Godbole S, Dong A, Garbin K, Bowler B E
Department of Chemistry and Biochemistry, University of Denver, Colorado 80208, USA.
Biochemistry. 1997 Jan 7;36(1):119-26. doi: 10.1021/bi961915m.
In this paper we report thermodynamic studies on a variant of yeast iso-1-cytochrome c in which a surface lysine residue at position 73 has been replaced with a histidine (H73). Guanidine hydrochloride denaturation studies monitored by circular dichroism spectroscopy indicated decreased thermodynamic stability (a lower delta G(o)(u)H20) and a smaller m value for the H73 protein as compared to the wild type (WT) protein. Further investigations to probe the causes for the thermodynamic stability differences between the two proteins involved guanidine hydrochloride and urea denaturations monitored by tryptophan fluorescence. The stability of heme ligation in the denatured state in the presence of either guanidine hydrochloride or urea was monitored by the spin-state transition of the heme iron induced by pH. None of these studies supported the hypothesis that the decreased m value was due to heme-His73 ligation in the denatured state. Guanidine hydrochloride denaturations monitored by the change in the extinction coefficient at 695 nm, which is sensitive to the presence of heme-Met80 ligation, revealed a native-like intermediate for the H73 protein, probably caused by displacement of the Met80 heme ligand by histidine 73 at guanidine hydrochloride concentrations much lower than required for full cooperative unfolding. Presence of the native-like intermediate is most likely the cause of the smaller m value and decreased thermodynamic stability for the CD-monitored H73 protein unfolding as compared to the unfolding of the WT protein. Guanidine hydrochloride denaturations in the presence of 200 mM imidazole provide further evidence in support of the proposed mechanism.
在本文中，我们报道了对酵母同工酶-1-细胞色素c变体的热力学研究，该变体中第73位的表面赖氨酸残基被组氨酸（H73）取代。通过圆二色光谱监测的盐酸胍变性研究表明，与野生型（WT）蛋白相比，H73蛋白的热力学稳定性降低（较低的ΔG(o)(u)H20）且m值较小。通过色氨酸荧光监测盐酸胍和尿素变性，进一步探究这两种蛋白之间热力学稳定性差异的原因。在盐酸胍或尿素存在下，通过pH诱导的血红素铁自旋态转变监测变性状态下血红素连接的稳定性。这些研究均不支持m值降低是由于变性状态下血红素-His73连接的假设。通过695 nm处消光系数的变化监测盐酸胍变性，该变化对血红素-Met80连接的存在敏感，结果显示H73蛋白存在类似天然态的中间体，这可能是由于在远低于完全协同展开所需浓度的盐酸胍作用下，组氨酸73取代了Met80血红素配体所致。与WT蛋白的展开相比，类似天然态中间体的存在很可能是CD监测的H73蛋白展开时m值较小和热力学稳定性降低的原因。在200 mM咪唑存在下的盐酸胍变性提供了进一步证据支持所提出的机制。
