McGee W A, Rosell F I, Liggins J R, Rodriguez-Ghidarpour S, Luo Y, Chen J, Brayer G D, Mauk A G, Nall B T
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Biochemistry. 1996 Feb 13;35(6):1995-2007. doi: 10.1021/bi951228f.
The relationship between structure and stability has been investigated for the folded forms and the unfolded forms of iso-2 cytochrome c and a variant protein with a stability-enhancing mutation, N52I iso-2. Differential scanning calorimetry has been used to measure the reversible unfolding transitions for the proteins in both heme oxidation states. Reduction potentials have been measured as a function of temperature for the folded forms of the proteins. The combination of measurements of thermal stability and reduction potential gives three sides of a thermodynamic cycle and allows prediction of the reduction potential of the thermally unfolded state. The free energies of electron binding for the thermally unfolded proteins differ from those expected for a fully unfolded protein, suggesting that residual structure modulates the reduction potential. At temperatures near 50 degrees C the N52I mutation has a small but significant effect on oxidation state-sensitive structure in the thermally unfolded protein. Inspection of the high-resolution X-ray crystallographic structures of iso-2 and N52I iso-2 shows that the effects of the N52I mutation and oxidation state on native protein stability are correlated with changes in the mobility of specific polypeptide chain segments and with altered hydrogen bonding involving a conserved water molecule. However, there is no clear explanation of oxidation state or mutation-induced differences in stability of the proteins in terms of observed changes in structure and mobility of the folded forms of the proteins alone.
已经对同工-2细胞色素c的折叠形式和未折叠形式以及具有稳定性增强突变的变体蛋白N52I同工-2的结构与稳定性之间的关系进行了研究。差示扫描量热法已被用于测量两种血红素氧化状态下蛋白质的可逆解折叠转变。已经测量了蛋白质折叠形式的还原电位随温度的变化。热稳定性和还原电位的测量组合给出了热力学循环的三个方面，并允许预测热未折叠状态的还原电位。热未折叠蛋白质的电子结合自由能与完全未折叠蛋白质预期的自由能不同，这表明残余结构调节还原电位。在接近50摄氏度的温度下，N52I突变对热未折叠蛋白质中氧化态敏感结构有微小但显著的影响。对同工-2和N52I同工-2的高分辨率X射线晶体结构的检查表明，N52I突变和氧化态对天然蛋白质稳定性的影响与特定多肽链段的流动性变化以及涉及一个保守水分子的氢键改变有关。然而，仅根据观察到的蛋白质折叠形式的结构和流动性变化，无法对蛋白质氧化态或突变引起的稳定性差异做出明确解释。
