MUTATIONS THAT STABILIZE FOLDING INTERMEDIATES OF PHAGE-P22 TAILSPIKE PROTEIN - FOLDING IN-VIVO AND IN-VITRO, STABILITY, AND STRUCTURAL CONTEXT
MUTATIONS THAT STABILIZE FOLDING INTERMEDIATES OF PHAGE-P22 TAILSPIKE PROTEIN - FOLDING IN-VIVO AND IN-VITRO, STABILITY, AND STRUCTURAL CONTEXT
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DOI：
10.1006/jmbi.1995.0288
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发表时间：
1995-05-26
影响因子：
5.6
通讯作者：
SECKLER, R
中科院分区：
文献类型：
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作者：
BEISSINGER, M;LEE, SC;SECKLER, R
三聚体噬菌体P22尾刺蛋白的折叠受到单氨基酸取代称为温度敏感折叠（tsf）突变的影响。它们的表型通过两个反复分离的全局抑制因子（su）突变（sm V331A和su A334V）和两个额外的替换（su V331G和su A334I）减轻，可通过定点诱变获得。我们研究了抑制基因突变对体外尾穗重折叠的影响，对其在体内高表达水平下成熟的影响，以及对天然蛋白热展开率的影响。ahsu突变提高了体外和体内的折叠效率，但334位取代的相对作用在体内更为明显，而331位取代的相对作用在体外更有效。V331G引起的重折叠产量的增加是所有单突变中最强的，并且与V331A/A334V双突变一样有效，其中两个单突变表现出加性效应。V331A和V331G均能延缓热变性，而A334V无影响，A334I加速展开。A334I是发现的第一个影响尾穗折叠和天然蛋白相反方向热稳定性的突变。根据最近确定的n端短尾尖的晶体结构，所观察到的效应可以合理化。由于Val331的主二面角（phi = -119度，psi = -142度）在非甘氨酸残基中是不寻常的，V331G和V331A可以去除位阻应变，从而稳定折叠中间体和天然蛋白。Val和Ile的p支侧链取代了蛋白质内部的Ala334，可能改善了大平行β -螺旋中的疏水残基堆栈。这在结构松散的早期折叠中间体中可能很重要，但在非常刚性的原生结构中就不重要了，在原生结构中，侧链很难被容纳。
The folding of the trimeric phage P22 tailspike protein is affected by single amino acid substitutions designated temperature-sensitive folding (tsf) mutations. Their phenotypes are alleviated by two repeatedly isolated global suppressor (su) mutations (sm V331A and su A334V) and by two additional substitutions (su V331G and su A334I), accessible through site-directed mutagenesis. We investigated the influence of the suppressor mutations on tailspike refolding in vitro, on its maturation at high expression levels in vivo, and on the rates of thermal unfolding of the native protein. Ah su mutations improved the folding efficiency in vitro and in vivo, but the relative effects of substitutions at position 334 were more pronounced in vivo, whereas the 331 substitutions were more effective in vitro. V331G caused the strongest increase in refolding yields of any single mutation, and was as effective as the V331A/A334V double mutation, where the two single mutations exhibited an additive effect. Both V331A and V331G retarded thermal denaturation, while A334V did not affect, and A334I accelerated unfolding. A334I is the first mutation found to affect the folding of the tailspike and the thermal stability of the native protein in opposite directions. The observed effects can be rationalized on the basis of the recently determined crystal structure of an N-terminally shortened tailspike. As the backbone dihedral angles of Val331 (phi = -119 degrees, psi = -142 degrees) are unusual for non-glycine residues, V331G and V331A may remove steric strain and thereby stabilize folding intermediates and the native protein. The P-branched side-chains of Val and Ile substituted for Ala334 in the interior of the protein may improve a hydrophobic stack of residues in the large parallel beta-helix. This is likely important in loosely structured early folding intermediates, but not in the very rigid native structure, where the side-chain of Ile carl hardly be accommodated.