Collaborative Research: Understanding Protein Mechanical Stability and its Impact on Secretion

许多细菌利用其表面的纳米注射器将蛋白质注射到宿主细胞中以促进感染。这些被注射的蛋白质，称为效应物，通常被折叠成执行其功能所需的特定三维结构。然而，它们需要被机械解折叠，才能通过针头分泌到宿主体内，在那里它们重新折叠成正常结构。为了促进它们的分泌，效应蛋白很容易机械解折叠-它们是机械可解折叠的-而机械坚固的蛋白不能分泌。然而，是什么使蛋白质在机械上不稳定或坚固却知之甚少。本项目旨在解决这一知识差距。一组精心挑选的模型蛋白和最先进的实验和计算工具将被用来阐明是什么使蛋白质分泌的细菌纳米注射器。这将确定一个基本的细菌感染机制，也可能允许未来的系统工程注入到宿主细胞的蛋白质。更一般地说，该项目将通过帮助定义蛋白质机械稳定性的规则来推进该领域。生物物理和计算方法的结合为物理和生物科学的研究生和本科生提供了一个出色的交叉培训机会。蛋白质的机械稳定性是如何编码的--机械力如何通过序列和结构来调节展开--目前还知之甚少。本项目通过研究细菌III型分泌系统（TTSS）分泌的蛋白质（称为效应子）作为模型系统来填补这一知识空白。TTSS机械地展开并分泌其效应子，而其他蛋白质则停滞在分泌装置中。PI的研究小组发现，TTSS效应子与其非效应子同源物相比在机械上是不稳定的。在这个项目中，他们探索了这样一个假设，即效应子已经进化到机械不稳定，因此它们可以通过弱TTSS解折叠酶解折叠，解释了它们与非效应子同源物的极端序列差异。该系统提供了一个自然发生的模型来理解机械稳定性如何通过序列进行调节。该合作方法结合了：（i）高精度单分子测定，以确定TTSS效应子及其非效应同源物的机械特性;（ii）操纵分子动力学模拟，以提供保守折叠内差异机械稳定性机制的理论模型;和（iii）活细胞成像，以测试TTSS分泌中不同机械稳定性的影响。这提供了一个全面的，定量的，该研究由生物科学理事会分子和细胞生物科学部的分子生物物理学项目资助。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响进行评估，被认为值得支持审查标准。

Many bacteria use a nanosyringe on their surface to inject proteins into host cells to facilitate infection.These injected proteins, called effectors, are normally folded into specific three-dimensional structuresrequired to carry out their functions. However, they need to be mechanically unfolded by the syringemachinery to be secreted through the needle and into the host where they refold to their normal structure.To facilitate their secretion, effector proteins are easy to unfold mechanically—they are mechanicallylabile—whereas proteins that are mechanically robust cannot be secreted. However, what makes proteinsmechanically labile or robust is poorly understood. This project addresses this knowledge gap. A carefullychosen set of model proteins and state of the art experimental and computational tools will be used toelucidate what makes a protein secretable by bacterial nanosyringes. This will define a fundamentalbacterial infection mechanism and may also allow future engineering of the system to inject proteins ofinterest into host cells. More generally, the project will advance the field by helping define the rules forprotein mechanical stability. The combination of biophysical and computational approaches provides anoutstanding cross-training opportunity for graduate and undergraduate students in the physical andbiological sciences.How protein mechanical stability is encoded—how unfolding by mechanical force is modulated bysequence and structure—is poorly understood. This project addresses this knowledge gap by examiningproteins secreted by the bacterial Type III Secretion System (TTSS), called effectors, as model systems.The TTSS mechanically unfolds and secretes its effectors while other proteins stall in the secretionapparatus. The PI’s team discovered that TTSS effectors are mechanically labile compared to their noneffectorhomologs. In this project, they explore the hypothesis that effectors have evolved to bemechanically labile, so they can be unfolded by a weak TTSS unfoldase, explaining their extreme sequencedivergence from their non-effector homologs. The system provides a naturally occurring model tounderstand how mechanical stability is modulated by sequence. The collaborative approach combines: (i)a high-precision single molecule assay to determine mechanical properties of TTSS effectors and their noneffectorhomologs; (ii) steered molecular dynamics simulations to provide a theoretical model of themechanisms of differential mechanical stability within a conserved fold; and (iii) live-cell imaging to testthe effect of different mechanical stabilities in TTSS secretion. This provides a comprehensive, quantitative,and physiologically validated model for how mechanical stability is encoded and it impact on TTSSsecretion.This research is funded by the Molecular Biophysics program in the Division of Molecular and Cellular Biosciences in the Directorate of Biological Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.