A PROTEIN PEPTIDE ASSAY USING PEPTIDE-RESIN ADDUCT - APPLICATION TO THE CALMODULIN/RS20 COMPLEX/

To obtain equilibrium and kinetic constants of a protein/peptide compl ex, we have developed a rapid procedure which uses peptides specifical ly linked to a resin. With this peptide-resin adduct, bound and free I -125-labeled protein could be easily separated by simple centrifugatio n. The feasibility of the method was demonstrated with the calmodulin/ RS20 complex, where RS20 is the putative calmodulin binding peptide of the smooth muscle myosin light chain kinase (smMLCK). In addition to the wild-type calmodulin (SYNCAM) expressed in Escherichia coli, we al so examined calmodulin mutants with charge reversals called SYNCAM12A (DEE 118-120 --> KKK) and SYNCAM18A (EEE 82-84 --> KKK and DEE 118-120 --> KKK). The kinetic analysis of the interaction between SYNCAM and RS20 associated with titration experiments allowed us to measure disso ciation constants (K-D) in the range of 10(-9) M, in good agreement wi th previously published data. Moreover, the binding assays showed that SYNCAM18A did not interact with RS20, whereas SYNCAM12A did with a K- D around 10(-8) M. The lack of binding of SYNCAM18A to RS20 provides a n explanation for the lack of smMLCK activation by SYNCAM18A. Altogeth er, these results demonstrate that peptide-resin can be used as a tool for separating bound from free protein, thus enabling a rapid and rel iable quantification of the protein/peptide interaction. (C) 1994 Acad emic Press, Inc.