Carbonell X, Villaverde A
Institut de Biologia Fonamental and Department de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Spain.
FEBS Lett. 1998 Aug 7;432(3):228-30. doi: 10.1016/s0014-5793(98)00876-x.
The tailspike protein (TSP) of bacteriophage P22 is a homotrimeric multifunctional protein responsible for cell attachment and hydrolysis of the Salmonella typhimurium host cell receptor. Despite the folding of TSP involves the formation of thermolabile intermediates, the mature protein is extremely resistant to heat and detergent denaturation. We have analyzed the thermal resistance and unfolding pathway of two mutant, functional TSPs carrying end-terminal peptide fusions. Whereas the C-terminal fusion has minor effects on the TSP stability, the presence of a 23-mer foreign peptide at the N terminus (protein ATSP) results in a significant enhancement of the thermal resistance by retarding the first transition step of the unfolding process. At 65 degrees C and in 2% SDS, the unfolding rate constant for the transition from the native to the unfolding intermediate is 9.3 x 10(-4) s(-1) for ATSP versus 1.7 x 10(-3) s(-1) for wild-type TSP. On the other hand, the electrophoretic mobility of ATSP intermediates is greatly affected, proving structural modifications induced by the fused peptide. These results suggest a critical participation of the N-terminal domain in the unfolding kinetic barriers generated during the TSP denaturation pathway.
噬菌体P22的尾刺蛋白（TSP）是一种同源三聚体多功能蛋白，负责与鼠伤寒沙门氏菌宿主细胞受体结合并水解该受体。尽管TSP的折叠涉及热不稳定中间体的形成，但其成熟蛋白对热和去污剂变性具有极强的抗性。我们分析了两种携带末端肽融合的功能性突变TSP的耐热性和去折叠途径。虽然C末端融合对TSP稳定性影响较小，但在N末端存在一个23肽的外源肽（蛋白ATSP）会通过延缓去折叠过程的第一步转变而显著增强耐热性。在65℃和2%十二烷基硫酸钠（SDS）条件下，从天然态转变为去折叠中间体的去折叠速率常数，ATSP为9.3×10⁻⁴ s⁻¹，而野生型TSP为1.7×10⁻³ s⁻¹。另一方面，ATSP中间体的电泳迁移率受到极大影响，这证明了融合肽诱导的结构修饰。这些结果表明N末端结构域在TSP变性途径中产生的去折叠动力学屏障中起关键作用。
