Thermodynamic Effects of Noncoded and Coded Methionine Substitutions in Calmodulin
Thermodynamic Effects of Noncoded and Coded Methionine Substitutions in Calmodulin
复制标题
DOI：
10.1016/j.bpj.2008.10.060
复制
发表时间：
2009-02-18
影响因子：
3.4
通讯作者：
Vogel, Hans J.
中科院分区：
文献类型：
--
作者：
Yamniuk, Aaron P.;Ishida, Hiroaki;Vogel, Hans J.
钙调节蛋白钙调素（CaM）中的甲硫氨酸残基在结构和功能上都很重要。它们被埋在apo-CaM的N-和C-结构域中，但在Ca 2 +-CaM中暴露于溶剂中，在那里它们与许多靶蛋白相互作用。以前的结构研究表明，蛋氨酸取代非编码氨基酸硒代蛋氨酸，乙硫氨酸，或正亮氨酸，或突变为亮氨酸不影响钙调素的主链结构。在这里，我们使用差示扫描量热法来显示这些取代增强了两个结构域的稳定性，其中在apo-C结构域中使用亮氨酸或正亮氨酸实现的解链温度（19-26摄氏度）的最大增加。核磁共振光谱实验还揭示了Leu取代的apo-C结构域中缓慢的构象交换过程的丧失。此外，等温滴定量热实验揭示了靶与apo-CaM和Ca 2 +-CaM结合的焓和熵的相当大的变化，但结合的自由能由于焓熵补偿而在很大程度上不受影响。总的来说，这些结果表明，非编码和编码的蛋氨酸取代可以容纳在钙调素，因为蛋白质的结构可塑性。然而，侧链包装和动力学的调整导致蛋白质稳定性和靶结合热力学的显着差异。
The methionine residues in the calcium (Ca2+) regulatory protein calmodulin (CaM) are structurally and functionally important. They are buried within the N- and C-domains of apo-CaM but become solvent-exposed in Ca2+-CaM, where they interact with numerous target proteins. Previous structural studies have shown that methionine substitutions to the noncoded amino acids selenomethionine, ethionine, or norleucine, or mutation to leucine do not impact the main chain structure of CaM. Here we used differential scanning calorimetry to show that these substitutions enhance the stability of both domains, with the largest increase in melting temperature (19-26 degrees C) achieved with leucine or norleucine in the apo-C-domain. Nuclear magnetic resonance spectroscopy experiments also revealed the loss of a slow conformational exchange process in the Leu-substituted apo-C-domain. In addition, isothermal titration calorimetry experiments revealed considerable changes in the enthalpy and entropy of target binding to apo-CaM and Ca2+-CaM, but the free energy of binding was largely unaffected due to enthalpy-entropy compensation. Collectively, these results demonstrate that noncoded and coded methionine substitutions can be accommodated in CaM because of the structural plasticity of the protein. However, adjustments in side-chain packing and dynamics lead to significant differences in protein stability and the thermodynamics of target binding.