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

影响热稳定性 P22 尾刺蛋白体内折叠中间体的氨基酸取代

• 批准号: 8704126
• 负责人: Jonathan King
• 金额: --
• 依托单位: Massachusetts Institute of Technology
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 1991-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8704126&HistoricalAwards=false
• 关键词: Amino; Acid; Substitutions; Affecting; Vivo

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.

对于确定三维蛋白质结构至关重要的一些氨基酸相互作用必须以某种方式指导折叠中间体的构象。 噬菌体 P22 的热稳定尾刺内含鼠李糖苷酶在其折叠和亚基关联途径中具有不耐热的早期中间体。 已分离出 100 多个温度敏感折叠突变，这些突变要么进一步破坏热不稳定中间体的稳定性，要么改变早期动力学步骤。 许多甘氨酸和亲水残基的取代位于天然结构的表面，可能与表面转动有关。 由于尾刺二级结构超过 50% β-折叠/转角，这些可能是 β 转角，并且突变识别出影响其形成的残基。 该奖项的具体目标是： 1) 确定其余 tsf 突变的位点和氨基酸取代； 2) 对于那些改变电荷的取代，确定哪些位于表面并因此成为候选者，并分析这些突变体中的折叠缺陷； 3) 分离这些缺陷的第二位点抑制子，以确定相互作用的残基是在链中的局部还是远处； 4) 从野生型和突变型感染细胞中分离并表征前体中间体； 5）分离和表征在高温下积累的聚集链，作为研究其形成的早期中间体的构象的手段； 6) 纯化 tsf 突变体和 tsf/抑制子-tsf 双突变体蛋白，用于协作 X 射线衍射、拉曼光谱、体外重折叠和差示扫描量热研究。