A protein/peptide assay using peptide-resin adduct: application to the calmodulin/RS20 complex.
To obtain equilibrium and kinetic constants of a protein/peptide complex, we have developed a rapid procedure which uses peptides specifically linked to a resin. With this peptide-resin adduct, bound and free 125I-labeled protein could be easily separated by simple centrifugation. 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 also 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 dissociation constants (KD) in the range of 10(-9) M, in good agreement with previously published data. Moreover, the binding assays showed that SYN-CAM18A did not interact with RS20, whereas SYN-CAM12A did with a KD around 10(-8) M. The lack of binding of SYNCAM18A to RS20 provides an explanation for the lack of smMLCK activation by SYNCAM18A. Altogether, these results demonstrate that peptide-resin can be used as a tool for separating bound from free protein, thus enabling a rapid and reliable quantification of the protein/peptide interaction.
Guimard
L
L
Centre de Recherches de Biochemie Macromoléculaire (CNRS UPR 9008 et INSERM U249), Montpellier, France.
Afshar
M
M
Haiech
J
J
Calas
B
B
D000595:Amino Acid Sequence; D002147:Calmodulin / Q000032:analysis*; D007700:Kinetics; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000032:analysis*; D012117:Resins, Synthetic; D012680:Sensitivity and Specificity
D002147:Calmodulin; D012117:Resins, Synthetic; D009219:Myosin-Light-Chain Kinase