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 capable of binding to peptides with very high affinity, and is therefore an interesting candidate for biotechnological applications and a good model system for studying how proteins associate. We have synthesized a complete series of peptides derived from the recognition sequence of skeletal muscle myosin light-chain kinase, corresponding to single-point amino acid mutations to alanine. These peptides bind to calmodulin with a biphasic kinetic: a fast association step followed by a slow intramolecular isomerisation. We have measured the isomerisation rate (k(isom)) of these peptides for calmodulin by stopped-flow analysis, and their association and dissociation kinetic constants (k(on) and k(off)) by real-time interaction analysis using surface plasmon resonance detection. In addition, k(off) constants were measured by competition experiments using a high-sensitivity luminescence analyser and native polyacrylamide gels. We have observed that all the alanine-scanning peptides bound to calmodulin with better affinity than the wild-type. In one case, a Asn-->Ala substitution resulted in a 1000-fold improvement in affinity, owing to a slower off-rate. Our results indicate that naturally occurring calmodulin binders may have evolved to have high affinities, but far from the maximum. Our affinity data are in contrast with recently published predictions of interactions responsible for high-affinity calmodulin binding based on modelling and energy calculations.
Montigiani
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Dipartimento di Biologia Molecolare, Università di Siena, Centro Didattico loc. Le Scotte, Italy.
Neri
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Neri
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D000409:Alanine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010455:Peptides; D009219:Myosin-Light-Chain Kinase; D000409:Alanine