Role of interchain alpha-helical hydrophobic interactions in Ca2+ affinity, formation, and stability of a two-site domain in troponin C.
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 Ca2+ affinity, respectively. Simultaneous substitution of both hydrophobes in the N- and C-terminal region resulted in a 1,000-fold decrease in Ca2+ 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 Ca2+ affinity.
Monera
O D
OD
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Shaw
G S
GS
Zhu
B Y
BY
Sykes
B D
BD
Kay
C M
CM
Hodges
R S
RS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D002942:Circular Dichroism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D014336:Troponin / Q000737:chemistry* / Q000378:metabolism*; D019209:Troponin C
D006146:Guanidines; D046911:Macromolecular Substances; D010455:Peptides; D014336:Troponin; D019209:Troponin C; D019791:Guanidine; D002118:Calcium