Amino Acid Substitutions Affecting in Vivo Folding Intermediates of the Thermostable P22 Tailspike Protein

某些氨基酸相互作用对确定三维蛋白质结构至关重要，必须以某种方式指导折叠中间体的构象。噬菌体P22的热稳定尾穗内啡肽酶在其折叠和亚基结合途径中具有热稳定的早期中间体。超过100个温度敏感的折叠突变已经被分离出来，这些突变要么进一步破坏了热稳定中间体的稳定性，要么改变了早期的动力学步骤。许多甘氨酸和亲水性残基的取代发生在天然结构的表面，可能与表面转动有关。由于尾穗二级结构超过50%的β -sheet/turn，这些可能是β - turn，突变识别影响其形成的残基。该奖项的具体目标是：1)确定剩余的tsf突变的位点和氨基酸替换；2)对于那些改变电荷的取代，确定哪些是在表面上的，从而成为候选突变体，并分析这些突变体的折叠缺陷；3)分离这些缺陷的第二位点抑制子，以确定相互作用残基是在链的局部还是远端；4)从野生型和突变型感染细胞中分离并鉴定中间引物；5)分离和表征在高温下积累的聚合链，作为研究其形成的早期中间体构象的一种手段；6)纯化tsf突变体和tsf/抑制因子-tsf双突变体蛋白，用于协同x射线衍射、拉曼光谱、体外重折叠和差示扫描量热法研究。

Some of the amino acid interactions critical for determining three dimensional protein structure must somehow direct the conformation of folding intermediates. The thermostable tailspike endorhamnosidase of phage P22 has a thermolabile early intermediate in its folding and subunit association pathway. Over 100 temperature sensitive folding mutations have been isolated which either further destabilize the thermolabile intermediate, or otherwise alter early kinetic steps. Many of the substitutions of glycines and hydrophilic residues are at the surface of the native structure, probably associated with surface turns. Since tailspike secondary structure is over 50% beta-sheet/turn, these are probably beta turns, and the mutations identify residues influencing their formation. The specific objectives of the award are to: 1) Identify the sites and amino acid substitutions for the remainder of the tsf mutations; 2) For those substitutions altering charge, determine which are at the surface and therefore turn candidates, and analyze the folding defect in these mutants; 3) Isolate second site suppressors of these defects, to determine whether the interacting residues are local or distant in the chain; 4) Isolate and characterize the protrimer intermediate from wild type and mutant infected cells; 5) Isolate and characterize the aggregated chains that accumulate at high temperature as a means of investigating the conformation of the early intermediate from which it forms; 6) Purify tsf mutant and tsf/suppressor-tsf double mutant proteins for collaborative X-ray diffraction, Raman spectroscopy, in vitro refolding, and differential scanning calorimetry studies.