Mutagenic analysis of the interior packing of an alpha/beta barrel protein. Effects on the stabilities and rates of interconversion of the native and partially folded forms of the alpha subunit of tryptophan synthase.
Mutagenic analysis of the interior packing of an alpha/beta barrel protein. Effects on the stabilities and rates of interconversion of the native and partially folded forms of the alpha subunit of tryptophan synthase.
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DOI：
10.1021/bi00072a011
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发表时间：
1993-06
期刊：
影响因子：
2.9
通讯作者：
T. Tsuji;B. Chrunyk;X. Chen;C. Matthews
中科院分区：
文献类型：
--
作者：
T. Tsuji;B. Chrunyk;X. Chen;C. Matthews
对鼠伤寒沙门氏菌色氨酸合成酶α亚基的4条β链和α/β桶蛋白进行了一系列的单链和双氨基酸替换，以研究桶的内包装并阐明其折叠机制。尿素诱导的阿尔法亚基的展开被认为涉及一个稳定的中间体，其中氨基折叠单元(残基1-188；螺旋0-5，链1-6)保持折叠，而羧基折叠单元(残基189-268；螺旋6-8，链7-8)变得无序[Bester，A.M.，&Matthews，C.R.(1985)BioChemical 24,3547；Miles，E.W.，Yutani，K.，&Ogarahara，K.(1982)BioChemical 21,2586]。在这两个折叠单元之间的1链(A18G和A18V)、6(Y175Q)、7(L209V)和8(G230A、G230V和I232V)链上的突变表明，对天然和中间构象稳定性的影响严重依赖于替换的位置。虽然所有这些突变都会降低天然构象的稳定性，但只有6链Y175Q上的替换，可能还有8链I232V上的替换，也会扰乱中间体。比较三对双突变体和单链突变体对稳定性的影响，发现链6和7在中间构象和天然构象中都相互作用，而链1和8只在天然构象中相互作用。展开的动力学研究表明，在天然构象中发生的相互作用发生在先前的过渡态。这些结果表明，与我们之前的解释相反，羧基折叠单元在中间体中采用了有组织的结构。其一般含义是，蛋白质的一个片段的折叠状态可能取决于另一个更稳定的结构元素的存在。
A series of single and double amino acid replacements in four beta strands of the alpha subunit of tryptophan synthase from Salmonella typhimurium, and alpha/beta barrel protein, was made to study the interior packing of the barrel and to clarify its folding mechanism. The urea-induced unfolding of the alpha subunit is thought to involve a stable intermediate in which the amino folding unit (residues 1-188; helices 0-5, strands 1-6) remains folded while the carboxy folding unit (residues 189-268; helices 6-8, strands 7-8) becomes disordered [Beasty, A. M., & Matthews, C. R. (1985) Biochemistry 24, 3547; Miles, E. W., Yutani, K., & Ogasahara, K. (1982) Biochemistry 21, 2586]. Mutations in strands 1 (A18G and A18V), 6 (Y175Q), 7 (L209V), and 8 (G230A, G230V, and I232V) at the interface between these two folding units show that the effects on the stabilities of the native and intermediate conformations critically depend on the site of the replacement. Although all of these mutations decrease the stability of the native conformation, only the replacements in strand 6, Y175Q, and possibly strand 8, I232V, also perturb the intermediate. Comparisons of the effects of three pairs of double mutants with the effects of the constituent single mutants on stability show that strands 6 and 7 interact in both the intermediate and native conformations, while strands 1 and 8 interact only in the native conformation. Kinetic studies of unfolding indicate that the interactions which occur in the native conformation arise in the preceding transition state. These results demonstrate that the carboxy folding unit adopts an organized structure in the intermediate, contrary to our previous interpretation. The general implication is that the state of folding of one segment of a protein can depend on the presence of another, more stable element of structure.