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.

一些氨基酸的相互作用对于决定 三维蛋白质结构必须以某种方式引导 折叠中间体的构象。 述热稳定性 噬菌体P22的尾穗内鼠李糖苷酶具有热不稳定性， 折叠和亚基结合的早期中间体 通路 超过100种温度敏感性折叠突变 已经被隔离，这要么进一步破坏了 不耐热中间体，或以其他方式改变早期动力学 步 许多甘氨酸和亲水性氨基酸的取代 残基可能位于天然结构的表面， 与曲面转弯相关。 自从尾穗中学 结构超过50% β-折叠/转角，这些可能是 β转角，并且突变识别影响 他们的形成。 该奖项的具体目标是：1）确定 的其余部分的位点和氨基酸取代 2）对于那些改变的替换， 在表面上，确定哪些是电荷， 候选人，并分析这些突变体中的折叠缺陷; 3)分离这些缺陷的第二部位抑制物， 确定相互作用的残基是局部的还是 4）分离和表征 来自感染的野生型和突变体的前三聚体中间体 5）分离并表征聚集的链 在高温下积累， 研究早期中间体的构象， 6）纯化tsf突变体和tsf/抑制子-tsf 用于协同X射线衍射的双突变蛋白质， 拉曼光谱、体外重折叠和差示 扫描量热法研究。