Effects of cardiac thin filament Ca2+: statistical mechanical analysis of a troponin C site II mutant.
Cardiac thin filaments contain many troponin C (TnC) molecules, each with one regulatory Ca2+ binding site. A statistical mechanical model for the effects of these sites is presented and investigated. The ternary troponin complex was reconstituted with either TnC or the TnC mutant CBMII, in which the regulatory site in cardiac TnC (site II) is inactivated. Regardless of whether Ca2+ was present, CBMII-troponin was inhibitory in a thin filament-myosin subfragment 1 MgATPase assay. The competitive binding of [3H]troponin and [14C]CBMII-troponin to actin.tropomyosin was measured. In the presence of Mg2+ and low free Ca2+ they had equal affinities for the thin filament. When Ca274+ was added, however, troponin's affinity for the thin filament was 2.2-fold larger for the mutant than for the wild type troponin. This quantitatively describes the effect of regulatory site Ca2+ on troponin's affinity for actin.tropomyosin; the decrease in troponin-thin filament binding energy is small. Application of the theoretical model to the competitive binding data indicated that troponin molecules bind to interdependent rather than independent sites on the thin filament. Ca2+ binding to the regulatory site of TnC has a long-range rather than a merely local effect. However, these indirect TnC-TnC interactions are weak, indicating that the cooperativity of muscle activation by Ca2+ requires other sources of cooperativity.
Huynh
Q
Q
Department of Internal Medicine, University of Iowa, Iowa City 52242, USA.
Butters
C A
CA
Leiden
J M
JM
Tobacman
L S
LS
D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D051379:Mice; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D014335:Tropomyosin / Q000378:metabolism; D014336:Troponin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019209:Troponin C
D000199:Actins; D046911:Macromolecular Substances; D014335:Tropomyosin; D014336:Troponin; D019209:Troponin C; D002118:Calcium