Supercomputing Institute Fellow, Professor David Thomas, of the Biochemistry, Molecular Biology, and Biophysics Department at the University of Minnesota has combined molecular spectroscopy and computer simulation to determine the molecular structure and mechanics of muscle contraction and relaxation. Graduate Student John Stamm and Assistant Professor Christine Karim have obtained a structural model of phospholamban (PLB), a 52 amino acid integral membrane protein that helps regulate calcium pumping in the heart. To test models for the pentameric structure of PLB and study its structure and molecular dynamics in solution, these researchers have characterized recombinant PLB and several of its mutants.
In mutagenesis studies, it was found that the key residues occur every seven amino acids, forming a heptad repeat of mutational sensitivity. On the basis of these results, a schematic model was proposed for the PLB pentamer as a left-handed coiled coil stabilized specifically by interhelical interactions between leucines at one heptad position with isoleucines at another (i.e., a leucine-isoleucine zipper). (This work is shown on the cover.) An alternative model was constructed for the transmembrane domain of PLB, from a schematic representation of molecular dynamics simulations, with constraints based on the effects of random mutagenesis. This atomic model also proposes a left-handed coiled coil of five helices, but the contacts between the helices are quite different and do not support the proposal of the leucine-isoleucine zipper.
Supercomputer simulation of a molecular motor. The myosin catalytic domain (red) binds to actin (purple) while the myosin light-chain domain (blue) transitions between two distinct angles of the myosin head, thus driving muscle contraction. This molecular motor movement was detected by electron paramagnetic resonance of spin labels attached to myosin light chains in contracting muscle.
Further work in Professor Thomas' group used electronic paramagnetic resonance (EPR) of spin-labeled muscle fibers, in conjunction with molecular dynamics simulations, to obtain the first direct evidence that the light-chain domain of myosin (the molecular motor within muscle) rotates upon muscle activation. (This can be seen at left.) For more than 30 years, the fundamental goal in molecular motility has been to resolve force-generating motor protein structural changes. Although low-resolution
Using EPR, researchers in the Thomas group (graduate researchers Josh Baker and Leslie LaConte and research associate Ingrid Brust-Mascher) observed two distinct orientations of a spin label attached specifically to a single site on the light chain domain of myosin in relaxed scallop muscle fibers. The two probe orientations did not change upon muscle activation, but the distribution between them changed substantially, indicating that a fraction of myosin heads undergoes a large axial rotation of the myosin light chain domain upon force generation and muscle contraction. The resulting model helps explain why this observation has remained so elusive and provides insight into the mechanisms by which motor protein structural transitions drive molecular motility.
Using ATP analogs, these researchers showed that the orientations of the light-chain domain in the pre- and post-hydrolysis states are not significantly different from each other. This surprising result suggests that the ATP hydrolysis step does not induce a major structural change in myosin; rather the major structural change occurs upon strong actin binding and phosphate release.
Other researchers in the Thomas laboratory used site-directed mutations and computational molecular dynamics simulations to analyze the spectroscopic signals in terms of specific molecular motions within myosin. Using the programs INSIGHT II and DISCOVER, undergraduate researchers Greg Wilde, Michael Enz, and Will Meland simulated the effects of mutations on molecular dynamics while graduate researcher Wendy Smith carried out mutagenesis to test the predictions of the virtual mutagenesis computer calculations. This approach is proving to be a powerful tool in understanding the working of these complex molecular motors.
