Mapping functional domains in myosin

Abstract

Two general approaches were taken to learn more about how structure relates to function in myosin. One approach involved the development of an expression system in which mutants of myosin could be generated and assayed. To this end, the Dictyostelium discoideum myosin heavy chain (MHC) was coexpressed in E. coli with the scallop myosin light chain 1 (MLC1) sequence resulting in coassembly with normal stoichiometry. To improve this system, the rat alpha cardiac MHC was expressed in E. coli to generate higher level expression of MHC sequences which facilitates biochemical assays of the expressed myosin.;To reconstitute a native interaction of the subunits of myosin, the predominant MLC1 sequence was isolated and characterized from a rat heart cDNA library. The expression of the rat ventricular MLC1 was shown to be developmentally regulated and abundantly expressed in slow but not fast skeletal muscle. The ventricular MLC1 does not change its level of expression in hypertensive animals.;The rat alpha cardiac MHC and the complete rat ventricular MLC1 were coexpressed in E. coli. In preliminary experiments, the E. coli expressed myosin was determined to be enzymatically active. The portion of the MHC that mediates MLC1 binding was mapped by expressing deletions of the MHC with the intact MLC1 in E. coli identifying a conserved 40 amino acid sequence. Site-directed mutagenesis was used to characterize this region further.;The second approach taken to map functional domains within myosin was to compare the rat alpha and beta cardiac MHC sequences. These two sequences are extremely homologous yet encode proteins which differ functionally. There are 131 differences of the 1938 amino acids. The differences between these two proteins map to regions which are thought to be involved in ATP binding, actin binding, MLC binding and force production. This comparison identifies residues which may contribute to these functions.;The combination of an expression system, in which enzymatically active mutant proteins can be generated, with the identification of sequences which are likely to mediate function, allows the possibility of testing these residues to map further the domains of myosin.