Denatured state thermodynamics: Residual structure, chain stiffness and scaling factors
Denatured state thermodynamics: Residual structure, chain stiffness and scaling factors
复制标题
DOI：
10.1006/jmbi.2001.4909
复制
发表时间：
2001-08-31
影响因子：
5.6
通讯作者：
Bowler, BE
中科院分区：
文献类型：
--
作者：
Hammack, BN;Smith, CR;Bowler, BE
一组九个变体的酵母异-1-细胞色素c与零或一个表面组氨酸已被改造，使N-末端氨基在体内乙酰化。N-末端乙酰化已通过完整和蛋白水解消化的蛋白质的质谱分析确认。在变性条件下（3 M盐酸胍），通过pH滴定法测定组氨酸-血红素成环平衡，提供各变体的实测pK（a）、pK（a）（obs）。N-末端乙酰化防止N-末端氨基-血红素结合平衡干扰组氨酸-血红素亲和力的测量。观察到pK（a）（obs）对所形成环中单体数量对数的线性依赖性存在显著偏差，这是无规卷曲变性状态的预期结果。最大.对于37个单体的环大小发生组氨酸-血红素亲和性。对于37-83个单体的环大小，组氨酸-血红素pK（a）（obs）值与比例因子-4.2 +/-0.3一致。该值远大于自由连接的无规卷曲的比例因子-1.5，其通常用于表示蛋白质变性状态的构象性质。对于9至22个单体的环大小，链刚度可能是造成组氨酸-血红素亲和力相对于37个环大小降低的原因。结果进行了讨论，在残余结构和序列组成的影响蛋白质的变性状态的构象特性。(C)北京：科学出版社.
A set of nine variants of yeast iso-1-cytochrome c with zero or one surface histidine have been engineered such that the N-terminal amino group is acetylated in vivo. N-terminal acetylation has been confirmed by mass spectral analysis of intact and proteolytically digested protein. The histidine-heme loop-forming equilibrium, under denaturing conditions (3 M guanidine hydrochloride), has been measured by pH titration providing an observed pK(a), pK(a)(obs), for each variant. N-terminal acetylation prevents the N-terminal amino group-heme binding equilibrium from interfering with measurements of histidine-heme affinity. Significant deviation is observed from the linear dependence of pK(a)(obs) on the log of the number of monomers in the loop formed, expected for a random coil denatured state. The maximum. histidine-heme affinity occurs for a loop size of 37 monomers. For loop sizes of 37-83 monomers, histidine-heme pK(a)(obs) values are consistent with a scaling factor of -4.2 +/-0.3. This value is much larger than the scaling factor of -1.5 for a freely jointed random coil, which is commonly used to represent the conformational properties of protein denatured states. For loop sizes of nine to 22 monomers, chain stiffness is likely responsible for the decreases in histidine-heme affinity relative to a loop size of 37. The results are discussed in terms of residual structure and sequence composition effects on the conformational properties of the denatured states of proteins. (C) 2001 Academic Press.