Dipolar Electron-electron Resonance Research Articles

MESMER: minimal ensemble solutions to multiple experimental restraints

Macromolecular structures and interactions are intrinsically heterogeneous, temporally adopting a range of configurations that can confound the analysis of data from bulk experiments. To obtain quantitative insights into heterogeneous systems, an ensemble-based approach can be employed, in which predicted data computed from a collection of models is compared to the observed experimental results. By simultaneously fitting orthogonal structural data (e.g. small-angle X-ray scattering, nuclear magnetic resonance residual dipolar couplings, dipolar electron-electron resonance spectra), the range and population of accessible macromolecule structures can be probed. We have developed MESMER, software that enables the user to identify ensembles that can recapitulate experimental data by refining thousands of component collections selected from an input pool of potential structures. The MESMER suite includes a powerful graphical user interface (GUI) to streamline usage of the command-line tools, calculate data from structure libraries and perform analyses of conformational and structural heterogeneity. To allow for incorporation of other data types, modular Python plugins enable users to compute and fit data from nearly any type of quantitative experimental data. Conformational heterogeneity in three macromolecular systems was analyzed with MESMER, demonstrating the utility of the streamlined, user-friendly software. https://code.google.com/p/mesmer/

Probing Conformational Change in the First Actin-Binding Domain of Dystrophin

We have used time-resolved EPR and fluorescence to resolve structural transitions of dystrophin upon actin binding. Dystrophin (Dys) is a muscle cytoskeletal protein that binds to filamentous actin (F-actin) and the dystroglycan complex in the sarcolemmal membrane. Dys acts to dissipate mechanical forces generated during the contraction and relaxation of muscle thereby maintaining sarcolemmal membrane integrity and protecting from tears. The protein-protein interactions and allostery underlying this function of Dys have not been well studied in the context of conformational change and thermodynamics, partly because acquisition of structural and thermodynamic detail on large and flexible proteins is difficult. Two techniques capable of measuring large-scale conformational changes are dipolar electron-electron resonance (DEER) and time-resolved fluorescence resonance energy transfer (TR-FRET). Using a combination of DEER and TR-FRET, we placed a single label (nitroxide or fluorescent) in each CH domain Dys ABD1 and subsequently measured the interprobe distance to assess conformational change upon association with F-actin. To probe the allosteric network of Dys ABD1, we also subjected the protein to differential scanning calorimetry.

EPR with Rigidly Bound Spin Labels used to Probe the Interaction of Calmodulin with the Ryanodine Receptor

We have used site-directed spin labeling and EPR spectroscopy to probe the structural dynamics of calmodulin bound to a ryanodine receptor target peptide. Calmodulin binds to a conserved 30-amino acid sequence on the sarcoplasmic reticulum calcium release channel (ryanodine receptor, RyR) and directly modulate its activity in muscle contraction. In order to characterize the structural details of this crucial interaction, a bifunctional spin label was used to rigidly label di-cysteine mutant calmodulin at positions 34 and 38. Solid-phase peptide synthesis was used to create a peptide corresponding to the calmodulin binding site on the ryanodine receptor channel (RyRp, residues 3614-3643 in RyR1) with a TOAC spin label rigidly coupled to the backbone. The intermolecular distance distribution between the two spin probes was determined by EPR, using both continuous wave dipolar broadening and dipolar electron-electron resonance (DEER). The center of the distance distribution was in good agreement with the crystal structure of the complex (18 A), but the width of the distribution was ∼ 1 nm, comparable to that observed previously within calmodulin itself. Thus calmodulin retains significant inter-lobe domain motion, even after RyRp binding. These results are consistent with a model in which calmodulin binds RyRp, bringing its two terminal lobes close together but preventing them from physical contact with each other, allowing flexibility in each of the lobe.

Backbone Orientation and Distance Measurements in Myosin II: Applications of High-Resolution EPR using a Bifunctional Spin Label

We have used electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) to obtain high-resolution measurements of individual structural elements within the myosin II catalytic domain (CD). Two complementary EPR techniques were employed to measure protein orientation (conventional EPR) and intra-protein distances (dipolar electron-electron resonance, DEER). The use of BSL greatly enhances the resolution of EPR, by virtue of its strongly immobilized and stereoselective bifunctional attachment to the protein backbone at two engineered Cys residues. Crucially, both techniques utilized here permit the elucidation of myosin structure while in complex with actin, generating relevant constraints for the refinement of actomyosin structural models. In the current work, Dictyostelium myosin II was used as our model system. We measured nucleotide-dependent structural transitions of three key helices within the myosin CD. Three double-Cys sites were engineered, with Cys pairs located on the relay helix, helix HK (upper 50kDa domain) and helix HW (lower 50kDa domain), respectively. BSL on a construct with one of these pairs was used to measure myosin orientation relative to oriented actin. BSL on a construct with two pairs was used to measure interprobe distances. The effect of ADP binding was clearly detected by EPR, and subsequently modeled using the orientation and distance measurements as constraints. We find that the structural change induced by ADP in the actin-bound myosin CD is clearly different from that predicted from actin-free crystal structures. This work was funded by grants from NIH (R01 AR32961, T32 AR07612, P30 AR0507220).

