Our goal is to understand the development of myofibrils in striated muscle. The project focuses on the myosin-binding proteins, MyBP-C and MyBP-H (C- and H-proteins, respectively) which are hypothesized to regulate A-band assembly through interactions with myosin and titin. To test this hypothesis, we propose three sets of interlocking experiments which span atomic structural studies to intra-embryonic gene delivery. In a subcontract with David Cowburn (Rockefeller University), we will complete the 3-D atomic structure of the 14 kDa myosin binding domains of MyBP-C and MyBP-H by high resolution NMR spectroscopy. These domains belong to the IgC2 family of proteins and share structural features with telokin, whose crystal structure has been established. Next we will develop a model for placement of MyBP-C in the thick filament based on the atomic structure of the 14 kDa domain and the site-directed mutagenic studies described in project 2. Site-directed mutagenesis of 14 kDa suggests that one 14 kDa domain binds two or more molecules of light meromyosin, explaining the myosin cross-linking properties of MyBP-C. We have identified two acidic residues in LMM which are potential candidates for interaction with the l4kDa peptide. These residues will be mutated to assess their effects on MyBP-C binding to LMM. Two cell-free systems will be used to analyze binding interactions between MyBP-C and MyBP-H and myosin. One involves the binding to LMM of mutated 14 kDa, synthesized in E. coli, using fluorescence emission of tryptophans in the 14 kDa domain to quantify binding. Since MyBP-C and -H bind to other A- band proteins (e.g. titin) besides myosin, we will study the in vitro incorporation of MyBP-C or -H into myofibrils and permeabilized myocytes using recombinant MyBP peptides tagged with myc or the Green Fluorescent Protein. In vivo expression of wild-type and mutant MyBPs will be examined in cultured and intraembryonic muscle, the latter with retroviral vectors co- expressing beta-galactosidase and a MyBP peptide. These experiments will assess functions of the MyBPs during myofibril assembly in vivo.