Heinz D W, Baase W A, Zhang X J, Blaber M, Dahlquist F W, Matthews B W
Institute of Molecular Biology, University of Oregon, Eugene 97403.
J Mol Biol. 1994 Feb 25;236(3):869-86. doi: 10.1006/jmbi.1994.1195.
One to four alanines were inserted by site-directed mutagenesis at three different locations within the alpha-helix comprising residues 39 to 50 in bacteriophage T4 lysozyme. All insertion mutants were correctly folded and catalytically active although the insertions led to a thermal destabilization by 1.1 to 4.2 kcal/mol when compared to wild-type. Variants that restored part of the loss in stability associated with the initial alanine insertions could be found by randomizing the inserted amino acids. In selected cases, directed mutagenesis of adjacent residues was also used to regain stability. Structural information obtained from X-ray crystallography and/or 2D-NMR for 10 different variants showed two distinct ways in which the protein responded to the amino acid insertions: (1) The inserted amino acids were incorporated into the helix by replacing preceding wild-type amino acids and causing a shift in register towards the N terminus. As a consequence, wild-type amino acids were translocated from the helix into the preceding loop. (2) Insertions caused a "looping out" within the alpha-helix. In this case the perturbation was confined to a minimal region in the immediate vicinity of the insertion. No change in the length of the helix was detected in either case. The structural response appears to be determined by the maintenance of the hydrophobic interface between the helix and the rest of the protein. This interface remains essentially intact in all variant structures. The results exemplify the plasticity and the adaptability of the protein structure which allows the incorporation of additional amino acids into a secondary structure element without large structural perturbations, as long as vital internal interactions are preserved. They also suggest that loops in proteins related by evolution can vary in length not only because of insertions within the loops themselves but also as a consequence of insertions within neighboring secondary structure elements.
通过定点诱变，在噬菌体T4溶菌酶中包含39至50位残基的α-螺旋内的三个不同位置插入了1至4个丙氨酸。所有插入突变体均正确折叠且具有催化活性，尽管与野生型相比，插入导致热稳定性降低了1.1至4.2千卡/摩尔。通过随机化插入的氨基酸，可以找到恢复与初始丙氨酸插入相关的部分稳定性损失的变体。在某些选定的情况下，还使用相邻残基的定向诱变来恢复稳定性。从10种不同变体的X射线晶体学和/或二维核磁共振获得的结构信息表明，蛋白质对氨基酸插入有两种不同的反应方式：（1）插入的氨基酸通过取代先前的野生型氨基酸并入螺旋中，并导致向N端的寄存器移位。结果，野生型氨基酸从螺旋转移到前面的环中。（2）插入导致α-螺旋内出现“环出”。在这种情况下，扰动仅限于插入紧邻区域的最小区域。在这两种情况下均未检测到螺旋长度的变化。结构反应似乎由螺旋与蛋白质其余部分之间疏水界面的维持所决定。该界面在所有变体结构中基本保持完整。结果例证了蛋白质结构的可塑性和适应性，只要重要的内部相互作用得以保留，就允许在不产生大的结构扰动的情况下将额外的氨基酸并入二级结构元件中。它们还表明，通过进化相关的蛋白质中的环长度可以变化，不仅是因为环本身内的插入，还因为相邻二级结构元件内的插入。
