Effect of Hydrophobic Residue Substitutions with Glutamine on Ca2+Binding and Exchange with the N-Domain of Troponin C

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Abstract
Troponin C (TnC) is an EF-hand Ca2+ binding protein that regulates skeletal muscle contraction. The mechanisms that control the Ca2+ binding properties of TnC and other EF-hand proteins are not completely understood. We individually substituted 27 Phe, Ile, Leu, Val, and Met residues with polar Gln to examine the role of hydrophobic residues in Ca2+ binding and exchange with the N-domain of a fluorescent TnCF29W. The global N-terminal Ca2+ affinities of the TnCF29W mutants varied ∼2340-fold, while Ca2+ association and dissociation rates varied less than 70-fold and more than 45-fold, respectively. Greater than 2-fold increases in Ca2+ affinities were obtained primarily by slowing of Ca2+ dissociation rates, while greater than 2-fold decreases in Ca2+ affinities were obtained by slowing of Ca2+ association rates and speeding of Ca2+ dissociation rates. No correlation was found between the Ca2+ binding properties of the TnCF29W mutants and the solvent accessibility of the hydrophobic amino acids in the apo state, Ca2+ bound state, or the difference between the two states. However, the effects of these hydrophobic mutations on Ca2+ binding were contextual possibly because of side chain interactions within the apo and Ca2+ bound states of the N-domain. These results demonstrate that a single hydrophobic residue, which does not directly ligate Ca2+, can play a crucial role in controlling Ca2+ binding and exchange within a coupled and functional EF-hand system.