Benjamin P. Binder

Redox-sensitive residue in the actin-binding interface of myosin.

We have examined the chemical and functional reversibility of oxidative modification in myosin. Redox regulation has emerged as a crucial modulator of protein function, with particular relevance to aging. We previously identified a single methionine residue in Dictyostelium discoideum (Dicty) myosin II (M394, near the myosin cardiomyopathy loop in the actin-binding interface) that is functionally sensitive to oxidation. We now show that oxidation of M394 is reversible by methionine sulfoxide reductase (Msr), restoring actin-activated ATPase activity. Sequence alignment reveals that M394 of Dicty myosin II is a cysteine residue in all human isoforms of skeletal and cardiac myosin. Using Dicty myosin II as a model for site-specific redox sensitivity of this Cys residue, the M394C mutant can be glutathionylated in vitro, resulting in reversible inhibition of actin-activated ATPase activity, with effects similar to those of methionine oxidation at this site. This work illustrates the potential for myosin to function as a redox sensor in both non-muscle and muscle cells, modulating motility/contractility in response to oxidative stress.

High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label

Significance The interaction between actin and myosin is responsible for driving a vast array of essential biological processes, including the production of force in contracting muscle. However, the structural behavior of the two proteins in complex is not well understood, because high-resolution atomic models are not yet available by traditional methods. We use a bifunctional spin label and site-directed electron paramagnetic resonance spectroscopy to determine orientations of individual α-helices within the complex. We thus quantify for the first time, to our knowledge, structural changes within the motor domain of actin-bound myosin on nucleotide binding and dissociation. Our results provide valuable insight into the mechanism of muscle contraction while showcasing a method with wide applicability to other oriented biological systems. Using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) bound stereospecifically to Dictyostelium myosin II, we determined with high resolution the orientation of individual structural elements in the catalytic domain while myosin is in complex with actin. BSL was attached to a pair of engineered cysteine side chains four residues apart on known α-helical segments, within a construct of the myosin catalytic domain that lacks other reactive cysteines. EPR spectra of BSL-myosin bound to actin in oriented muscle fibers showed sharp three-line spectra, indicating a well-defined orientation relative to the actin filament axis. Spectral analysis indicated that orientation of the spin label can be determined within <2.1° accuracy, and comparison with existing structural data in the absence of nucleotide indicates that helix orientation can also be determined with <4.2° accuracy. We used this approach to examine the crucial ADP release step in myosin’s catalytic cycle and detected reversible rotations of two helices in actin-bound myosin in response to ADP binding and dissociation. One of these rotations has not been observed in myosin-only crystal structures.

Bifunctional Spin Labeling of Muscle Proteins

While EPR allows for the characterization of protein structure and function due to its exquisite sensitivity to spin label dynamics, orientation, and distance, these measurements are often limited in sensitivity due to the use of labels that are attached via flexible monofunctional bonds, incurring additional disorder and nanosecond dynamics. In this chapter, we present methods for using a bifunctional spin label (BSL) to measure muscle protein structure and dynamics. We demonstrate that bifunctional attachment eliminates nanosecond internal rotation of the spin label, thereby allowing the accurate measurement of protein backbone rotational dynamics, including microsecond-to-millisecond motions by saturation transfer EPR. BSL also allows for accurate determination of helix orientation and disorder in mechanically and magnetically aligned systems, due to the labels stereospecific attachment. Similarly, labeling with a pair of BSL greatly enhances the resolution and accuracy of distance measurements measured by double electron-electron resonance (DEER). Finally, when BSL is applied to a protein with high helical content in an assembly with high orientational order (e.g., muscle fiber or membrane), two-probe DEER experiments can be combined with single-probe EPR experiments on an oriented sample in a process we call BEER, which has the potential for ab initio high-resolution structure determination.

