculation protocol improves local RMSD, and allows for refinement of the orientation of helices. Supported by U.S. DOE Contract DE-FG02-02ER15354 to AIS and NSERC Discovery Grants RGPIN-2014-04547 to VL and RGPIN-2013-250202 to LSB.

230/115 GHz Electron Paramagnetic Resonance/Double Electron-Electron Resonance Spectroscopy.

Electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) spectroscopies are powerful and versatile tools for studying local structures and dynamic properties of biological molecules. Similar to nuclear magnetic resonance (NMR) spectroscopy, EPR/DEER spectroscopies become more advantageous at higher frequencies and higher magnetic fields because of better spectral resolution as well as higher spin polarization. Here, we describe development of a high-frequency (HF) EPR/DEER spectrometer operating in the frequency range of 107-120 and 215-240 GHz and in the magnetic field range of 0-12.1 T, which has unique experimental capabilities such as enabling the complete spin polarization and wide-band DEER spectroscopy. Emphasis is given on the application of HF EPR/DEER techniques, and specific examples of HF EPR spectroscopy to drastically increase spin coherence in nanodiamonds as well as HF DEER spectroscopy to extract spin concentration in a diamond crystal are presented.

Uncertainty Quantification in Deer Spectroscopy using Bayesian Statistical Inversion Methods

Double electron-electron resonance (DEER) spectroscopy is a powerful electron paramagnetic resonance experiment that provides 2-8nm distance measurements between spin-labels in systems varying from small, soluble proteins, to large, membrane-bound complexes. A key strength of DEER is that it reports a distance distribution corresponding to conformational ensembles in frozen solutions. However, in practice its usable information is often limited to the dominant spin-spin distance. Peak widths and shapes, which describe conformational heterogeneity, are highly sensitive to time-domain noise, regularization parameters, and spin-label conformations. A robust method for quantifying uncertainty in DEER distributions does not currently exist, complicating interpretation. Here we demonstrate the use of Bayesian statistical inversion methods to generate conditional covariance estimates and Bayesian credibility sets in DEER data analysis. To decouple true uncertainty from spin-label conformations, we attached spin labels featuring varying flexibilities to maltose binding protein (MBP) and measured using DEER under varying conditions. By determining the contributions to DEER data from both the spin-label and uncertainty, we have taken an important step towards robust assignment of distance distribution features to protein conformations.

Pores of No Return

In this issue, Bleicken et al. (2014) use double electron-electron resonance (DEER) spectroscopy to propose a new model for the active form of Bax at membranes that differs significantly from those previously proposed.

Determining the Oligomeric Structure of Proteorhodopsin by Gd3+-Based Pulsed Dipolar Spectroscopy of Multiple Distances

The structural organization of the functionally relevant, hexameric oligomer of green-absorbing proteorhodopsin (G-PR) was obtained from double electron-electron resonance (DEER) spectroscopy utilizing conventional nitroxide spin labels and recently developed Gd3+ -based spin labels. G-PR with nitroxide or Gd3+ labels was prepared using cysteine mutations at residues Trp58 and Thr177. By combining reliable measurements of multiple interprotein distances in the G-PR hexamer with computer modeling, we obtained a structural model that agrees with the recent crystal structure of the homologous blue-absorbing PR (B-PR) hexamer. These DEER results provide specific distance information in a membrane-mimetic environment and across loop regions that are unresolved in the crystal structure. In addition, the X-band DEER measurements using nitroxide spin labels suffered from multispin effects that, at times, compromised the detection of next-nearest neighbor distances. Performing measurements at high magnetic fields with Gd3+ spin labels increased the sensitivity considerably and alleviated the difficulties caused by multispin interactions.