High-Resolution Measurement of Distance and Orientation in Myosin: EPR of a Bifunctional Spin Label

We present a method for obtaining high-resolution information on protein backbone structure and dynamics using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) and molecular modeling. Two complementary EPR techniques were employed to measure protein orientation (conventional EPR) and intra-protein distances (dipolar electron-electron resonance, DEER). BSL attaches at Cys positions i and i+4 on a helix, greatly reducing probe mobility relative to the peptide backbone, compared to monofunctional labels. Accurate modeling of BSL provides the coordinates required to directly relate spectroscopic data to backbone structure (both orientation and distance), and dynamics (rotational motion). In the current work, the motor protein Dictyostelium myosin II was used to demonstrate this approach. We measured nucleotide-dependent structural transitions of two key helices within the myosin catalytic domain (CD). Two double-Cys sites were engineered, with one Cys pair located on the relay helix, and the other on a stable helix in the upper 50kD domain. BSL on a construct with one of these pairs was used to measure myosin orientation relative to oriented actin. BSL on a construct with two pairs was used to measure interprobe distances. The effect of ADP binding on both orientation and distance was clearly detected with BSL, but not with a monofunctional label. The significance of this work is twofold: (1) A structural transition in the relay helix upon ADP binding was clearly defined with high resolution. (2) BSL spectra demonstrate superior resolution, compared to monofunctional spin labels, making it possible to directly translate spectroscopic data to protein structure and dynamics. This work was funded by grants from NIH (R01 AR32961, T32 AR07612, P30 AR0507220).

Structure-Function Relationships of M124Q Calmodulin, a Mutant that Mimics Oxidative Insults

Calmodulin (CaM) is a calcium-sensitive regulatory protein that interacts with numerous target proteins, including the ryanodine receptor (RyR). The RyR is a calcium channel responsible for releasing Ca2+ from the sarcoplasmic reticulum to induce muscle contraction. Due to its high methionine content, CaM is highly susceptible to oxidation by reactive oxygen species produced by normal metabolism. It has been shown that the oxidation level of CaM increases with aging, and contributes to muscle degeneration. Oxidized forms of CaM also have a decreased ability to regulate RyR function, thus disrupting the regulation of muscle contraction. The M124Q CaM mutant mimics the effects of a methionine sulfoxide at position 124, a key residue for CaM's interaction with the RyR. Here, we used dipolar electron-electron resonance (DEER) and NMR to characterize the structural dynamics of the M124Q-CaM. DEER analysis of M124Q-CaM suggests that the mutant exhibits open and closed conformations in micromolar Ca2+, but shifts to the more open conformation in nanomolar Ca2+. Differences in the population distributions were also observed for M124Q-CaM in the presence and absence of a RyR1 peptide that forms part of the CaM binding site. Finally, NMR analysis of wild-type and M124Q-CaM also indicated structural shifts due to the mutation and describe how both the wild-type and M124Q CaM respond to changes in Ca2+ concentration. These initial results give important insights on the structural effects of deleterious oxidative processes on the RyR regulation by CaM.

Large Scale Opening of the N Terminal Actin-Binding Domain of Dystrophin Detected by Dipolar Electron-Electron Resonance (DEER)

We have used dipolar electron-electron resonance (DEER) to observe the structural dynamics of the calponin homology (CH) domains within the N-terminal actin binding domain (ABD1) of dystrophin, in order to determine the conformation of the ABD1 while free in solution and while actin-bound. Loss of functional dystrophin causes Duchenne (DMD) and Becker (BMD) muscular dystrophies, prompting a need for structural information about dystrophin to aid in engineering gene therapy constructs. We have previously shown that the ABD1 of dystrophin's autosomal homologue utrophin opens upon actin binding via an induced fit mechanism, suggesting a similar mechanism for the dystrophin ABD1. Site-directed spin labeling and DEER were used to measure the distance between labels on the two CH domains, in the presence or absence of actin and at varying ionic strength. DEER spectra showed that like utrophin, the actin-bound dystrophin ABD1 occupied a single well-ordered conformation in which the CH domains were much more open than when the ABD1 is free in solution. Unlike utrophin, the dystrophin ABD1 was much more disordered when free in solution, an effect that was exacerbated by increasing the ionic strength. We conclude that, similarly to utrophin, the dystrophin ABD1 undergoes a closed-to-open transition upon binding to actin. In contrast to utrophin, the dystrophin ABD1 is much more disordered when not bound to actin, suggesting that dystrophin is intrinsically less stable than utrophin. These results provide the needed structural foundation for studying disease-causing mutations in ABD1, and they provide a rationale for using utrophin in place of dystrophin in DMD therapy. This work was funded by NIH Grants AR032961, AR057220 Core C, and AG026160 (to D.D.T.), MDA Grant 4322 (to D.D.T.), and NIH grant F30AG034033 (to A.Y.L.).

