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.

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.

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.

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.

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.