Linske-O'Connell L I, Sherman F, McLendon G
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642, USA.
Biochemistry. 1995 May 30;34(21):7094-102. doi: 10.1021/bi00021a022.
Position 52 of iso-1-cytochrome c in the yeast Saccharomyces cerevisiae was systematically replaced with all possible amino acids to investigate the molecular basis of the "global suppressor" activity for the N52I mutation. Isogenic strains containing the variant proteins were made with a mixed oligonucleotide-directed mutagenesis technique in vivo. A relationship between thermodynamic protein stability and cellular protein levels was established by comparing direct spectroscopic measurements of cytochrome c levels in vivo with the thermodynamic parameters from guanidine hydrochloride denaturation and microcalorimetric measurements. Reversible denaturation data show that, within a large group of amino acid side chains, the thermodynamic stabilization at position 52 is related to the hydrophobicity of the side chain at that site.
在酿酒酵母中，将异-1-细胞色素c的第52位系统地替换为所有可能的氨基酸，以研究N52I突变的“全局抑制”活性的分子基础。利用体内混合寡核苷酸定向诱变技术构建了含有变体蛋白的同基因菌株。通过比较体内细胞色素c水平的直接光谱测量结果与盐酸胍变性和微量量热测量得到的热力学参数，建立了蛋白质热力学稳定性与细胞内蛋白质水平之间的关系。可逆变性数据表明，在一大组氨基酸侧链中，第52位的热力学稳定性与该位点侧链的疏水性有关。
