ALANINE SUBSTITUTIONS IN CALMODULIN-BINDING PEPTIDES RESULT IN UNEXPECTED AFFINITY ENHANCEMENT

Calmodulin is a calcium-binding protein that regulates a wide range of enzymes. It is also one of the few examples of a small protein capabl e of binding to peptides with very high affinity, and is therefore an interesting candidate for biotechnological applications and a good mod el system for studying how proteins associate. We have synthesized a c omplete series of peptides derived from the recognition sequence of sk eletal muscle myosin light-chain kinase, corresponding to single-point amino acid mutations to alanine. These peptides bind to calmodulin wi th a biphasic kinetic: a fast association step followed by a slow intr amolecular isomerisation. We have measured the isomerisation rate (k(i som)) of these peptides for calmodulin by stopped-flow analysis, and t heir association and dissociation kinetic constants (k(on) and k(off)) by real-time interaction analysis using surface plasmon resonance det ection. In addition, k(off) constants were measured by competition exp eriments using a high-sensitivity luminescence analyser and native pol yacrylamide gels. We have observed that all the alanine-scanning pepti des bound to calmodulin with better affinity than the wild-type. In on e case, a Asn --> Ala substitution resulted in a 1000-fold improvement in affinity, owing to a slower off-rate. Our results indicate that na turally occurring calmodulin binders may have evolved to have high aff inities, but far from the maximum. Our affinity data are in contrast w ith recently published predictions of interactions responsible for hig h-affinity calmodulin binding based on modelling and energy calculatio ns. (C) 1996 Academic Press Limited