Campbell A P, Cachia P J, Sykes B D
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Biochem Cell Biol. 1991 Sep;69(9):674-81. doi: 10.1139/o91-101.
We have used 19F nuclear magnetic resonance spectroscopy to study the interaction of the inhibitory region of troponin (TnI) with apo- and calcium(II)-saturated turkey skeletal troponin C (TnC), using the synthetic TnI analogue N alpha-acetyl[19FPhe106]TnI(104-115)amide. Dissociation constants of Kd = (3.7 +/- 3.1) x 10(-5) M for the apo interaction and Kd = (4.8 +/- 1.8) x 10(-5) M for the calcium(II)-saturated interaction were obtained using a 1:1 binding model of peptide to protein. The 19F NMR chemical shifts for the F-phenylalanine of the bound peptide are different from the apo- and calcium-saturated protein, indicating a different environment for the bound peptide. The possibility of 2:1 binding of the peptide to Ca(II)-saturated TnC was tested by calculating the fit of the experimental titration data to a series of theoretical binding curves in which the dissociation constants for the two hypothetical binding sites were varied. We obtained the best fit for 0.056 mM less than or equal to Kd1 less than or equal to 0.071 mM and 0.5 mM less than or equal to Kd2 less than or equal to 2.0 mM. These results allow the possibility of a second peptide binding site on calcium(II)-saturated TnC with an affinity 10- to 20-fold weaker than that of the first site.
我们使用了19F核磁共振光谱法，以合成的肌钙蛋白I类似物Nα-乙酰基[19F苯丙氨酸106]肌钙蛋白I(104 - 115)酰胺为研究对象，来研究肌钙蛋白抑制区（TnI）与脱辅基和钙（II）饱和的火鸡骨骼肌肌钙蛋白C（TnC）之间的相互作用。采用肽与蛋白质的1:1结合模型，得出脱辅基相互作用的解离常数Kd = (3.7 ± 3.1) × 10⁻⁵ M，钙（II）饱和相互作用的解离常数Kd = (4.8 ± 1.8) × 10⁻⁵ M。结合肽的F - 苯丙氨酸的19F NMR化学位移与脱辅基和钙饱和蛋白不同，表明结合肽所处环境不同。通过计算实验滴定数据与一系列理论结合曲线的拟合度来测试肽与钙（II）饱和TnC的2:1结合可能性，其中两个假设结合位点的解离常数是变化的。我们得出，当0.056 mM ≤ Kd1 ≤ 0.071 mM且0.5 mM ≤ Kd2 ≤ 2.0 mM时拟合效果最佳。这些结果表明，在钙（II）饱和的TnC上可能存在第二个肽结合位点，其亲和力比第一个位点弱10至20倍。