High-Resolution EPR of a Bifunctional Spin Label Reveals Structural Transitions within Myosin's Catalytic Domain

We present a method for obtaining high-resolution information on protein backbone structure and dynamics using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) and molecular modeling. Two complementary EPR techniques were employed to measure protein orientation (conventional EPR) and intra-protein distances (dipolar electron-electron resonance, DEER). BSL attaches at Cys positions i and i+4 on a helix, greatly reducing probe mobility relative to the peptide backbone, compared to monofunctional labels. Accurate modeling of BSL provides the coordinates required to directly relate spectroscopic data to backbone structure (both orientation and distance), and dynamics (rotational motion). In the current work, the motor protein Dictyostelium myosin II was used to demonstrate this approach. We measured nucleotide-dependent structural transitions of two key helices within the myosin catalytic domain (CD). Two double-Cys sites were engineered, with one Cys pair located on the relay helix, and the other on a stable helix in the upper 50kD domain. BSL on a construct with one of these pairs was used to measure myosin orientation relative to oriented actin. BSL on a construct with two pairs was used to measure interprobe distances. The effect of ADP binding on both orientation and distance was clearly detected with BSL, but not with a monofunctional label. The significance of this work is twofold: (1) A structural transition in the relay helix upon ADP binding was clearly defined with high resolution. (2) BSL spectra demonstrate superior resolution, compared to monofunctional spin labels, making it possible to directly translate spectroscopic data to protein structure and dynamics.

High-Resolution Myosin Structure from DEER Using a Bifunctional Spin Label

We are using site-directed spin-labeling (SDSL) and dipolar electron-electron resonance (DEER) to study the structural dynamics of proteins involved in muscle function. Muscle proteins are ideal systems for DEER applications, since these proteins form large dynamic assemblies that are inaccessible to crystallography and NMR. DEER is a pulsed ELDOR technique that detects the spin-spin dipolar interaction as a decay and modulation of a spin echo signal. While the CW EPR spectrum is sensitive only to distances on the order of 1 to 2 nm, DEER is sensitive from 1.5 to 5 nm and more. DEER is sensitive to the width of the distance distribution and can resolve multiple structural populations simultaneously. However, a limitation of DEER is the use of spin probes with flexible linkers, which adds uncertainty to the locations of protein backbone atoms. A bifunctional spin label (BSL), which attaches at position (i, i+4) of a helix, was used to measure the distance between two di-Cys pairs. BSL is rigidly coupled to the protein backbone and is a breakthrough for high resolution measurement of orientation, dynamics, and distance. These distance measurements were compared to conventional labeling of cysteine residues with MSL/MSL pairs.

Characterizing the Phospholamban-SERCA Complex by Pulsed EPR Spectroscopy

Muscle contraction and relaxation are controlled through the release and reuptake of Ca2+ stored in the sarcoplasmic reticulum (SR). Relaxation is mediated by the SR Ca2+ ATPase (SERCA), a pump that drives Ca2+ against its concentration gradient while hydrolyzing ATP. Cardiac SERCA is regulated by phospholamban (PLB), a small membrane protein that inhibits the pump except when phosphorylated at Ser16. PLB phosphorylation restores SERCA activity without dissociating the two proteins, instead inducing a structural change within the PLB-SERCA complex. Although a number of studies have investigated the interaction of these proteins, the relationship between phosphorylation, structure, and activity remains unresolved. We have used dipolar electron-electron resonance (DEER) spectroscopy, a technique capable of measuring distances from 2-7nm, to characterize large conformational changes within PLB upon phosphorylation and SERCA binding. Our results show that the transmembrane and cytoplasmic helices of PLB draw closer upon SERCA binding, with subsequent phosphorylation compacting the structure still further. However, relative distances between the cytoplasmic domains of PLB and SERCA remain largely constant before and after phosphorylation, suggesting that the observed structural change occurs in the transmembrane domain of PLB. Ultimately, our goal is to make exhaustive distance measurements within the SERCA-PLB complex in order to understand the structural basis of phosphorylation-mediated inhibition relief.

Structural Basis for Uncoupling of Force Generation in the F506A Dictyostelium Myosin Revealed by Time-Resolved EPR and FRET

We have used dipolar electron-electron resonance (DEER) and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the role of the myosin relay helix in coupling between the active site and the force-generating region of myosin II. Two double-Cys Dictyostelium myosin constructs have been engineered, and the structure of the relay helix was monitored by measuring interprobe distances in MSL/MSL or IAEDANS/Dabcyl-labeled myosin. Experiments were performed on WT myosin and on F506A, a functional mutant that has close to normal enzymatic activity but completely lacks motor functions (e.g.