Beissinger M, Lee S C, Steinbacher S, Reinemer P, Huber R, Yu M H, Seckler R
Institut für Biophysik, Universität Regensburg, Germany.
J Mol Biol. 1995 May 26;249(1):185-94. doi: 10.1006/jmbi.1995.0288.
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 (su 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. All 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 beta-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 can hardly be accommodated.
三聚体噬菌体P22尾刺蛋白的折叠受单个氨基酸取代的影响，这些取代被称为温度敏感折叠（tsf）突变。它们的表型可通过两个反复分离得到的全局抑制（su）突变（su V331A和su A334V）以及另外两个取代（su V331G和su A334I）得到缓解，后者可通过定点诱变获得。我们研究了抑制突变对尾刺蛋白体外重折叠、其在体内高表达水平下成熟以及天然蛋白热解折叠速率的影响。所有的su突变都提高了体外和体内的折叠效率，但334位取代的相对影响在体内更为明显，而331位取代在体外更有效。V331G导致任何单个突变的重折叠产率增加最为显著，并且与V331A/A334V双突变一样有效，其中这两个单突变表现出加性效应。V331A和V331G都延缓了热变性，而A334V没有影响，A334I加速了解折叠。A334I是第一个被发现以相反方向影响尾刺蛋白折叠和天然蛋白热稳定性的突变。基于最近确定的N端缩短的尾刺蛋白晶体结构，可以解释观察到的效应。由于Val331的主链二面角（φ = -119°，ψ = -142°）对于非甘氨酸残基来说是不寻常的，V331G和V331A可能消除了空间位阻，从而稳定了折叠中间体和天然蛋白。在蛋白质内部取代Ala334的Val和Ile的β分支侧链可能改善了大平行β螺旋中残基的疏水堆积。这在结构松散的早期折叠中间体中可能很重要，但在非常刚性的天然结构中可能不重要，因为Ile的侧链几乎无法容纳。
