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 light-chain kinase (M13). This hybrid molecule appears to have high thermal stability (Tm > 75 degrees C in the presence of Ca2+) as well as unusual Ca(2+)-binding properties: (i) a wide-range biphasic Ca(2+)-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, 191-204]. The biphasic Ca(2+)-binding curve can be interpreted using a linear combination of two Hill binding curves with significantly different dissociation constants (2 x 10(-9) 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 Ca(2+)-binding affinities using the known CaM structures as a template.
Porumb
T
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Department of Medical Biophysics, University of Toronto, Ontario, Canada.
Yau
P
P
Harvey
T S
TS
Ikura
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D004926:Escherichia coli; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D009219:Myosin-Light-Chain Kinase; D002118:Calcium