Gagné S M, Tsuda S, Spyracopoulos L, Kay L E, Sykes B D
Medical Research Council Group in Protein Structure and Function, University of Alberta, Edmonton, T6G 2H7, Canada.
J Mol Biol. 1998 May 8;278(3):667-86. doi: 10.1006/jmbi.1998.1723.
The N-terminal domain (residues 1 to 90) of chicken skeletal troponin C (NTnC) regulates muscle contraction upon the binding of a calcium ion to each of its two calcium binding loops. In order to characterize the backbone dynamics of NTnC in the apo state (NTnC-apo), we measured and carefully analyzed 15N NMR relaxation parameters T1, T2 and NOE at 1H NMR frequencies of 500 and 600 MHz. The overall rotational correlation time of NTnC-apo at 29.6 degrees C is 4.86 (+/-0.15) ns. The experimental data indicate that the rotational diffusion of NTnC-apo is anisotropic with a diffusion anisotropy, D parallel/D perpendicular, of 1.10. Additionally, the dynamic properties of side-chains having a methyl group were derived from 2H relaxation data of CH2D groups of a partially deuterated sample. Based on the dynamic characteristics of TnC, two different levels of "fine tuning" of the calcium affinity are presented. Significantly lower backbone order parameters (S2), were observed for calcium binding site I relative to site II and the contribution of the bond vector fluctuations to the conformational entropy of sites I and II was calculated. The conformational entropy loss due to calcium binding (DeltaDeltaSp) differs by 1 kcal/mol between sites I and II. This is consistent with the different dissociation constants previously measured for sites I and II of 16 microM and 1. 7 microM, respectively. In addition to the direct role of binding loop dynamics, the side-chain methyl group dynamics play an indirect role through the energetics of the calcium-induced structural change from a closed to an open state. Our results show that the side-chains which will be exposed upon calcium binding have reduced motion in the apo state, suggesting that conformational entropic contributions can be used to offset the free energy cost of exposing hydrophobic groups. It is clear from this work that a complete determination of their dynamic characteristics is necessary in order to fully understand how TnC and other proteins are fine tuned to appropriately carry out their function.
鸡骨骼肌肌钙蛋白C（NTnC）的N端结构域（第1至90位氨基酸残基）在钙离子与其两个钙结合环中的每一个结合时调节肌肉收缩。为了表征无钙状态下NTnC（NTnC-apo）的主链动力学，我们在500和600 MHz的1H NMR频率下测量并仔细分析了15N NMR弛豫参数T1、T2和NOE。在29.6摄氏度时，NTnC-apo的整体旋转相关时间为4.86（±0.15）纳秒。实验数据表明，NTnC-apo的旋转扩散是各向异性的，扩散各向异性D平行/D垂直为1.10。此外，具有甲基的侧链的动力学性质来自部分氘代样品的CH2D基团的2H弛豫数据。基于肌钙蛋白C的动力学特征，提出了两种不同水平的钙亲和力“微调”。相对于位点II，在钙结合位点I观察到明显更低的主链序参数（S2），并计算了键矢量波动对位点I和II构象熵的贡献。位点I和II之间由于钙结合导致的构象熵损失（ΔΔSp）相差1千卡/摩尔。这与之前分别测量的位点I和II的16微摩尔和1.7微摩尔的不同解离常数一致。除了结合环动力学的直接作用外，侧链甲基动力学通过钙诱导的从闭合状态到开放状态的结构变化的能量学发挥间接作用。我们的结果表明，在无钙状态下，钙结合时将暴露的侧链运动减少，这表明构象熵贡献可用于抵消暴露疏水基团的自由能成本。从这项工作可以清楚地看出，为了充分理解肌钙蛋白C和其他蛋白质如何被微调以适当地发挥其功能，有必要完整地确定它们的动力学特征。
