Prajapati Ravindra Singh, Das Mili, Sreeramulu Sridhar, Sirajuddin Minhajuddin, Srinivasan Sankaranarayanan, Krishnamurthy Vaishnavi, Ranjani Ranganathan, Ramakrishnan C, Varadarajan Raghavan
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Proteins. 2007 Feb 1;66(2):480-91. doi: 10.1002/prot.21215.
The amino acid Pro is more rigid than other naturally occurring amino acids and, in proteins, lacks an amide hydrogen. To understand the structural and thermodynamic effects of Pro substitutions, it was introduced at 13 different positions in four different proteins, leucine-isoleucine-valine binding protein, maltose binding protein, ribose binding protein, and thioredoxin. Three of the maltose binding protein mutants were characterized by X-ray crystallography to confirm that no structural changes had occurred upon mutation. In the remaining cases, fluorescence and CD spectroscopy were used to show the absence of structural change. Stabilities of wild type and mutant proteins were characterized by chemical denaturation at neutral pH and by differential scanning calorimetry as a function of pH. The mutants did not show enhanced stability with respect to chemical denaturation at room temperature. However, 6 of the 13 single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, 2 mutants showed no change, and 5 were destabilized. In five of the six cases, the stabilization was because of reduced entropy of unfolding. However, the magnitude of the reduction in entropy of unfolding was typically several fold larger than the theoretical estimate of -4 cal K(-1) mol(-1) derived from the relative areas in the Ramachandran map accessible to Pro and Ala residues, respectively. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were nonadditive.
氨基酸脯氨酸（Pro）比其他天然存在的氨基酸更具刚性，并且在蛋白质中缺乏酰胺氢。为了了解脯氨酸取代的结构和热力学效应，在四种不同的蛋白质（亮氨酸 - 异亮氨酸 - 缬氨酸结合蛋白、麦芽糖结合蛋白、核糖结合蛋白和硫氧还蛋白）的13个不同位置引入了脯氨酸。对三个麦芽糖结合蛋白突变体进行了X射线晶体学表征，以确认突变后没有发生结构变化。在其余情况下，使用荧光和圆二色光谱来表明没有结构变化。通过在中性pH下的化学变性以及作为pH函数的差示扫描量热法来表征野生型和突变型蛋白质的稳定性。这些突变体在室温下的化学变性方面没有表现出增强的稳定性。然而，13个单突变体中的6个在热解折叠自由能方面显示出0.3 - 2.4千卡/摩尔范围内的小幅但显著增加，2个突变体没有变化，5个则不稳定。在6个案例中的5个中，稳定性增强是由于解折叠熵的降低。然而，解折叠熵降低的幅度通常比分别从脯氨酸和丙氨酸残基可及的拉氏图相对面积得出的理论估计值 -4卡K⁻¹摩尔⁻¹大几倍。构建了两个双突变体。在这两种情况下，单突变对热解折叠自由能的影响是非加和性的。
