Abstract：
To investigate the characteristics of the postulated carboxy terminal chain-folding initiation site in bovine pancreatic ribonuclease A (RNase A) (residues 106-118), important in the early stages of the folding pathway, we have engineered by site-directed mutagenesis a set of 14 predominantly conservative hydrophobic variants of the protein. The stability of each variant has been compared by pressure and temperature-induced unfolding, monitored by fourth derivative UV absorbance spectroscopy. Apparently simple two-state, reversible unfolding transitions are observed, suggesting that the disruption of tertiary structure of each protein at high pressure or temperature is strongly cooperative. Within the limits of the technique, we are unable to detect significant differences between the two processes of denaturation. Both steady-state kinetic parameters for the enzyme reaction and UV CD spectra of each RNase A variant indicate that truncation of hydrophobic side chains in this region has, in general, little or no effect on the native structure and function of the enzyme. Furthermore, the decreases in free energy of unfolding upon pressure and thermal denaturation of all the variants, particularly those modified at residues 106 and 108, suggest that the hydrophobic residues and side chain packing interactions of this region play an important role in maintaining the conformational stability of RNase A. We also demonstrate the potential of Tyr115 replacement by Trp as a non-destabilizing fluorescence probe of conformational changes local to the region.
摘要：
研究牛胰腺核糖核酸酶A（RNaseA）（残基106-118）中假定的羧基末端链折叠起始位点的特征，在折叠途径的早期阶段很重要，我们通过定点诱变设计了14种主要保守的疏水性蛋白质变体。通过压力和温度诱导的展开比较了每个变体的稳定性，通过四次导数紫外吸收光谱法监测。显然是简单的两个国家，观察到可逆的展开转变，表明在高压或高温下每种蛋白质的三级结构的破坏是强烈的协同作用。在技术的限制范围内,我们无法检测到两种变性过程之间的显着差异。酶反应的稳态动力学参数和每个RNaseA变体的UVCD光谱都表明该区域疏水性侧链的截短具有，总的来说,对酶的天然结构和功能几乎没有影响。此外，所有变体在压力和热变性时解折叠的自由能降低，特别是在残基106和108处修饰的那些，表明该区域的疏水残基和侧链填充相互作用在维持RNaseA的构象稳定性中起重要作用。我们还证明了Trp替代Tyr115作为该区域局部构象变化的非不稳定荧光探针的潜力。
