Interdepartmental Program in Biomolecular Science and Engineering, University of California, Santa Barbara, Santa Barbara, CA 93106, USA.
J Mol Biol. 2010 Oct 29;403(3):446-58. doi: 10.1016/j.jmb.2010.08.049. Epub 2010 Sep 15.
The folding rates of two-state single-domain proteins are generally resistant to small-scale changes in amino acid sequence. For example, having surveyed here over 700 single-residue substitutions in 24 well-characterized two-state proteins, we find that the majority (55%) of these substitutions affect folding rates by less than a factor of 2, and that only 9% affect folding rates by more than a factor of 8. Among those substitutions that significantly affect folding rates, we find that accelerating substitutions are an order of magnitude less common than those that decelerate the process. One of the most extreme outliers in this data set, an arginine-to-phenylalanine substitution at position 48 (R48F) of chymotrypsin inhibitor 2 (CI2), accelerates the protein's folding rate by a factor of 36 relative to that of the wild-type protein and is the most accelerating substitution reported to date in a two-state protein. In order to better understand the origins of this anomalous behavior, we have characterized the kinetics of multiple additional substitutions at this position. We find that substitutions at position 48 in CI2 fall into two distinct classes. The first, comprising residues that ablate the charge of the wild-type arginine but retain the hydrophobicity of its alkane chain, accelerate folding by at least 10-fold. The second class, comprising all other residues, produces folding rates within a factor of two of the wild-type rate. A significant positive correlation between hydrophobicity and folding rate across all of the residues we have characterized at this position suggests that the hydrophobic methylene units of the wild-type arginine play a significant role in stabilizing the folding transition state. Likewise, studies of the pH dependence of the histidine substitution indicate a strong correlation between folding rate and charge state. Thus, mutations that ablate the arginine's positive charge while retaining the hydrophobic contacts of its methylene units tend to dramatically accelerate folding. Previous studies have suggested that arginine 48 plays an important functional role in CI2, which may explain why it is highly conserved despite the anomalously large deceleration it produces in the folding of this protein.
两种状态单域蛋白质的折叠速率通常不受氨基酸序列小范围变化的影响。例如，在这里对 24 种经过充分研究的两种状态蛋白质中的 700 多个单残基取代进行了调查，我们发现这些取代中的大多数（55%）对折叠速率的影响小于 2 倍，而只有 9%对折叠速率的影响大于 8 倍。在那些显著影响折叠速率的取代中，我们发现加速取代比减速过程的取代要少见一个数量级。在这个数据集的最极端异常值之一中，糜蛋白酶抑制剂 2（CI2）位置 48 的精氨酸到苯丙氨酸取代（R48F）使蛋白质的折叠速率相对于野生型蛋白质提高了 36 倍，这是迄今为止在两种状态蛋白质中报道的最加速取代。为了更好地理解这种异常行为的起源，我们已经对该位置的多个其他取代进行了动力学特性分析。我们发现，CI2 位置 48 的取代分为两类。第一类包括消除野生型精氨酸电荷但保留其烷链疏水性的残基，至少加速 10 倍折叠。第二类包括所有其他残基，其折叠速率与野生型速率相差不超过两倍。我们在该位置所研究的所有残基中，疏水性与折叠速率之间存在显著的正相关性，这表明野生型精氨酸的疏水性亚甲基单元在稳定折叠过渡态方面发挥了重要作用。同样，对组氨酸取代的 pH 依赖性研究表明，折叠速率与电荷状态之间存在很强的相关性。因此，消除精氨酸正电荷同时保留其亚甲基单元疏水性接触的突变往往会显著加速折叠。先前的研究表明，精氨酸 48 在 CI2 中发挥着重要的功能作用，这可能解释了为什么尽管它对该蛋白质的折叠产生了异常大的减速作用，但它仍高度保守。
