Ventura Salvador, Vega Maria Cristina, Lacroix Emmanuel, Angrand Isabelle, Spagnolo Laura, Serrano Luis
European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Nat Struct Biol. 2002 Jun;9(6):485-93. doi: 10.1038/nsb799.
We have designed de novo 13 divergent spectrin SH3 core sequences to determine their folding properties. Kinetic analysis of the variants with stability similar to that of the wild type protein shows accelerated unfolding and refolding rates compatible with a preferential stabilization of the transition state. This is most likely caused by conformational strain in the native state, as deletion of a methyl group (Ile-->Val) leads to deceleration in unfolding and increased stability (up to 2 kcal x mol(-1)). Several of these Ile-->Val mutants have negative phi(-U) values, indicating that some noncanonical phi(-U) values might result from conformational strain. Thus, producing a stable protein does not necessarily mean that the design process has been entirely successful. Strained interactions could have been introduced, and a reduction in the buried volume could result in a large increase in stability and a reduction in unfolding rates.
我们从头设计了13个不同的血影蛋白SH3核心序列，以确定它们的折叠特性。对稳定性与野生型蛋白相似的变体进行动力学分析，结果显示其解折叠和重折叠速率加快，这与过渡态的优先稳定相一致。这很可能是由天然状态下的构象应变引起的，因为甲基的缺失（异亮氨酸→缬氨酸）会导致解折叠减速并增加稳定性（高达2千卡·摩尔⁻¹）。其中几个异亮氨酸→缬氨酸突变体具有负的φ（-U）值，表明一些非典型的φ（-U）值可能是由构象应变导致的。因此，产生一个稳定的蛋白质并不一定意味着设计过程完全成功。可能引入了应变相互作用，并且埋藏体积的减少可能导致稳定性大幅增加和解折叠速率降低。
