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

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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.