Calcium-induced dimerization of troponin C: mode of interaction and use of trifluoroethanol as a denaturant of quaternary structure.
Protein aggregation can be a problem, especially as a large number of proteins become available for structural studies at fairly high concentrations using solution techniques such as NMR spectroscopy. The muscle regulatory protein troponin C (TnC) undergoes a calcium-induced dimerization at neutral pH with a dissociation constant for the dimerization of 0.4 mM at 20 degrees C. The present study indicates that the mode of dimerization involves the N-domain of one monomer interacting with the N-domain of another monomer. Addition of the solvent trifluoroethanol (TFE) to a concentration of 15%, v/v, results in a 10-fold increase in the dimer dissociation constant of calcium-saturated TnC (4 mM at 20 degrees C), making TnC predominantly a monomer for spectroscopic studies. Further, TFE, at the concentrations used herein, acts to perturb the quaternary structure of TnC without adversely affecting the secondary or tertiary structure as evidenced by minimal changes to its CD spectra and 1H, 13C, and 15N NMR chemical shifts.
Slupsky
C M
CM
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Kay
C M
CM
Reinach
F C
FC
Smillie
L B
LB
Sykes
B D
BD
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology*; D002247:Carbon Isotopes; D002645:Chickens; D002942:Circular Dichroism; D017931:DNA Primers; D006859:Hydrogen; D007700:Kinetics; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000378:metabolism; D009587:Nitrogen Isotopes; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D015183:Restriction Mapping; D014270:Trifluoroethanol; D014336:Troponin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D019209:Troponin C
D002247:Carbon Isotopes; D017931:DNA Primers; D046911:Macromolecular Substances; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D014270:Trifluoroethanol; D006859:Hydrogen; D002118:Calcium