Protein Science, Vol 1, Issue 7 945-955, Copyright © 1992 by Cold Spring Harbor Laboratory Press

ARTICLE

Role of interchain {alpha}-helical hydrophobic interactions in Ca(2+) affinity, formation, and stability of a two-site domain in troponin C

O. D. MONERA, G. S. SHAW, B. Y. ZHU, B. D. SYKES, C. M. KAY and R. S. HODGES
Department of Biochemistry, the Medical Research Council of Canada Group in Protein Structure and Function, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

We have previously shown that a 34-residue synthetic peptide representing the calcium-binding site III of troponin C formed a symmetric two-site dimer consisting of two helix-loop-helix motifs arranged in a head-to-tail fashion (Shaw, G.S., Hodges, R.S., & Sykes, B.D., 1990, Science 249, 280-283). In this study the hydrophobicities of the {alpha}-helices were altered by replacing L-98 and F-102 in the N-terminal region and/or I-121 and L-122 in the C-terminal region with alanine residues. Our results showed that substitution of hydrophobic residues either in the N- or C-terminal region have little effect on {alpha}-helix formation but resulted in a 100- and 300-fold decrease in Ca(2+) affinity, respectively. Simultaneous substitution of both hydrophobes in the N- and C-terminal region resulted in a 1,000-fold decrease in Ca(2+) affinity. Data from guanidine hydrochloride denaturation studies suggested that intermolecular interactions occur and that the less hydrophobic analogs had a lower overall conformational stability. These data support the contention that the hydrophobic residues are important in the formation of the two-site domain in troponin C, and this hydrophobic association stabilizes Ca(2+) affinity.
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