The acrosomal process of Limulus sperm is a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102 kDa protein, scruin, in a 1:1 molar ratio with actin. Based on secondary structure analysis and proteolysis results, scruin is organized into two domains connected by a neck region. Image reconstruction of electron micrographs of the filaments has shown that two large domains of scruin bind to actin on subdomains I and 3. This proposal will study the binding sites involved in the scruin/actin interaction using biochemical and molecular biological approaches. Chemical modification, chemical protection as well as peptide binding assays will be used to map the putative binding sites, the relevant amino acids or peptides on both scruin and actin. Subsequently, site-direct mutagenesis will be utilized to identify the exact amino acids at the scruin-actin interface.
