Cobos Eva S, Filimonov Vladimir V, Vega Maria Cristina, Mateo Pedro L, Serrano Luis, Martínez Jose C
Department of Physical Chemistry and Institute of Biotechnology, Faculty of Sciences, University of Granada, 1807 Granada, Spain.
J Mol Biol. 2003 Apr 18;328(1):221-33. doi: 10.1016/s0022-2836(03)00273-0.
The folding thermodynamics and kinetics of the alpha-spectrin SH3 domain with a redesigned hydrophobic core have been studied. The introduction of five replacements, A11V, V23L, M25V, V44I and V58L, resulted in an increase of 16% in the overall volume of the side-chains forming the hydrophobic core but caused no remarkable changes to the positions of the backbone atoms. Judging by the scanning calorimetry data, the increased stability of the folded structure of the new SH3-variant is caused by entropic factors, since the changes in heat capacity and enthalpy upon the unfolding of the wild-type and mutant proteins were identical at 298 K. It appears that the design process resulted in an increase in burying both the hydrophobic and hydrophilic surfaces, which resulted in a compensatory effect upon the changes in heat capacity and enthalpy. Kinetic analysis shows that both the folding and unfolding rate constants are higher for the new variant, suggesting that its transition state becomes more stable compared to the folded and unfolded states. The phi(double dagger-U) values found for a number of side-chains are slightly lower than those of the wild-type protein, indicating that although the transition state ensemble (TSE) did not change overall, it has moved towards a more denatured conformation, in accordance with Hammond's postulate. Thus, the acceleration of the folding-unfolding reactions is caused mainly by an improvement in the specific and/or non-specific hydrophobic interactions within the TSE rather than by changes in the contact order. Experimental evidence showing that the TSE changes globally according to its hydrophobic content suggests that hydrophobicity may modulate the kinetic behaviour and also the folding pathway of a protein.
对具有重新设计疏水核心的α-血影蛋白SH3结构域的折叠热力学和动力学进行了研究。引入五个替换位点，即A11V、V23L、M25V、V44I和V58L，导致形成疏水核心的侧链总体积增加了16%，但主链原子的位置没有显著变化。根据扫描量热法数据判断，新的SH3变体折叠结构稳定性的增加是由熵因素引起的，因为在298K时野生型和突变型蛋白质展开时的热容和焓变是相同的。似乎设计过程导致疏水和亲水表面的埋藏都增加了，这导致了热容和焓变变化的补偿效应。动力学分析表明，新变体的折叠和展开速率常数都更高，这表明与折叠态和未折叠态相比，其过渡态变得更稳定。一些侧链的φ（双 dagger-U）值略低于野生型蛋白质，这表明尽管过渡态系综（TSE）总体上没有变化，但根据哈蒙德假说，它已朝着更变性的构象移动。因此，折叠-展开反应的加速主要是由TSE内特异性和/或非特异性疏水相互作用的改善引起的，而不是由接触顺序的变化引起的。实验证据表明，TSE根据其疏水含量整体发生变化，这表明疏水性可能调节蛋白质的动力学行为以及折叠途径。
