Distance and dynamics determination by W-band DEER and W-band ST-EPR

To explore high-field EPR in biological applications we have compared measurements of dynamics with X-band (9 GHz) and W-band (94 GHz) saturation transfer EPR (ST-EPR) and distance determination by X and W-band DEER. A fourfold increase of sensitivity was observed for W-band ST-EPR compared with X-band. The distance measurements at both fields showed very good agreement in both the average distances and in the distance distributions. Multifrequency EPR thus provides an additional experimental dimension to facilitate extraction of distance populations. However, the expected orientational selectivity of W-band DEER to determine the relative orientation of spins has not been realized, most likely because of the large orientational disorder of spin labels on the protein surface.     

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

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

Pulsed electron-electron double resonance: beyond nanometre distance measurements on biomacromolecules

PELDOR (or DEER; pulsed electron-electron double resonance) is an EPR (electron paramagnetic resonance) method that measures via the dipolar electron-electron coupling distances in the nanometre range, currently 1.5-8 nm, with high precision and reliability. Depending on the quality of the data, the error can be as small as 0.1 nm. Beyond mere mean distances, PELDOR yields distance distributions, which provide access to conformational distributions and dynamics. It can also be used to count the number of monomers in a complex and allows determination of the orientations of spin centres with respect to each other. If, in addition to the dipolar through-space coupling, a through-bond exchange coupling mechanism contributes to the overall coupling both mechanisms can be separated and quantified. Over the last 10 years PELDOR has emerged as a powerful new biophysical method without size restriction to the biomolecule to be studied, and has been applied to a large variety of nucleic acids as well as proteins and protein complexes in solution or within membranes. Small nitroxide spin labels, paramagnetic metal ions, amino acid radicals or intrinsic clusters and cofactor radicals have been used as spin centres.     

Simulation vs. reality: a comparison of in silico distance predictions with DEER and FRET measurements

Site specific incorporation of molecular probes such as fluorescent- and nitroxide spin-labels into biomolecules, and subsequent analysis by F?rster resonance energy transfer (FRET) and double electron-electron resonance (DEER) can elucidate the distance and distance-changes between the probes. However, the probes have an intrinsic conformational flexibility due to the linker by which they are conjugated to the biomolecule. This property minimizes the influence of the label side chain on the structure of the target molecule, but complicates the direct correlation of the experimental inter-label distances with the macromolecular structure or changes thereof. Simulation methods that account for the conformational flexibility and orientation of the probe(s) can be helpful in overcoming this problem. We performed distance measurements using FRET and DEER and explored different simulation techniques to predict inter-label distances using the Rpo4/7 stalk module of the M. jannaschii RNA polymerase. This is a suitable model system because it is rigid and a high-resolution X-ray structure is available. The conformations of the fluorescent labels and nitroxide spin labels on Rpo4/7 were modeled using in vacuo molecular dynamics simulations (MD) and a stochastic Monte Carlo sampling approach. For the nitroxide probes we also performed MD simulations with explicit water and carried out a rotamer library analysis. Our results show that the Monte Carlo simulations are in better agreement with experiments than the MD simulations and the rotamer library approach results in plausible distance predictions. Because the latter is the least computationally demanding of the methods we have explored, and is readily available to many researchers, it prevails as the method of choice for the interpretation of DEER distance distributions.     

Toward the fourth dimension of membrane protein structure: insight into dynamics from spin-labeling EPR spectroscopy

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 ?-80 ?) 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.     

Determination of interspin distances between spin labels attached to insulin: comparison of electron paramagnetic resonance data with the X-ray structure

A method was developed to determine the interspin distances of two or more nitroxide spin labels attached to specific sites in proteins. This method was applied to different conformations of spin-labeled insulins. The electron paramagnetic resonance (EPR) line broadening due to dipolar interaction is determined by fitting simulated EPR powder spectra to experimental data, measured at temperatures below 200 K to freeze the protein motion. The experimental spectra are composed of species with different relative nitroxide orientations and interspin distances because of the flexibility of the spin label side chain and the variety of conformational substates of proteins in frozen solution. Values for the average interspin distance and for the distance distribution width can be determined from the characteristics of the dipolar broadened line shape. The resulting interspin distances determined for crystallized insulins in the R6 and T6 structure agree nicely with structural data obtained by x-ray crystallography and by modeling of the spin-labeled samples. The EPR experiments reveal slight differences between crystal and frozen solution structures of the B-chain amino termini in the R6 and T6 states of hexameric insulins. The study of interspin distances between attached spin labels can be applied to obtain structural information on proteins under conditions where other methods like two-dimensional nuclear magnetic resonance spectroscopy or x-ray crystallography are not applicable.     

