CONSERVED CORE RESIDUES AND CYTOCHROME C FOLDING

保守的核心残基和细胞色素 C 折叠

• 批准号: 2669175
• 负责人: HEINRICH RODER
• 金额: $ 3.39万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-03-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2669175
• 关键词: biochemical evolution; cytochrome c; fluorescence; high performance liquid chromatography; polymerase chain reaction; protein folding; protein purification; protein sequence; protein structure function; recombinant proteins; site directed mutagenesis; tryptophan; ultrafiltration; ultraviolet spectrometry

DESCRIPTION PCR based mutagenesis will be used to selectively alter the amino acid sequence of wild type horse cytochrome c and a variant, H33N, which lacks a non-native histidine ligand of the heme iron and thus exhibits a simplified folding mechanism. Twelve evolutionarily conserved sites in the structural core of the protein, which are unlikely to contribute to function, have been identified for mutagenesis. Mutant proteins will be expressed using a yeast strain which lacks cytochrome c. Recombinant protein purified by NaCI-ethyl acetate precipitation, Amberlite CG-50 chromatography, ultrafiltration and FPLC, will be examined for solubility, stability, and folding - unfolding equilibrium. Tryptophan fluorescence will assess structural stability. This method provides an indication of the overall dimensions of cytochrome c since the single tryptophan in the polypeptide is fluorescent in the unfolded state but loses fluorescence in the native state as a result of energy transfer to heme. Far UV CD spectroscopy will monitor the equilibria of folding and unfolding under various conditions of denaturation and temperature.

描述 基于聚合酶链式反应的突变将被用于选择性地改变氨基酸 野生型马细胞色素c的序列和一个缺失a的变异体H33N 非天然组氨酸配基的血红素铁，因此表现出简化的 折叠机构。12个进化保守的遗址在结构上 蛋白质的核心，不太可能对功能有贡献，一直是 被确认为诱变基因。突变蛋白将使用酵母表达 一株缺乏细胞色素c.NaCI-乙基纯化重组蛋白的菌株 醋酸盐沉淀，Amberlite CG-50层析，超滤和 将检查FPLC的溶解性、稳定性和折叠-展开 均衡。色氨酸荧光将评估结构稳定性。 这种方法提供了细胞色素c的整体尺寸的指示。 由于多肽中的单一色氨酸在 未折叠状态，但由于以下原因而在本征状态中失去荧光 能量转移到亚铁血红素。远紫外圆二色光谱将监测平衡 在不同的变性条件下折叠和展开 温度。