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HpmA is a hemolytic protein secreted by the bacterium Proteus mirabilis. HpmA is an example of a class of hemolytic proteins that are exported using a two-partner secretion (TPS) pathway. This pathway utilizes a B-component to recognize and transport the A-component across the outer membrane of gram-negative bacteria. During secretion by the B-component, the A-component also adopts its functional folded structure. The partner for HpmA is HpmB. This project aims to understand how the energetics of folding and the interactions between HpmA and HpmB impact the secretion of HpmA. A working model for HpmA secretion invokes a Brownian Ratchet mechanism. The Brownian portion refers to the thermal motion of the unfolded HpmA protein which results in its transport through the pore of HpmB. This is followed by folding of HpmA, the structure of which prevents reverse transport (the ratchet). Previous work showed that the TPS domain of HpmA consists of three structural domains that fold sequentially. These domains may represent individual ratchet pause points during secretion. It is thought that the full-length structure of HpmA continues the β-helix found in the TPS domain. To test the Brownian Ratchet model for secretion of the HpmA, we have created and isolated proteins that extend the TPS domain by multiples of two residues. To determine if these longer constructs have affected the domains and thus the pause points, we have determined their stability via CD-monitored Protein Denaturation. Longer proteins revealed a new denaturation transition, suggesting a new pause point for extended helices in agreement with the ratchet model. To determine which constructs best act as templates to catalyze the folding of the remainder of the proteinas it is secreted, we determined their activity via Template Assisted Hemolytic Activity (TAHA) Assays. Together, template length and location of cutoff influenced folding interactions. HpmA recognition by HpmB is determined by the TPS domain which comprises the first ∼15% of the HpmA protein (265 amino acids). HpmB is thought to identify HpmAvia polypeptide-transport-associated (POTRA) domains. These interactions are specific, as the HpmB POTRA domains recognize only HpmA and no other TPS proteins. To test which portions of HpmA lead to this specificity, we are determining the strength of interactions with POTRA domains using Surface Plasmon Resonance (SPR) experiments.These experiments allow us to determine which portions have the fastest association rates, and therefore preferentially bind to HpmB's POTRA domains. Understanding the preference and strength of HpmA-HpmB interactions will aid in determining the process of HpmA secretion. This work was funded and supported by the University of Wisconsin La Crosse Undergraduate Research and Creativity Awards.
Phelps et al. (Fri,) studied this question.