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Folding of horse cytochrome c in the reduced state

机译：还原状态下的马细胞色素c折叠

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Equilibrium and kinetic folding studies of horse cytochrome c in the reduced state have been carried out under strictly anaerobic conditions at neutral pH, 10 degreesC, in the entire range of aqueous solubility of guanidinium hydrochloride (GdnHCl). Equilibrium unfolding transitions observed by Soret heme absorbance, excitation energy transfer from the lone tryptophan residue to the ferrous heme, and far-UV circular dichroism (CD) are all biphasic and superimposable, implying no accumulation of structural intermediates. The thermodynamic parameters obtained by two-state analysis of these transitions yielded DeltaG(H2O) = 18.8(+/-1.45) kcal mol(-1), and C-m = 5.1(+/-0.15) M GdnHCl, indicating unusual stability of reduced cytochrome c. These results have been used in conjunction with the redox potential of native cytochrome c and the known stability of oxidized cytochrome c to estimate a value of -164 mV as the redox potential of the unfolded protein. Stopped-flow kinetics of folding and unfolding have been recorded by Soret heme absorbance, and tryptophan fluorescence as observables. Th refolding kinetics are monophasic in the transition region, but become biphasic as moderate to strongly native-like conditions are approached. There also is a burst folding reaction unobservable in the stopped-flow time window. Analyses of the two observable rates and their amplitudes indicate that the faster of the two rates corresponds to apparent two-state folding (U <----> N) of 80-90 % of unfolded molecules with a time constant in the range 190-550 ps estimated by linear extrapolation and model calculations. The remaining 10-20%. of the population folds to an off-pathway intermediate, 1, which is required to unfold first to the initial unfolded state, U, in order to refold correctly to the native state, N (I <----> U <----> N). The slower of the two observable rates, which has a positive slope in the linear functional dependence on the denaturant concentration indicating that an unfolding process under native-like conditions indeed exists, originates from the unfolding of I to U, which rate-limits the overall folding of these 10-20 % of molecules. Both fast and slow rates are independent of protein concentration and pH of the refolding milieu, suggesting that the off-pathway intermediate is not a protein aggregate or trapped by heme misligation. The nature or type of unfolded-state heme ligation does not interfere with refolding. Equilibrium pH titration of the unfolded state yielded coupled ionization of the two non-native histidine ligands, H26 and H33, with a pK(a) value of 5.85. A substantial fraction of the unfolded population persists as the six-coordinate form even at low pH, suggesting ligation of the two methionine residues, M65 and M80. These results have been used along with the known ligand-binding properties of unfolded cytochrome c to propose a model for heme ligation dynamics. In contrast to refolding kinetics, the unfolding kinetics of reduced cytochrome c recorded by observation of Soret absorbance and tryptophan fluorescence are all slow, simple, and single-exponential. In the presence of 6.8 M GdnHCl, the unfolding time constant is similar to 300(+/- 125) ms. There is no burst unfolding reaction. Simulations of the observed folding-unfolding kinetics by numerical solutions of the rate equations corresponding to the three-state I <----> U <----> N scheme have yielded the microscopic rate constants.
机译：在盐酸胍（GdnHCl）的整个水溶性范围内，在严格的厌氧条件下，中性pH，10摄氏度下，对还原状态下的马细胞色素c进行了平衡和动力学折叠研究。 Soret血红素吸光度观察到的平衡展开过渡，激发能量从孤色氨酸残基转移到亚铁血红素以及远紫外圆二色性（CD）都是两相且可叠加的，这意味着没有结构中间体的积累。通过对这些转变进行二态分析获得的热力学参数产生DeltaG（H2O）= 18.8（+/- 1.45）kcal mol（-1），Cm = 5.1（+/- 0.15）M GdnHCl，表明还原的异常稳定性细胞色素c。这些结果已与天然细胞色素c的氧化还原电势和氧化细胞色素c的已知稳定性结合使用，以估计-164 mV的值作为未折叠蛋白的氧化还原电势。 Soret血红素吸光度记录了折叠和展开的停流动力学，色氨酸荧光可观察到。 Th复性动力学在过渡区域是单相的，但随着接近中度至强烈的天然样条件而变为双相。在停止流时间窗口中还观察到突发折叠反应。对两个可观察速率及其幅度的分析表明，这两个速率中的较快值对应于80-90％的未折叠分子的明显二态折叠（U <----> N），时间常数在190范围内通过线性外推法和模型计算可估算-550 ps。其余的10-20％。的种群折叠到一个非中间途径的中间位1，该位点需要首先展开到初始展开状态U，以便正确地重新折叠到原始状态N（I <----> U <- -> N）。两种可观察到的速率中较慢的速率，在线性函数对变性剂浓度的依赖性上呈正斜率，表明确实存在天然条件下的展开过程，这是由I到U的展开引起的，该速率限制了整体折叠这些10-20％的分子。快速和慢速都与蛋白质浓度和重折叠环境的pH无关，这表明非通路中间体不是蛋白质聚集体，也不是被血红素错配捕获的。展开状态血红素连接的性质或类型不干扰重新折叠。展开状态的平衡pH滴定产生两个非天然组氨酸配体H26和H33的耦合电离，pK（a）值为5.85。即使在低pH值下，大部分未折叠种群仍以六坐标形式存在，这表明两个蛋氨酸残基M65和M80可以连接。这些结果与未折叠的细胞色素c的已知配体结合特性一起用于提出血红素连接动力学的模型。与重折叠动力学相反，通过观察Soret吸光度和色氨酸荧光记录的还原的细胞色素c的展开动力学都是缓慢，简单和单指数的。在存在6.8 M GdnHCl的情况下，展开时间常数类似于300（+/- 125）ms。没有爆发展开反应。通过对应于三态I <----> U <----> N方案的速率方程的数值解对观察到的折叠-展开动力学进行模拟，得出了微观速率常数。

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