Relative stabilities of synthetic peptide homo- and heterodimeric troponin-C domains.
It has previously been shown that synthetic peptides corresponding to calcium-binding sites III (SCIII) and IV (SCIV) from troponin-C can undergo a calcium-induced dimerization to form the respective homodimers (Shaw GS, Hodges RS, Sykes BD, 1990, Science 249:280-283; Shaw GS et al., 1992a, J Am Chem Soc 114:6258-6259). In addition, an equimolar mixture of SCIII and SCIV has been shown to form preferentially the SCIII/SCIV heterodimer (Shaw GS et al., 1992a, J Am Chem Soc 114:6258-6259). The stabilities of these dimers have been investigated by using 1H-NMR and circular dichroism spectroscopies to follow temperature- and guanidine hydrochloride (GuHCl)-induced denaturations. It has been found that the most stable species, the SCIII/SCIV heterodimer (delta GuH2O = -64.8 kJ/mol), is about 13 kJ/mol more stable than the least stable species, the SCIV homodimer, while the SCIII homodimer is of intermediate stability. This trend of free energies agrees well with the trend of delta G0 values derived from the products of the dissociation constants for calcium binding and peptide association determined from earlier calcium-titration studies. These observations provide evidence that calcium affinity and the association of 2-calcium binding sites are tightly linked. However, it was noted that in all cases delta G0 was considerably more negative than delta GuH2O determined from GuHCl experiments. This difference increased as the stability of the peptide complex increased, providing evidence that linear extrapolation of GuHCl data for very stable proteins may significantly underestimate the value for delta G0.
Shaw
G S
GS
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Hodges
R S
RS
Kay
C M
CM
Sykes
B D
BD
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D004355:Drug Stability; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000737:chemistry*; D017510:Protein Folding; D013816:Thermodynamics; D014336:Troponin / Q000737:chemistry*; D019209:Troponin C
D006146:Guanidines; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014336:Troponin; D019209:Troponin C; D019791:Guanidine; D002118:Calcium