CONSERVED CORE RESIDUES AND CYTOCHROME C FOLDING

描述 基于PCR的诱变将用于选择性地改变氨基酸 野生型马细胞色素c和变体H33 N的序列，该变体缺乏 非天然组氨酸配体的血红素铁，因此表现出简化的 折叠机构 结构中的12个进化保守位点 蛋白质的核心，这是不太可能有助于功能，已被 鉴定为诱变。 突变蛋白将使用酵母表达， 缺乏细胞色素C的菌株。NaCl-乙基纯化的重组蛋白 乙酸盐沉淀，Amberlite CG-50色谱，超滤和 将检查FPLC的溶解度、稳定性和折叠-解折叠 均衡 色氨酸荧光将评估结构稳定性。 该方法提供了细胞色素c的总体尺寸的指示 由于多肽中的单个色氨酸在多肽中是荧光的， 未折叠状态，但由于 能量转移到血红素。 远紫外CD光谱将监测平衡 在不同的变性条件下折叠和展开， 温度

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.