Wunderlich Michael, Max Klaas E A, Roske Yvette, Mueller Uwe, Heinemann Udo, Schmid Franz X
Laboratorium für Biochemie und Bayreuther Zentrum für Molekulare Biowissenschaften, Universität Bayreuth, D-95440 Bayreuth, Germany.
J Mol Biol. 2007 Oct 26;373(3):775-84. doi: 10.1016/j.jmb.2007.08.004. Epub 2007 Aug 19.
Computational design and in vitro evolution are major strategies for stabilizing proteins. For the four critical positions 16, 18, 25, and 29 of the B domain of the streptococcal protein G (Gbeta1), they identified the same optimal residues at positions 16 and 25, but not at 18 and 29. Here we analyzed the energetic contributions of the residues from these two approaches by single and double mutant analyses and determined crystal structures for a variant from the calculation (I16/L18/E25/K29) and from the selection (I16/I18/E25/F29). The structural analysis explains the observed differences in stabilization. Residues 16, 18, and 29 line an invagination, which results from a packing defect between the helix and the beta-sheet of Gbeta1. In all stabilized variants, residues with larger side-chains occur at these positions and packing is improved. In the selected variant, packing is better optimized than in the computed variant. Such differences in side-chain packing strongly affect stability but are difficult to evaluate by computation.
计算设计和体外进化是稳定蛋白质的主要策略。对于链球菌蛋白G（Gβ1）B结构域的四个关键位置16、18、25和29，他们在位置16和25处确定了相同的最佳残基，但在位置18和29处并非如此。在这里，我们通过单突变和双突变分析，分析了这两种方法中残基的能量贡献，并确定了计算变体（I16/L18/E25/K29）和选择变体（I16/I18/E25/F29）的晶体结构。结构分析解释了观察到的稳定性差异。残基16、18和29排列在内陷处，这是由Gβ1的螺旋和β折叠之间的堆积缺陷导致的。在所有稳定的变体中，这些位置出现了侧链较大的残基，堆积得到改善。在选择的变体中，堆积比计算变体得到了更好的优化。侧链堆积的这种差异强烈影响稳定性，但很难通过计算进行评估。
