ALANINE SCANNING MUTAGENESIS OF THE ALPHA-HELIX-115-123 OF PHAGE-T4 LYSOZYME - EFFECTS ON STRUCTURE, STABILITY AND THE BINDING OF SOLVENT
ALANINE SCANNING MUTAGENESIS OF THE ALPHA-HELIX-115-123 OF PHAGE-T4 LYSOZYME - EFFECTS ON STRUCTURE, STABILITY AND THE BINDING OF SOLVENT
复制标题
DOI：
10.1006/jmbi.1994.0087
复制
发表时间：
1995-02-17
影响因子：
5.6
通讯作者：
MATTHEWS, BW
中科院分区：
文献类型：
--
作者：
BLABER, M;BAASE, WA;MATTHEWS, BW
在噬菌体T4溶菌酶的螺旋区115至123中构建了一系列单独的丙氨酸突变，以评估该区域内不同侧链对蛋白质稳定性的贡献。构建成对丙氨酸突变和具有7个丙氨酸取代的组合突变体以评估对结构和稳定性的加性效应：该区域内只有3个残基（Ser 117、Leu 118和Leu 121）对稳定性具有实质性影响（展开自由能的变化大于1.0 kcal/mol）。用丙氨酸替换Ser 117导致蛋白质稳定性增加1.27 kcal/mol，这显然是由于野生型蛋白质中存在的菌株的释放。取代被掩埋的残基Leu 118和Leu 121是不稳定的。用丙氨酸取代其余6个残基对稳定性的影响相对较小。这与以前的研究结果一致，即T4溶菌酶的两亲性螺旋中只有20 - 30%的残基对稳定性至关重要。对于一些成对的丙氨酸突变体，稳定性的影响是累加的。然而，对于这些突变体中的大多数，存在轻微的（类似于0.15至0.25 kcal/mol）非加和性，使得双突变体比构成单突变体的总和更稳定。对于具有位置i、i + 4的残基，即相邻但在螺旋的连续转角中，一致地观察到这种效应，表明这些氨基酸残基之间存在弱但显著的相互作用。在7-丙氨酸组合突变体中观察到更明显的非加和性（类似于0.5 kcal/mol）。这种非加和性是由于对于组合突变体（尤其是多丙氨酸突变体）发生的适度的“折叠"或”重新包装"，但对于单一置换是不可能的。一些侧链的截短允许主链酰胺和羰基的溶剂可及性增加。这种效应对于七-丙氨酸组合突变体是最明显的，其中两个溶剂分子，不存在于野生型中，氢键结合到螺旋中间区域的主链羰基。有人认为，这种水分子的结合可能代表了溶剂介导的α-螺旋解折叠的第一步。然而，有序溶剂的出现似乎对稳定性的影响很小（小于或接近0.2千卡/摩尔）。
A series of individual alanine mutations has been constructed in the helical region 115 to 123 in phage T4 lysozyme in order to evaluate the contribution to protein stability of the different side-chains within this region. Pairwise alanine mutations and a combination mutant with seven alanine substitutions were constructed to evaluate the additive effects upon structure and stability: Only three residues within this region (Ser117, Leu118 and Leu121) have a substantial influence upon stability (change in free energy of unfolding greater than 1.0 kcal/mol). Replacement of Ser117 with alanine results in an increase in protein stability of 1.27 kcal/mol, apparently due to the release of strain present in the wild-type protein. Replacement of the buried residues Leu118 and Leu121 is destabilizing. Substitution of the remaining six residues with alanine has relatively Little effect on stability This is consistent with prior studies showing that only 20 to 30% ofthe residues in amphipathic helices in T4 lysozyme are critical for stability.For some of the pairwise alanine mutants the effects on stability are additive. For most of these mutants, however, there is a slight (similar to 0.15 to 0.25 kcal/mol) non-additivity such that the double mutant is more stable than the sum of the constituent single mutants. This effect is consistently observed for residues with positions i, i + 4; i.e. adjacent, but in consecutive turns of the helix, suggesting a weak but significant interaction between these amino acid residues. A more pronounced non-additivity (similar to 0.5 kcal/mol) is seen in the seven-alanine combination mutant. This non-additivity is due to a modest ''collapse'' or ''repacking'' that occurs for the combination mutants (especially the multiple alanine mutant) but is not possible for the single replacements. The truncation of some side-chains permits an increase in solvent accessibility of main-chain amide and carbonyl groups. This effect is most pronounced for the seven-alanine combination mutant, where two solvent molecules, not present in wild-type, hydrogen bond to main-chain carbonyl groups in the middle region of the helix. It has been suggested that the binding of such water molecules might represent the first step in solvent-mediated unfolding of an alpha-helix. The appearance of ordered solvent, however, appears to have very little effect on stability (less than or similar to 0.2 kcal/mol).