A CALMODULIN-TARGET PEPTIDE HYBRID MOLECULE WITH UNIQUE CALCIUM-BINDING PROPERTIES

This paper describes the production and properties of a hybrid protein comprising the full length of the Xenopus laevis calmodulin (CaM) sequence, followed, through a glycylglycine linker, by the 26-residue CaM-binding region of myosin lightchain kinase (M13). This hybrid molecule appears to have high thermal stability (T(m) > 75-degrees-C in the presence of Ca2+) as well as unusual Ca2+-binding properties: (i) a wide-range biphasic Ca2+-binding response (extending over pCa 4.8-7.4) and (ii) a high apparent binding constant (pCa50% = 6.3, a 10-fold increase from that of wild-type CaM). NMR and CD data indicate that the CaM - M13 hybrid molecule exists in equilibrium in an approximate 1:1 ratio between two major conformations, one of which is similar to the compact globular structure of the CaM-M13 complex [M.Ikura, G.M.Clore, A.M.Gronenborn, G.Zhu, C.B.Klee and A.Bax (1992) Science, 256, 632 - 638] and the other to the dumb-bell-like structure of the wild type CaM [Y.S.Babu, C.E.Bugg and W.J.Cook (1988) J. Mol. Biol., 204, 191-2041. The biphasic Ca2+-binding curve can be interpreted using a linear combination of two Hill binding curves with significantly different dissociation constants (2 x 10(-6) M and 8 x 10(-8) M), which can be attributed to the two conformations in equilibrium. The present study has opened an avenue to engineer proteins with higher Ca2+-binding affinities using the known CaM structures as a template.