Sánchez Ignacio E, Kiefhaber Thomas
Department of Biophysical Chemistry, Biozentrum der Universität Basel, Klingelberstrasse 70, CH-4056 Basel, Switzerland.
J Mol Biol. 2003 Dec 12;334(5):1077-85. doi: 10.1016/j.jmb.2003.10.016.
phi(f)-value analysis is one of the most common methods to characterize the structure of protein folding transition states. It compares the effects of mutations on the folding kinetics with the respective effects on equilibrium stability. The interpretation of the results usually focuses on a few unusual phi(f)-values, which are either particularly high or which are larger than 1 or smaller than 0. These mutations are believed to affect the most important regions for the folding process. A major uncertainty in experimental phi(f)-values is introduced by the commonly used analysis of only a single mutant at various positions in a protein (two-point analysis). To test the reliability of two-point phi(f)-values we used reference data from three positions in two different proteins at which multiple mutations have been introduced. The results show that two-point phi(f)-values are highly inaccurate if the difference in stability between two variants is less than 7 kJ/mol, corresponding to a 20-fold difference in equilibrium constant. Comparison with reported phi(f)-values for 11 proteins shows that most unusual phi(f)-values are observed in mutants which show changes in protein stability that are too small to allow a reliable analysis. The results argue against specific nucleation sites in protein folding and give a picture of transition states as distorted native states for the major part of a protein or for large substructures.
φ(f)值分析是表征蛋白质折叠过渡态结构最常用的方法之一。它将突变对折叠动力学的影响与对平衡稳定性的相应影响进行比较。结果的解释通常集中在一些异常的φ(f)值上，这些值要么特别高，要么大于1或小于0。这些突变被认为会影响折叠过程中最重要的区域。实验φ(f)值的一个主要不确定性是由常用的仅对蛋白质中不同位置的单个突变体进行分析（两点分析）引入的。为了测试两点φ(f)值的可靠性，我们使用了来自两种不同蛋白质中三个位置的参考数据，在这些位置引入了多个突变。结果表明，如果两个变体之间的稳定性差异小于7 kJ/mol，对应于平衡常数20倍的差异，两点φ(f)值就会非常不准确。与报道的11种蛋白质的φ(f)值比较表明，大多数异常的φ(f)值出现在蛋白质稳定性变化太小而无法进行可靠分析的突变体中。这些结果反对蛋白质折叠中有特定的成核位点，并给出了一幅过渡态的图景，即对于蛋白质的主要部分或大的亚结构来说，过渡态是扭曲的天然态。
