The long term objective is to understand the mechanism whereby the Ca2+ - ATPase of sarcoplasmic reticulum transports Ca2+ to luminal spaces. The expression and mutagenesis of full length cDNA encoding the Ca2+ -ATPase, followed by assay of the function of the product, is being used to address questions of structure/function relationships in the protein. Further research is proposed under four headings: I. Development of an expression system for large scale production of normal and mutant forms of the Ca2+ -pump: In order to complement the present expression/mutagenesis system in which Ca-ATPase cDNA is expressed in COS-1 cells, development of a Baculovirus expression system that will allow production of large amounts of protein is proposed. Purification of the expressed mutant Ca2+-ATPase protein in one step using a monoclonal antibody affinity column with the IgM antibody A20 is also proposed. II. Determination of the topology of the transmembrane domain of the Ca2+ -ATPase. Luminal and cytoplasmic epitopes in the Ca2+-ATPase have been distinguished with monoclonal antibodies against defined sequences. This analysis will be extended to other sequences predicted to lie on lumenal or cytoplasmic sides. Alternatively, a specific epitope from the fast-twitch Ca2+-ATPase will be inserted at various sites in the sequence of the slow- twitch Ca2+-ATPase in order to determine topology. The boundaries of membrane spanning sequences will be determined by insertion of basic clusters at each end. III. Site-specific mutagenesis to define amino acids critical to Ca2+ transport. Mutation is proposed of: (a) residues in transmembrane sequences 4,5,6 and 8 to determine how they relate to Ca2+ binding and Ca2+ transport; (b) residues throughout the proposed nucleotide binding domain to identify those involved in ATP binding; (c) residues potentially involved in conformational changes in the molecule; (d) residues that will permit measurement of spatial arrangements in the Ca2+-ATPase; (e) Gly, Pro, Cys and Trp residues throughout the molecule. IV. Kinetic analysis of mutant function. Since many mutants retain partial reactions of Ca2+ transport, equilibrium and kinetic analyses of these partial reactions will be carried out. Ca2+ and ATP binding constants, rates of formation and breakdown of phosphorylated intermediates from ATP and Pi, and rates for overall Ca2+ transport will be measured where possible.
| Rice, W J; Green, N M; MacLennan, D H (1997) Site-directed disulfide mapping of helices M4 and M6 in the Ca2+ binding domain of SERCA1a, the Ca2+ ATPase of fast twitch skeletal muscle sarcoplasmic reticulum. J Biol Chem 272:31412-9 |
| Rice, W J; MacLennan, D H (1996) Scanning mutagenesis reveals a similar pattern of mutation sensitivity in transmembrane sequences M4, M5, and M6, but not in M8, of the Ca2+-ATPase of sarcoplasmic reticulum (SERCA1a). J Biol Chem 271:31412-9 |
