Godbole S, Hammack B, Bowler B E
Department of Chemistry and Biochemistry, University of Denver, 2190 East Iliff Avenue, Denver, CO 80208-2436, USA.
J Mol Biol. 2000 Feb 11;296(1):217-28. doi: 10.1006/jmbi.1999.3454.
The changes in the free energy of the denatured state of a set of yeast iso-1-cytochrome c variants with single surface histidine residues have been measured in 3 M guanidine hydrochloride. The thermodynamics of unfolding by guanidine hydrochloride is also reported. All variants have decreased stability relative to the wild-type protein. The free energy of the denatured state was determined in 3 M guanidine hydrochloride by evaluating the strength of heme-histidine ligation through determination of the pK(a) for loss of histidine binding to the heme. The data are corrected for the presence of the N-terminal amino group which also ligates to the heme under similar solution conditions. Significant deviations from random coil behavior are observed. Relative to a variant with a single histidine at position 26, residual structure of the order of -1.0 to -2.5 kcal/mol is seen for the other variants studied. The data explain the slower folding of yeast iso-1-cytochrome c relative to the horse protein. The greater number of histidines and the greater strength of ligation are expected to slow conversion of the histidine-misligated forms to the obligatory aquo-heme intermediate during the ligand exchange phase of folding. The particularly strong association of histidine residues at positions 54 and 89 may indicate regions of the protein with strong energetic propensities to collapse against the heme during early folding events, consistent with available data in the literature on early folding events for cytochrome c.
在3M盐酸胍中测定了一组具有单个表面组氨酸残基的酵母异-1-细胞色素c变体变性状态的自由能变化。还报道了盐酸胍诱导的去折叠热力学。所有变体相对于野生型蛋白的稳定性均有所降低。通过测定组氨酸与血红素结合丧失的pK(a)来评估血红素-组氨酸连接强度，从而在3M盐酸胍中确定变性状态的自由能。数据针对N端氨基的存在进行了校正，在相似的溶液条件下，N端氨基也会与血红素结合。观察到与无规卷曲行为存在显著偏差。相对于在26位有单个组氨酸的变体，其他所研究的变体存在约-1.0至-2.5千卡/摩尔的残余结构。这些数据解释了酵母异-1-细胞色素c相对于马细胞色素c折叠较慢的原因。在折叠的配体交换阶段，组氨酸数量较多且连接强度较大，预计会减缓组氨酸错配形式向必需的水合血红素中间体的转化。54位和89位组氨酸残基的特别强的结合可能表明在早期折叠事件中，蛋白质的这些区域具有很强的能量倾向，会在早期折叠事件中与血红素相互作用而发生折叠，这与文献中关于细胞色素c早期折叠事件的现有数据一致。
