Zarrine-Afsar Arash, Dahesh Samira, Davidson Alan R
Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S-1A8.
J Mol Biol. 2007 Oct 26;373(3):764-74. doi: 10.1016/j.jmb.2007.07.059. Epub 2007 Aug 9.
Structural database-derived propensities for amino acids to adopt particular local protein structures, such as alpha-helix and beta-strand, have long been recognized and effectively exploited for the prediction of protein secondary structure. However, the experimental verification of database-derived propensities using mutagenesis studies has been problematic, especially for beta-strand propensities, because local structural preferences are often confounded by non-local interactions arising from formation of the native tertiary structure. Thus, the overall thermodynamic stability of a protein is not always altered in a predictable manner by changes in local structural propensity at a single position. In this study, we have undertaken an investigation of the relationship between beta-strand propensity and protein folding kinetics. By characterizing the effects of a wide variety of amino acid substitutions at two different beta-strand positions in an SH3 domain, we have found that the observed changes in protein folding rates are very well correlated to beta-strand propensities for almost all of the substitutions examined. In contrast, there is little correlation between propensities and unfolding rates. These data indicate that beta-strand conformation is well formed in the structured portion of the SH3 domain transition state, and that local structure propensity strongly influences the stability of the transition state. Since the transition state is known to be packed more loosely than the native state and likely lacks many of the non-local stabilizing interactions seen in the native state, we suggest that folding kinetics studies may generally provide an effective means for the experimental validation of database-derived local structural propensities.
长期以来，人们已经认识到从结构数据库得出的氨基酸形成特定局部蛋白质结构（如α-螺旋和β-链）的倾向，并有效地利用这些倾向来预测蛋白质二级结构。然而，利用诱变研究对数据库得出的倾向进行实验验证一直存在问题，特别是对于β-链倾向，因为局部结构偏好常常被天然三级结构形成过程中产生的非局部相互作用所混淆。因此，蛋白质的整体热力学稳定性并不总是以可预测的方式因单个位置局部结构倾向的变化而改变。在本研究中，我们对β-链倾向与蛋白质折叠动力学之间的关系进行了研究。通过表征SH3结构域中两个不同β-链位置上多种氨基酸取代的影响，我们发现几乎所有所检测取代的蛋白质折叠速率的观察变化都与β-链倾向高度相关。相比之下，倾向与解折叠速率之间几乎没有相关性。这些数据表明，β-链构象在SH3结构域过渡态的结构化部分中形成良好，并且局部结构倾向强烈影响过渡态的稳定性。由于已知过渡态比天然态堆积更松散，并且可能缺乏天然态中存在的许多非局部稳定相互作用，我们认为折叠动力学研究通常可能为数据库得出的局部结构倾向的实验验证提供一种有效手段。
