Protasevich I, Ranjbar B, Lobachov V, Makarov A, Gilli R, Briand C, Lafitte D, Haiech J
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.
Biochemistry. 1997 Feb 25;36(8):2017-24. doi: 10.1021/bi962538g.
Scanning microcalorimetry and circular dichroism were used to study conformational state and heat denaturation of Ca2+-free synthetic calmodulin (SynCaM) and three charge reversal mutants. We produced evidence for the major role of the electrostatic potential in the stability and flexibility of SynCaM. The substitution of 118DEE120 by 118KKK120 (SynCaM12A) does not influence the flexibility of the protein; the replacement of 82EEE84 by 82KKK84 (SynCaM8) decreases its level, while the combination of these two mutations in SynCaM18A significantly increases the flexibility. The heat denaturation of apoSynCaM and its mutants is well approximated by two two-state transitions with the lower-temperature transition corresponding to C-terminal lobe melting and the higher-temperature one to N-terminal lobe melting. The difference in transition temperatures for the two lobes decreases in SynCaM8 and increases in SynCaM18A, suggesting a modification in the influence of one lobe to the other. The electrostatic mutations change the parameters of thermal denaturation of SynCaM lobes in a similar way as pH conditions affect thermal transition parameters of multidomain proteins, leading to a linear temperature dependence of transition enthalpy. One domain of the N-terminal lobe in apoSynCaM18A is unfolded in the native state. Near-UV CD spectra point out the invariability of the local structure of aromatic residues upon mutations, although the secondary structure undergoes striking transformations. Cacodylate ions strongly and specifically alter the helical content of SynCaM. Our data unambiguously demonstrate that the two lobes are not independent, and interactions between the lobes are mediated by the electrostatic potential of the molecule.
采用扫描量热法和圆二色性研究了无钙合成钙调蛋白（SynCaM）及其三个电荷反转突变体的构象状态和热变性。我们提供了证据证明静电势在SynCaM的稳定性和灵活性中起主要作用。将118DEE120替换为118KKK120（SynCaM12A）不影响蛋白质的灵活性；将82EEE84替换为82KKK84（SynCaM8）会降低其灵活性，而在SynCaM18A中这两个突变的组合则显著增加了灵活性。脱辅基SynCaM及其突变体的热变性可以很好地用两个两态转变来近似，较低温度的转变对应于C末端叶的熔化，较高温度的转变对应于N末端叶的熔化。两个叶的转变温度差异在SynCaM8中减小，在SynCaM18A中增加，这表明一个叶对另一个叶的影响发生了改变。静电突变改变SynCaM叶的热变性参数的方式与pH条件影响多结构域蛋白质的热转变参数的方式相似，导致转变焓与温度呈线性关系。脱辅基SynCaM18A的N末端叶的一个结构域在天然状态下是未折叠的。近紫外圆二色光谱指出，尽管二级结构发生了显著变化，但突变后芳香族残基的局部结构不变。二甲胂酸离子强烈且特异性地改变了SynCaM的螺旋含量。我们的数据明确表明，两个叶不是独立的，叶之间的相互作用是由分子的静电势介导的。
