Miyazawa S, Jernigan R L
Gunma University, Faculty of Technology, Japan.
Protein Eng. 1994 Oct;7(10):1209-20. doi: 10.1093/protein/7.10.1209.
The stability changes caused by single amino acid substitutions are studied by a simple, empirical method which takes account of the free energy change in the compact denatured state as well as in the native state. The conformational free energy is estimated from effective inter-residue contact energies, as evaluated in our previous study. When this method is applied, with a simple assumption about the compactness of the denatured state, for single amino acid replacements at Glu49 of the tryptophan synthase alpha subunit and at Ile3 of bacteriophage T4 lysozyme, the estimates of the unfolding Gibbs free energy changes correlate well with observed values, especially for hydrophobic amino acids, and it also yields the same magnitudes of energy as the observed values for both proteins. When it is also applied for amino acid replacements at various positions to estimate the average number of contacts at each position in the denatured state from the observed value of unfolding free energy change, those values for replacements with Gly and Ala at the same residue position in staphylococcal nuclease correlate well with each other. The estimated numbers of contacts indicate that the protein is not fully expanded in the denatured state and also that the compact denatured state may have a substantially native-like topology, like the molten globule state, in that there is a weak correlation between the estimated average number of contacts at each residue position in the denatured state and the number of contacts in the native structure. These results provide some further evidence that the inter-residue contact energies as applied here (i) properly reflect actual inter-residue interactions and (ii) can be considered to be a pairwise hydrophobicity scale. Also, the results indicate that characterization of the denatured state is critical to understanding the folding process.
通过一种简单的经验方法研究了由单个氨基酸取代引起的稳定性变化，该方法考虑了紧密变性状态以及天然状态下的自由能变化。构象自由能由有效的残基间接触能估算得出，如我们先前研究中所评估的那样。当应用此方法，并对变性状态的紧密性做一个简单假设，来研究色氨酸合酶α亚基的Glu49和噬菌体T4溶菌酶的Ile3处的单个氨基酸替换时，展开吉布斯自由能变化的估算值与观测值相关性良好，尤其是对于疏水氨基酸而言，并且对于这两种蛋白质，该方法得出的能量大小也与观测值相同。当将此方法应用于不同位置的氨基酸替换，以从展开自由能变化的观测值估算变性状态下每个位置的平均接触数时，葡萄球菌核酸酶中相同残基位置被Gly和Ala替换时的那些值彼此相关性良好。估算的接触数表明，该蛋白质在变性状态下并未完全伸展，并且紧密变性状态可能具有与天然状态非常相似的拓扑结构，类似于熔球态，因为变性状态下每个残基位置的估算平均接触数与天然结构中的接触数之间存在微弱的相关性。这些结果进一步证明了此处应用的残基间接触能（i）正确反映了实际的残基间相互作用，（ii）可被视为一种成对疏水性标度。此外，结果表明，对变性状态的表征对于理解折叠过程至关重要。
