BIOGRAPHY:

Dr. Padrón is Senior Investigator, Structural Biology Laboratory, Department of Structural Biology, at the Venezuelan Institute for Scientific Research (IVIC), Caracas. He received a Ph.D. in physiology and biophysics from this institute and conducted postdoctoral research at the Medical Research Council Laboratory of Molecular Biology in Cambridge, United Kingdom. He has been a Visiting Scientist at the European Molecular Biology Laboratory (EMBL) in Hamburg, at the Science and Engineering Research Council and at the Medical Research Council in the United Kingdom, and the University of Massachusetts Medical School. Dr. Padrón's honors include CONICIT biology prizes, a CONICIT PPI Fellowship, and the 1991 Polar Science (Biology) Prize. This is his second HHMI International Research Scholar award. His current project involves the structure and function of the myosin filaments of striated muscle.

RESEARCH ABSTRACT SUMMARY:

Helical Order in Myosin Filaments Requires the Closed Conformation of the Myosin Head

Myosin heads are helically ordered on the thick filament surface in relaxed muscle. In mammalian and avian filaments, this helical arrangement is dependent on temperature, and it has been suggested that helical order is related to ATP hydrolysis by the heads. To test this hypothesis, we used electron microscopy and image analysis to study the ability and temperature dependence of analogs of ATP and ADP.Pi to induce helical order in arthropod thick filaments. ATP or analogs were added to rigor myofibrils or purified thick filaments at 22°C and 4°C, and the samples were negatively stained. The ADP.Pi analogs ADP.AlF₄ and ADP.Vi and the ATP analogs ADP.BeF_x, AMPPNP, and ATPgammaNH₂ all induced helical order in these thick filaments, independent of temperature. In the absence of nucleotide or in the presence of ADP or the ATP analogue ATPgammaS there was no helical ordering. According to crystallographic and tryptophan fluorescence studies, all these analogues except ATPgammaS and ADP induce the closed conformation of the myosin head. We suggest that helical order requires the closed state of the myosin head, but not ATP hydrolysis. It appears that helical order is required for the normal relaxed state, given that it is found in all animals at their normal physiological temperatures. This helical organization may maintain the heads close to the filament backbone, helping to maintain the OFF state. Our results demonstrate that, to obtain an atomic model for the filament, it is necessary to dock the atomic structure of the head in the closed conformation to the three-dimensional filament reconstruction, which we are now carrying out. Activation of thick filaments by phosphorylation of the myosin regulatory light chain (RLC) results in disordering of the helical array. Using EM and the in vitro motility assay, we are currently studying the functional implications of RLC mutations that occur in the midventricular chamber phenotype of hypertrophic cardiomyopathy.

Photo: Dominic Chaplin, Pine Creek Pictures

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