The general aim of this proposal is to use cellular, biochemical and molecular techniques to understand the structure and function of caldesmon, an actin, tropomyosin, calmodulin and myosin binding protein present in smooth muscle and non-muscle cells. This protein is believed to participate in the regulation of actomyosin interactions and may be involved in the maintaining of tension in tonic smooth muscles in the 'latch' state. We have recently completed the sequencing of one full length caldesmon cDNA. The predicted protein sequence confirms our initial speculation that the actin-calmodulin-tropomyosin multi-site binding domain is on the C-terminus of caldesmon and also shows a middle, possibly helical region consisting of a 15 amino acid sequence repeated eight times. The multi-site domain contains a sequence similar to a tropomyosin binding region of skeletal muscle troponin T. The sequence is consistent with an elongated molecule that binds at one end to actin filaments and can bind to myosin at its other end.
Our specific aims are to use this cloned caldesmon cDNA to: a)Establish the structural relationship between the high and low molecular weight forms of smooth muscle and non-muscle caldesmons. b)Determine the sequence of the different caldesmon isoforms and whether they are products of different genes. c)Continue domain mapping studies using recombinant DNA methods. d)Initiate studies on caldesmon function using bacterial and eukaryotic expression vectors. e)Produce large quantities of the C-terminal actin-calmodulin-binding caldesmon fragment for crystallization studies.
Showing the most recent 10 out of 22 publications
