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

许多细菌使用表面的纳米注射器将蛋白质注入宿主细胞，以促进感染。这些被注射的蛋白质被称为效应器，通常被折叠成执行其功能所需的特定三维结构。然而，它们需要通过注射器机械地展开，通过针头分泌到宿主体内，在那里它们重新折叠成正常的结构。为了促进它们的分泌，效应蛋白很容易机械地展开——它们在机械上是不稳定的——而在机械上健壮的蛋白质则不能分泌。然而，是什么使蛋白质在机械上不稳定或健壮，人们知之甚少。本项目解决了这一知识鸿沟。一组精心挑选的模型蛋白质和最先进的实验和计算工具将被用来阐明是什么使细菌纳米注射器分泌蛋白质。这将定义一个基本的细菌感染机制，也可能允许未来的工程系统将感兴趣的蛋白质注入宿主细胞。更广泛地说，该项目将通过帮助定义蛋白质机械稳定性的规则来推动该领域的发展。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训练机会。蛋白质的机械稳定性是如何被编码的——机械力的展开是如何被序列和结构调节的——人们知之甚少。该项目通过检查细菌III型分泌系统（TTSS）分泌的蛋白质（称为效应器）作为模型系统来解决这一知识差距。当其他蛋白质在分泌装置中停滞时，TTSS机械地展开并分泌其效应物。PI的团队发现，与非效应同系物相比，TTSS效应物在机械上是不稳定的。在这个项目中，他们探索了效应器已经进化到机械不稳定的假设，因此它们可以被弱的TTSS展开酶展开，解释了它们与非效应器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是如何被序列调节的。合作方法包括：(i)高精度单分子分析，以确定TTSS效应物及其非效应同源物的机械特性；（ii）引导分子动力学模拟，为保守褶皱内的微分机械稳定性机制提供理论模型；（iii）活细胞成像检测不同机械稳定性对TTSS分泌的影响。这为机械稳定性如何编码及其对ttss分泌的影响提供了一个全面、定量和生理验证的模型。本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。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.