A calmodulin-target peptide hybrid molecule with unique calcium-binding properties

This paper describes the production and properties of a hybridprotein comprising the full length of the Xenopus laevis cabnodulin(CaM) sequence, followed, through a gh/cylgh/dne linker, bythe 26-residue CaM-binding region of myosin light-chain kinase(M13). This hybrid molecule appears to have high thermal stability(T_m > 75°C in the presence of Ca²⁺) as well as unusualCa²⁺-binding properties: (i) a wide-range biphasic Ca²⁺-bindingresponse (extending over pCa 4.8-7.4) and (ii) a high apparentbinding constant (pCa_50% = 6.3, a 10-fold increase from thatof wild-type CaM). NMR and CD data indicate that the CaM-M13hybrid molecule exists in equilibrium in an approximate 1:1ratio between two major conformations, one of which is similarto the compact globular structure of the CaM-M13 complex [M.Daira,G.M.Clore, A.M.Gronenborn, G.Zhu, C.B.Klee and A.Bax (1992)Science, 256, 632-638] and the other to the dumbbell-like structureof the wild type CaM [Y.S.Babu, C.E.Bugg and W.J.Cook (1988)J. Mol. Biol., 204, 191-204]. The biphasic Ca²⁺-binding curvecan be interpreted using a linear combination of two Hill bindingcurves with significantly different dissociation constants (2x 10^-6 M and 8 x 10^-6 M), which can be attributed to the twoconformations in equilibrium. The present study has opened anavenue to engineer proteins with higher Ca²⁺-binding affinitiesusing the known CaM structures as a template Received April 23, 1993; revised August 5, 1993; accepted August 25, 1993.