A bifunctional spin label reports the structural topology of phospholamban in magnetically-aligned bicelles

We have applied a bifunctional spin label and EPR spectroscopy to determine membrane protein structural topology in magnetically-aligned bicelles, using monomeric phospholamban (PLB) as a model system. Bicelles are a powerful tool for studying membrane proteins by NMR and EPR spectroscopies, where magnetic alignment yields topological constraints by resolving the anisotropic spectral properties of nuclear and electron spins. However, EPR bicelle studies are often hindered by the rotational mobility of monofunctional Cys-linked spin labels, which obscures their orientation relative to the protein backbone. The rigid and stereospecific TOAC label provides high orientational sensitivity but must be introduced via solid-phase peptide synthesis, precluding its use in large proteins. Here we show that a bifunctional methanethiosulfonate spin label attaches rigidly and stereospecifically to Cys residues at i and i+4 positions along PLBs transmembrane helix, thus providing orientational resolution similar to that of TOAC, while being applicable to larger membrane proteins for which synthesis is impractical. Computational modeling and comparison with NMR data shows that these EPR experiments provide accurate information about helix tilt relative to the membrane normal, thus establishing a robust method for determining structural topology in large membrane proteins with a substantial advantage in sensitivity over NMR.

Bifunctional Spin Labeling of Muscle Proteins: Accurate Rotational Dynamics, Orientation, and Distance by EPR

While EPR allows for the characterization of protein structure and function due to its exquisite sensitivity to spin label dynamics, orientation, and distance, these measurements are often limited in sensitivity due to the use of labels that are attached via flexible monofunctional bonds, incurring additional disorder and nanosecond dynamics. In this chapter, we present methods for using a bifunctional spin label (BSL) to measure muscle protein structure and dynamics. We demonstrate that bifunctional attachment eliminates nanosecond internal rotation of the spin label, thereby allowing the accurate measurement of protein backbone rotational dynamics, including microsecond-to-millisecond motions by saturation transfer EPR. BSL also allows for accurate determination of helix orientation and disorder in mechanically and magnetically aligned systems, due to the labels stereospecific attachment. Similarly, labeling with a pair of BSL greatly enhances the resolution and accuracy of distance measurements measured by double electron-electron resonance (DEER). Finally, when BSL is applied to a protein with high helical content in an assembly with high orientational order (e.g., muscle fiber or membrane), two-probe DEER experiments can be combined with single-probe EPR experiments on an oriented sample in a process we call BEER, which has the potential for ab initio high-resolution structure determination.

Structural Impact of Phosphorylation and Dielectric Constant Variation on Synaptotagmin’s IDR

We used time-resolved Förster resonance energy transfer, circular dichroism, and molecular dynamics simulation to investigate the structural dependence of synaptotagmin 1s intrinsically disordered region (IDR) on phosphorylation and dielectric constant. We found that a peptide corresponding to the full-length IDR sequence, a ∼60-residue strong polyampholyte, can sample structurally collapsed states in aqueous solution, consistent with its κ-predicted behavior, where κ is a sequence-dependent parameter that is used to predict IDR compaction. In implicit solvent simulations of this same sequence, lowering the dielectric constant to more closely mimic the environment near a lipid bilayer surface promoted further sampling of collapsed structures. We then examined the structural tendencies of central region residues of the IDR in isolation. We found that the exocytosis-modulating phosphorylation of Thr112 disrupts a local disorder-to-order transition induced by trifluoroethanol/water mixtures that decrease the solution dielectric constant and stabilize helical structure. Implicit solvent simulations on these same central region residues testing the impact of dielectric constant alone converge on a similar result, showing that helical structure is formed with higher probability at a reduced dielectric. In these helical conformers, lysine-aspartic acid salt bridges contribute to stabilization of transient secondary structure. In contrast, phosphorylation results in formation of salt bridges unsuitable for helix formation. Collectively, these results suggest a model in which phosphorylation and compaction of the IDR sequence regulate structural transitions that in turn modulate neuronal exocytosis.

Structural Transitions in Myosin I Detected by Conventional and Pulsed EPR of a Bifunctional Spin Label

We have employed electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) to develop high-resolution constraints for the myosin catalytic domain in the presence and absence of actin. Two complementary EPR techniques were employed to measure protein orientation (continuous-wave EPR, CW-EPR) and intra-protein distances (double electron-electron resonance, DEER). The use of BSL greatly enhances the resolution of both techniques, by virtue of its strongly immobilized and stereospecific 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, and providing insight for structural changes induced by formation of the complex. 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 MgADP binding was clearly detected by EPR, and subsequently modeled using the orientation and distance measurements as constraints. This work was funded by grants from NIH (R01 AR32961, T32 AR07612, P30 AR0507220).