Hickey D R, McLendon G, Sherman F
Department of Biochemistry, University of Rochester Medical School, New York 14642.
J Biol Chem. 1988 Dec 5;263(34):18298-305.
Iso-1-cytochromes c having lysine 32 replaced by leucine, glutamine, tyrosine, and tryptophan were prepared from strains of bakers' yeast, Saccharomyces cerevisiae, and chemically blocked at cysteine 107 with methyl methanethiolsulfonate to prevent dimerization. These modified ferricytochromes c were guanidine denatured, and the unfolding thermodynamics were determined by circular dichroism and fluorescence measurements. Thermal unfolding was also monitored by absorbance measurements. The guanidine denaturation midpoints for the altered proteins are smaller than the wild type, while the orders of stability from unfolding free energy changes are: Lys-32 (wild type) approximately Leu-32 approximately Gln-32 (circular dichroism), greater than Gln-32 (fluorescence) greater than Tyr-32 approximately Trp-32. Midpoints and differences in free energy changes for thermal unfolding parallel the fluorescence free energy changes for guanidine-induced unfolding. Thus, the blocked Leu-32 and Lys-32 proteins are equally stable with respect to both chemical and thermal denaturation. The reported data indicate that single replacements may significantly modify protein stability, and that substitution for an evolutionarily retained residue in normal cytochrome c structures does not always destabilize the protein. In addition, in vitro thermal stabilities approximately correlate with in vivo specific activities.
通过面包酵母酿酒酵母菌株制备了赖氨酸32被亮氨酸、谷氨酰胺、酪氨酸和色氨酸取代的异-1-细胞色素c，并在半胱氨酸107处用甲硫醇磺酸甲酯进行化学封闭以防止二聚化。这些修饰的高铁细胞色素c用胍变性，通过圆二色性和荧光测量确定其去折叠热力学。热去折叠也通过吸光度测量进行监测。改变后的蛋白质的胍变性中点比野生型小，而从去折叠自由能变化得出的稳定性顺序为：赖氨酸-32（野生型）约等于亮氨酸-32约等于谷氨酰胺-32（圆二色性），大于谷氨酰胺-32（荧光）大于酪氨酸-32约等于色氨酸-32。热去折叠的中点和自由能变化差异与胍诱导去折叠的荧光自由能变化平行。因此，封闭的亮氨酸-32和赖氨酸-32蛋白质在化学和热变性方面同样稳定。所报道的数据表明，单个取代可能会显著改变蛋白质稳定性，并且在正常细胞色素c结构中取代进化上保留的残基并不总是会使蛋白质不稳定。此外，体外热稳定性与体内比活性大致相关。
