Rotational dynamics of phospholamban determined by multifrequency electron paramagnetic resonance.

We have used multifrequency electron paramagnetic resonance to define the multistate structural dynamics of an integral membrane protein, phospholamban (PLB), in a lipid bilayer. PLB is a key regulator of cardiac calcium transport, and its function requires transitions between distinct states of intramolecular dynamics. Monomeric PLB was synthesized with the TOAC spin label at positions 11 (in the cytoplasmic domain) and 46 (in the transmembrane domain) and reconstituted into lipid bilayers. Unlike other protein spin labels, TOAC reports directly the motion of the peptide backbone, so quantitative analysis of its dynamics is worthwhile. Electron paramagnetic resonance spectra at 9.4 GHz (X-band) and 94 GHz (W-band) were analyzed in terms of anisotropic rotational diffusion of the two domains. Motion of the transmembrane domain is highly restricted, while the cytoplasmic domain exhibits two distinct conformations, a major one with moderately restricted nanosecond dynamics (T) and another with nearly unrestricted subnanosecond motion (R). The global analysis of spectra at two frequencies yielded values for the rotational correlation times and order parameters that were much more precisely determined than at either frequency alone. Multifrequency EPR is a powerful approach for analysis of complex rotational dynamics of proteins.

Calculating slow-motional electron paramagnetic resonance spectra from molecular dynamics using a diffusion operator approach.

A number of groups have utilized molecular dynamics (MD) to calculate slow-motional electron paramagnetic resonance (EPR) spectra of spin labels attached to biomolecules. Nearly all such calculations have been based on some variant of the trajectory method introduced by Robinson, Slutsky and Auteri (J. Chem. Phys. 1992,96, 2609-2616). Here we present an alternative approach that is specifically adapted to the diffusion operator-based stochastic Liouville equation (SLE) formalism that is also widely used to calculate slow-motional EPR line shapes. Specifically, the method utilizes MD trajectories to derive diffusion parameters such as the rotational diffusion tensor, diffusion tilt angles, and expansion coefficients of the orienting potential, which are then used as direct inputs to the SLE line shape program. This approach leads to a considerable improvement in computational efficiency over trajectory-based methods, particularly for high frequency, high field EPR. It also provides a basis for deconvoluting the effects of local spin label motion and overall motion of the labeled molecule or domain: once the local motion has been characterized by this approach, the label diffusion parameters may be used in conjunction with line shape analysis at lower EPR frequencies to characterize global motions. The method is validated by comparison of the MD predicted line shapes to experimental high frequency (250 GHz) EPR spectra.

ESR reveals the mobility of the neck linker in dimeric kinesin.

Conventional kinesin is a highly processive motor that converts the chemical energy of ATP hydrolysis into the unidirectional motility along microtubules. The processivity is thought to depend on the coordination between ATPase cycles of two motor domains and their neck linkers. Here we have used site-directed spin labeling electron spin resonance (SDSL-ESR) to determine the conformation of the neck linker in kinesin dimer in the presence and absence of microtubules. The spectra show that the neck linkers co-exist in both docked and disordered conformations, which is consistent with the results of monomeric kinesin. In all nucleotide states, however, the neck linkers are well ordered when dimeric kinesin is bound to the microtubule. This result suggests that the orientation of each neck linker that is fixed rigidly controls the kinesin motion along microtubule tracks.

Functional and spectroscopic studies of a familial hypertrophic cardiomyopathy mutation in Motif X of cardiac myosin binding protein-C.

Familial hypertrophic cardiomyopathy is an autosomal dominant genetic disorder caused by mutations in cardiac sarcomeric proteins. One such mutation is a six amino acid duplication of residues 1248-1253 in the C-terminal immunoglobulin domain of cardiac myosin binding protein-C, referred to as Motif X. Motif X binds the myosin rod and titin. Here we investigate the structural and functional alteration in the mutant Motif X protein to understand how sarcomeric dysfunction may occur. The cDNA encoding Motif X was cloned, mutated and expressed as wild-type and mutant proteins in a bacterial expression system. Circular dichroism spectroscopy confirmed that the normal and mutant Motif X exhibited a high beta-content, as predicted for immunoglobulin domains. Thermal denaturation curves showed that Motif X unfolded with at least two structural transitions, with the first transition occurring at 63 degrees C in the wild-type but at 40 degrees C in the mutant, consistent with the mutant being structurally less stable. Sedimentation binding studies with synthetic myosin filaments revealed no significant difference in binding to myosin between the wild-type and the mutant Motif X. Molecular modeling of this duplication mutation onto an homologous IgI structure (telokin) revealed that the duplicated residues lie within the F strand of the immunoglobulin fold, on a surface of Motif X distant from residues previously implicated in myosin binding. Taken together, these data suggest that the Motif X mutation may interfere with other, as yet unidentified, functional interactions.

Structural determination of spin label immobilization and orientation: a Monte Carlo minimization approach.

Electron paramagnetic resonance (EPR) is often used in the study of the orientation and dynamics of proteins. However, there are two major obstacles in the interpretation of EPR signals: (a) most spin labels are not fully immobilized by the protein, hence it is difficult to distinguish the mobility of the label with respect to the protein from the reorientation of the protein itself; (b) even in cases where the label is fully immobilized its orientation with respect to the protein is not known, which prevents interpretation of probe reorientation in terms of protein reorientation. We have developed a computational strategy for determining whether or not a spin label is immobilized and, if immobilized, predicting its conformation within the protein. The method uses a Monte Carlo minimization algorithm to search the conformational space of labels within known atomic level structures of proteins. To validate the method a series of spin labels of varying size and geometry were docked to sites on the myosin head catalytic and regulatory domains. The predicted immobilization and conformation compared well with the experimentally determined mobility and orientation of the label. Thus, probes can now be targeted to report on various modes of molecular dynamics: immobilized probes to report on protein backbone and domain dynamics or floppy probes to report on the extent of steric restriction experienced by the side chain.