Probing the relationship between alpha-helix formation and calcium affinity in troponin C: 1H NMR studies of calcium binding to synthetic and variant site III helix-loop-helix peptides.
Three 34-residue peptides corresponding to the high-affinity calcium-binding site III and two variant sequences from the muscle protein troponin C (TnC) were synthesized by solid-phase techniques. The two variant 34-residue peptides had amino acid modifications at either the coordinating positions or both the coordinating and noncoordinating positions, which corresponded to the residues found in the low-affinity calcium-binding site II of TnC. High-field 1H NMR spectroscopy was used to monitor calcium binding to each peptide to determine the effect these amino acid substitutions had on calcium affinity. The dissociation constant of the native site III peptide (SCIII) was 3 x 10(-6) M, smaller than that of the peptide incorporating the ligands from site II (LIIL), 8 x 10(-6) M, and that with the entire site II loop (LII), 3 x 10(-3) M, which bound calcium very weakly. These calcium dissociation constants demonstrate that very minor amino acid substitutions have a significant effect on the dissociation constant and give some insight into why the dissociation constants for site III and IV in TnC are 100-fold smaller than those for sites I and II. The results suggest that the differences in coordinating ligands between sites II and III have very little effect on Ca2+ affinity and that the noncoordinating residues in the site II loop are responsible for the low affinity of site II compared to the high affinity of site III in TnC.
Shaw
G S
GS
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Hodges
R S
RS
Sykes
B D
BD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002645:Chickens; D014644:Genetic Variation; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014336:Troponin / Q000737:chemistry*; D019209:Troponin C
D002135:Calcium-Binding Proteins; D010455:Peptides; D014336:Troponin; D019209:Troponin C