Siivari K, Zhang M, Palmer A G, Vogel H J
Department of Biological Sciences, University of Calgary, Alberta, Canada.
FEBS Lett. 1995 Jun 12;366(2-3):104-8. doi: 10.1016/0014-5793(95)00504-3.
Calmodulin (CaM) is a ubiquitous Ca(2+)-binding protein that can regulate a wide variety of cellular events. The protein contains 9 Met out of a total of 148 amino acid residues. The binding of Ca2+ to CaM induces conformational changes and exposes two Met-rich hydrophobic surfaces which provide the main protein-protein contact areas when CaM interacts with its target enzymes. Two-dimensional (1H,13C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy was used to study selectively 13C-isotope labelled Met methyl groups in apo-CaM, Ca(2+)-CaM and a complex of CaM with the CaM-binding domain of skeletal muscle Myosin Light Chain Kinase (MLCK). The resonance assignment of the Met methyl groups in these three functionally different states were obtained by site-directed mutagenesis (Met-->Leu). Chemical shift changes indicate that the methyl groups of the Met residues are in different environments in apo-, calcium-, and MLCK-bound-CaM. The T1 relaxation rates of the individual Met methyl carbons in the three forms of CaM indicate that those in Ca(2+)-CaM have the highest mobility. Our results also suggest that the methyl groups of the unbranched Met sidechains in general are more flexible than those of aliphatic amino acid residues such as Leu and Ile.
钙调蛋白（CaM）是一种普遍存在的钙结合蛋白，可调节多种细胞活动。该蛋白在总共148个氨基酸残基中含有9个甲硫氨酸。钙离子与钙调蛋白的结合会诱导构象变化，并暴露出两个富含甲硫氨酸的疏水表面，当钙调蛋白与其靶酶相互作用时，这些表面提供主要的蛋白质-蛋白质接触区域。二维（1H，13C）异核多量子相干（HMQC）核磁共振光谱用于选择性研究脱辅基钙调蛋白、钙离子-钙调蛋白以及钙调蛋白与骨骼肌肌球蛋白轻链激酶（MLCK）的钙调蛋白结合结构域形成的复合物中13C同位素标记的甲硫氨酸甲基基团。通过定点诱变（甲硫氨酸→亮氨酸）获得了这三种功能不同状态下甲硫氨酸甲基基团的共振归属。化学位移变化表明，在脱辅基、结合钙离子和结合MLCK的钙调蛋白中，甲硫氨酸残基的甲基处于不同环境。三种形式的钙调蛋白中单个甲硫氨酸甲基碳的T1弛豫速率表明，钙离子-钙调蛋白中的甲基具有最高的流动性。我们的结果还表明，一般来说，直链甲硫氨酸侧链的甲基比亮氨酸和异亮氨酸等脂肪族氨基酸残基的甲基更具灵活性。