Kinetics of Fast Changing Intramolecular Distance Distributions Obtained by Combined Analysis of FRET Efficiency Kinetics and Time-Resolved FRET Equilibrium Measurements

Detailed studies of the mechanisms of macromolecular conformational transitions such as protein folding are enhanced by analysis of changes of distributions for intramolecular distances during the transitions. Time-resolved Förster resonance energy transfer (FRET) measurements yield such data, but the more readily available kinetics of mean FRET efficiency changes cannot be analyzed in terms of changes in distances because of the sixth-power dependence on the mean distance. To enhance the information obtained from mean FRET efficiency kinetics, we combined the analyses of FRET efficiency kinetics and equilibrium trFRET experiments. The joint analysis enabled determination of transient distance distributions along the folding reaction both in cases where a two-state transition is valid and in some cases consisting of a three-state scenario. The procedure and its limits were tested by simulations. Experimental data obtained from stopped-flow measurements of the refolding of Escherichia coli adenylate kinase were analyzed. The distance distributions between three double-labeled mutants, in the collapsed transient state, were determined and compared to those obtained experimentally using the double-kinetics technique. The proposed method effectively provides information on distance distributions of kinetically accessed intermediates of fast conformational transitions induced by common relaxation methods.     

Kinetics of Fast Changing Intramolecular Distance Distributions Obtained by Combined Analysis of FRET Efficiency Kinetics and Time-Resolved FRET Equilibrium Measurements

Detailed studies of the mechanisms of macromolecular conformational transitions such as protein folding are enhanced by analysis of changes of distributions for intramolecular distances during the transitions. Time-resolved Förster resonance energy transfer (FRET) measurements yield such data, but the more readily available kinetics of mean FRET efficiency changes cannot be analyzed in terms of changes in distances because of the sixth-power dependence on the mean distance. To enhance the information obtained from mean FRET efficiency kinetics, we combined the analyses of FRET efficiency kinetics and equilibrium trFRET experiments. The joint analysis enabled determination of transient distance distributions along the folding reaction both in cases where a two-state transition is valid and in some cases consisting of a three-state scenario. The procedure and its limits were tested by simulations. Experimental data obtained from stopped-flow measurements of the refolding of Escherichia coli adenylate kinase were analyzed. The distance distributions between three double-labeled mutants, in the collapsed transient state, were determined and compared to those obtained experimentally using the double-kinetics technique. The proposed method effectively provides information on distance distributions of kinetically accessed intermediates of fast conformational transitions induced by common relaxation methods.     

Structural studies on membrane proteins using non-linear spin label EPR spectroscopy

Non-linear electron spin resonance (EPR) techniques suitable for measuring proximity relationships in membranes are reviewed. These were developed during the past decade in order to measure changes sensitively in the spin-lattice relaxation time (T1) of nitroxyl spin labels covalently attached to membrane lipids or proteins. In combination with paramagnetic quenching agents and double spin-labelling, the methods were further developed for distance measurements. Selected examples are given to illustrate different methods, and types of data obtained for both integral and peripheral membrane proteins.     

生物大分子相互作用检测技术新进展———三色荧光级联荧光共振能量转移技术

荧光共振能量转移(fluorescenceresonanceenergytransfer,FRET),是指能量从一种受激发的荧光基团(fluorophore)以非辐射的方式转移到另一种荧光基团的物理现象.FRET的能量转移效率是两个荧光基团间距离的函数,并对此距离十分敏感,它的有效响应距离一般在1～10nm之间,因而可被用于测定原子间及分子间的距离.这一特点使FRET技术在大分子构象变化、大分子之间相互作用、细胞信号通路等研究中发挥重要作用,成为生物医学研究中的重要方法.但细胞内的生物学过程常常涉及多于两个的大分子间相互作用,二色荧光基团的FRET技术不能满足这种生物学研究的需求.最近,两个研究小组在这方面取得突破,建立了分别基于共聚焦显微镜和流式细胞仪的三色荧光级联FRET技术.这一技术的出现将会极大地促进生物学及相关研究领域的发展.     

Structure and dynamics of a DNA-based model system for the study of electron spin-spin interactions

We report on the structure and dynamics of a model system for measuring long-range distances in biological macromolecules by saturation-recovery EPR. Four DNA duplexes that incorporate a paramagnetic dysprosium ion (Dy(III)) and a nitroxide spin-label were examined by electron paramagnetic resonance (EPR), circular dichroism (CD), and ultra-violet absorbance (UV) spectroscopy. Dy(III) is chelated by the modified base deoxythymidine-EDTA, (dT-EDTA). Electron spin-spin interactions between the Dy(III) ion and the nitroxide radical are observed at distances as great as ∼5.3 nm. A slight change in the conformation of those nucleotides lying between the EDTA(Dy(III)) complex and the nitroxide spin-label results in a “stiffening” of the DNA helix on the EPR time scale. Changes in conformation and helix dynamics are due to the binding of the EDTA(Dy(III)) complex to the phosphodiester backbone of the complementary strand. Molecular mechanics calculations indicate that binding occurs in the 5′ direction on the complementary strand, at a position 3 or 4 phosphates distant from the dT-EDTA(Dy(III)) * dA base pair.     

