Dawson Jennifer E, Nicholson Linda K
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA .
Protein Sci. 2008 Jul;17(7):1109-19. doi: 10.1110/ps.034223.107.
In order to infect their hosts, many Gram-negative bacteria translocate agents of infection, called effector proteins, through the type III secretion system (TTSS) into the host cytoplasm. This process is thought to require at least partial unfolding of these agents, raising the question of how an effector protein might unfold to enable its translocation and then refold once it reaches the host cytoplasm. AvrPto is a well-studied effector protein of Pseudomonas syringae pv tomato. The presence of a readily observed unfolded population of AvrPto in aqueous solution and the lack of a known secretion chaperone make it ideal for studying the kinetic and thermodynamic characteristics that facilitate translocation. Application of Nzz exchange spectroscopy revealed a global, two-state folding equilibrium with 16% unfolded population, a folding rate of 1.8 s(-1), and an unfolding rate of 0.33 s(-1) at pH 6.1. TrAvrPto stability increases with increasing pH, with only 2% unfolded population observed at pH 7.0. The R(1) relaxation of TrAvrPto, which is sensitive to both the global anisotropy of folded TrAvrPto and slow exchange between folded and unfolded conformations, provided independent verification of the global kinetic rate constants. Given the acidic apoplast in which the pathogen resides and the more basic host cytoplasm, these results offer an intriguing mechanism by which the pH dependence of stability and slow folding kinetics of AvrPto would allow efficient translocation of the unfolded form through the TTSS and refolding into its functional folded form once inside the host.
为了感染宿主，许多革兰氏阴性菌通过III型分泌系统（TTSS）将感染因子（称为效应蛋白）转运到宿主细胞质中。据认为，这一过程要求这些因子至少部分展开，这就引出了一个问题：效应蛋白如何展开以实现其转运，然后在到达宿主细胞质后又如何重新折叠。AvrPto是丁香假单胞菌番茄致病变种中一个经过充分研究的效应蛋白。在水溶液中很容易观察到AvrPto存在未折叠状态，且缺乏已知的分泌伴侣蛋白，这使其成为研究促进转运的动力学和热力学特征的理想对象。应用Nzz交换光谱法揭示了一种全局的两态折叠平衡，在pH 6.1时，未折叠状态的比例为16%，折叠速率为1.8 s⁻¹，解折叠速率为0.33 s⁻¹。TrAvrPto的稳定性随pH升高而增加，在pH 7.0时，仅观察到2%的未折叠状态。TrAvrPto的R(1)弛豫对折叠态TrAvrPto的全局各向异性以及折叠态和未折叠态构象之间的缓慢交换均敏感，它为全局动力学速率常数提供了独立验证。鉴于病原体所在的酸性质外体和碱性更强的宿主细胞质，这些结果提供了一种有趣的机制，即AvrPto稳定性的pH依赖性和缓慢的折叠动力学允许未折叠形式通过TTSS有效转运，并在进入宿主后重新折叠成其功能性折叠形式。
