Laboratorium für Biochemie und Bayreuther Zentrum für Molekulare Biowissenschaften, Universität Bayreuth, D-95440 Bayreuth, Germany.
Proc Natl Acad Sci U S A. 2009 Dec 1;106(48):20282-7. doi: 10.1073/pnas.0909544106. Epub 2009 Nov 17.
The cis/trans isomerization of peptide bonds before proline (prolyl bonds) is a rate-limiting step in many protein folding reactions, and it is used to switch between alternate functional states of folded proteins. Several prolyl isomerases of the FK506-binding protein family, such as trigger factor, SlyD, and FkpA, contain chaperone domains and are assumed to assist protein folding in vivo. The prolyl isomerase activity of FK506-binding proteins strongly depends on the nature of residue Xaa of the Xaa-Pro bond. We confirmed this in assays with a library of tetrapeptides in which position Xaa was occupied by all 20 aa. A high sequence specificity seems inconsistent with a generic function of prolyl isomerases in protein folding. Accordingly, we constructed a library of protein variants with all 20 aa at position Xaa before a rate-limiting cis proline and used it to investigate the performance of trigger factor and SlyD as catalysts of proline-limited folding. The efficiencies of both prolyl isomerases were higher than in the tetrapeptide assays, and, intriguingly, this high activity was almost independent of the nature of the residue before the proline. Apparently, the almost indiscriminate binding of the chaperone domain to the refolding protein chain overrides the inherently high sequence specificity of the prolyl isomerase site. The catalytic performance of these folding enzymes is thus determined by generic substrate recognition at the chaperone domain and efficient transfer to the active site in the prolyl isomerase domain.
在脯氨酸（脯氨酰键）之前，肽键的顺/反异构化是许多蛋白质折叠反应的限速步骤，它用于在折叠蛋白质的交替功能状态之间切换。FK506 结合蛋白家族的几种脯氨酰异构酶，如触发因子、SlyD 和 FkpA，含有伴侣结构域，被认为在体内协助蛋白质折叠。FK506 结合蛋白的脯氨酰异构酶活性强烈依赖于 Xaa-Pro 键中残基 Xaa 的性质。我们在含有所有 20 种氨基酸的四肽文库中进行的测定中证实了这一点。高序列特异性似乎与脯氨酰异构酶在蛋白质折叠中的通用功能不一致。因此，我们构建了一个文库，其中在限速顺式脯氨酸之前的位置 Xaa 处有所有 20 种氨基酸，并用它来研究触发因子和 SlyD 作为脯氨酸限制折叠的催化剂的性能。两种脯氨酰异构酶的效率都高于四肽测定中的效率，而且，有趣的是，这种高活性几乎与脯氨酸之前的残基的性质无关。显然，伴侣结构域对重折叠蛋白链的几乎无差别结合掩盖了脯氨酰异构酶位点固有的高序列特异性。因此，这些折叠酶的催化性能取决于伴侣结构域的通用底物识别和在脯氨酰异构酶结构域中有效的转移。