Single molecule conformational analysis of DNA G-quadruplexes

Single molecule fluorescence resonance energy transfer (FRET) can be employed to study conformational heterogeneity and real-time dynamics of biological macromolecules. Here we present single molecule studies on human genomic DNA G-quadruplex sequences that occur in the telomeres and in the promoter of a proto-oncogene. The findings are discussed with respect to the proposed biological function(s) of such motifs in living cells.     

Protein dynamics and monomer-monomer interactions in AntR activation by electron paramagnetic resonance and double electron-electron resonance

The Anthracis repressor (AntR) is a Mn(II)-activated DNA binding protein that is involved in the regulation of Mn(II) homeostasis in Bacillus anthracis. AntR is structurally and functionally homologous to Mn(II)-activated repressor from Bacillus subtillis (MntR). Our studies on AntR focus on metal-regulated activation of the protein. Line shape analysis of continuous wave electron paramagnetic resonance (EPR) spectra showed that metal binding resulted in a general reduction of backbone dynamics and that there were no further changes in backbone motion upon DNA binding. Double electron-electron resonance (DEER) pulsed EPR spectroscopy was used to measure distances between nitroxide spin labels strategically placed in dimeric AntR. The DEER data were analyzed assuming Gaussian distributions for discrete populations of spins. A structural model for AntR was built from homology to MntR, and the experimentally measured distances were simulated to distinguish between spin label and backbone motions. Together with the computational analysis, the DEER results for apo-AntR indicated relatively narrow conformational distributions for backbone residues at the dimer interface and near the metal binding site. No significant changes were observed on these sites in the presence of metal or DNA. On the other hand, the distribution of the conformers and the distances between the putative DNA binding helices decreased upon metal binding. These results suggest that the DNA binding region of AntR shows large amplitude backbone motions in the absence of metal, which may preclude sequence-specific binding to promoter sites. Metal binding narrows the range of conformations accessible in this region and shortens the mean distance between the DNA binding helices, probably resulting in alignment that optimizes promoter recognition and binding.     

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

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

A computational tool for designing FRET protein biosensors by rigid-body sampling of their conformational space

Biosensors relying on the fluorescence resonance energy transfer (FRET) between fluorescent proteins have been used for live-cell imaging of cellular events including Ca(2+) signaling. The efficiency of energy transfer between the donor and acceptor fluorescent proteins depends on the relative distance and orientation between them, which become altered by conformational changes of a fused sensory protein caused by a cellular event. In this way, changes in FRET efficiency of Ca(2+) biosensors can be correlated with Ca(2+) concentrations. The design of these FRET biosensors can be improved by modeling conformational changes before and after a cellular event. Hence, a computational tool called FPMOD was developed to predict FRET efficiency changes by constructing FRET biosensors and sampling their conformational space through rigid-body rotation. We showed with FPMOD that our computational modeling approach can qualitatively predict the FRET efficiencies of a range of biosensors, which had strong agreement with experimental results.     

Ensemble models of proteins and protein domains based on distance distribution restraints

Conformational ensembles of intrinsically disordered peptide chains are not fully determined by experimental observations. Uncertainty due to lack of experimental restraints and due to intrinsic disorder can be distinguished if distance distributions restraints are available. Such restraints can be obtained from pulsed dipolar electron paramagnetic resonance (EPR) spectroscopy applied to pairs of spin labels. Here, we introduce a Monte Carlo approach for generating conformational ensembles that are consistent with a set of distance distribution restraints, backbone dihedral angle statistics in known protein structures, and optionally, secondary structure propensities or membrane immersion depths. The approach is tested with simulated restraints for a terminal and an internal loop and for a protein with 69 residues by using sets of sparse restraints for underlying well‐defined conformations and for published ensembles of a premolten globule‐like and a coil‐like intrinsically disordered protein. Proteins 2016; 84:544–560. © 2016 Wiley Periodicals, Inc.     

Site-selective labeling and electron paramagnetic resonance studies of human cannabinoid receptor CB2

Integral membrane G protein-coupled receptors (GPCR) regulate multiple physiological processes by transmitting signals from extracellular milieu to intracellular proteins and are major targets of pharmaceutical drug development. Since GPCR are inherently flexible proteins, their conformational dynamics can be studied by spectroscopic techniques such as electron paramagnetic resonance (EPR) which requires selective chemical labeling of the protein. Here, we developed protocols for selective chemical labeling of the recombinant human cannabinoid receptor CB₂ by judiciously replacing naturally occurring reactive cysteine residues and introducing a new single cysteine residue in selected positions. The majority of the 47 newly generated single cysteine constructs expressed well in E. coli cells, and more than half of them retained high functional activity. The reactivity of newly introduced cysteine residues was assessed by incorporating nitroxide spin label and EPR measurement. The conformational transition of the receptor between the inactive and activated form were studied by EPR of selectively labeled constructs in the presence of either a full agonist CP-55,940 or an inverse agonist SR-144,528. We observed evidence for higher mobility of labels in the center of internal loop 3 and a structural change between agonist vs. inverse agonist-bound CB₂ in the extracellular tip of transmembrane helix 6. Our results demonstrate the utility of EPR for studies of conformational dynamics of CB₂.