Effect of hydrophobic residue substitutions with glutamine on Ca(2+) binding and exchange with the N-domain of troponin C.
Troponin C (TnC) is an EF-hand Ca(2+) binding protein that regulates skeletal muscle contraction. The mechanisms that control the Ca(2+) 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 Ca(2+) binding and exchange with the N-domain of a fluorescent TnC(F29W). The global N-terminal Ca(2+) affinities of the TnC(F29W) mutants varied approximately 2340-fold, while Ca(2+) association and dissociation rates varied less than 70-fold and more than 45-fold, respectively. Greater than 2-fold increases in Ca(2+) affinities were obtained primarily by slowing of Ca(2+) dissociation rates, while greater than 2-fold decreases in Ca(2+) affinities were obtained by slowing of Ca(2+) association rates and speeding of Ca(2+) dissociation rates. No correlation was found between the Ca(2+) binding properties of the TnC(F29W) mutants and the solvent accessibility of the hydrophobic amino acids in the apo state, Ca(2+) bound state, or the difference between the two states. However, the effects of these hydrophobic mutations on Ca(2+) binding were contextual possibly because of side chain interactions within the apo and Ca(2+) bound states of the N-domain. These results demonstrate that a single hydrophobic residue, which does not directly ligate Ca(2+), can play a crucial role in controlling Ca(2+) binding and exchange within a coupled and functional EF-hand system.
Tikunova
Svetlana B
SB
Department of Molecular and Cellular Biochemistry and Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH 43210, USA.
Rall
Jack A
JA
Davis
Jonathan P
JP
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D005973:Glutamine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D019209:Troponin C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D019209:Troponin C; D005973:Glutamine; D010649:Phenylalanine; D002118:Calcium