CAREER: Secondary Active Membrane Transporters: Determining Protein Structure and Transport Mechanisms with a New Hybrid Simulation

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INTELLECTUAL MERIT Cell membranes are a crucial component of all biological organisms. They can protect the cell or compartments within a cell that have their own membranes from harmful compounds. Membrane transport proteins that span cell membranes can act as gatekeepers that control the influx of helpful molecules and enable the efflux of harmful molecules. Membrane transport proteins are grouped into classes based on their function; one such class, secondary active transporters (SATs), is of specific interest to this project. SATs couple the movement of a small primary substrate (protons or ions) to that of a larger substrate (sugars, amino acids, peptides). These proteins use the energy gain from the downhill movement (high to low concentration) of the primary substrate to facilitate the uphill movement (low to high concentration) of a larger substrate. Although atomic-level structures have been determined for several SATs, for a given protein typically only a single conformation in the transport cycle is known. However, transport of substrates involves significant protein structural changes that are not captured by a single conformation. The main objective of this research is to investigate the transport mechanisms and multiple protein conformational changes of several SATs with atomic-level simulations. A new atomic resolution simulation approach known as "implicit-explicit membrane simulation" will be used to enhance conformational sampling while preserving the natural transition between conformational states in SATs. Initially, this new method will be tested on the sodium-hydantoin transporter (Mhp1) because several structures in its transport cycle have been determined from x-ray diffraction. In collaboration with experimentalists, transitions between Mhp1 conformations facing inside and outside the cell will be researched, as will possible mutations that stabilize new structures, such as the inward-facing occluded structure. Studies on Mhp1 will lay the groundwork for using this new method on other SATs, especially those with only a single known protein conformation, such as lactose permease. This new simulation tool ultimately will allow studies of previously unknown transport cycle states in SATs and give experimental structural biologists insight into how these states may be stabilized. Moreover, studies of these SATs may lead to better understanding of how similar proteins in mammals, plants, and single-celled organisms transport substrates and change conformations.BROADER IMPACTS High school instructional aids will be developed to promote active student engagement and interest in molecular biology. Specifically, an educational website will be developed that focuses on three general topics, namely, proteins, cell membranes, and function of membrane proteins. This website will include text, figures, movies, and interactive applets that describe each topic. In addition, this project will continue the development of a joint undergraduate and graduate course, Molecular Modeling Methods, intended to introduce students to chemical and biomolecular aspects of molecular simulation methods. Additionally, undergraduate and graduate students, particularly those from backgrounds underrepresented in research, will be recruited for training in computational biology techniques. Key to these educational goals is proper assessment of the impact of the educational website, teaching and mentoring. Collaboration with an educational researcher experienced in developing educational tools, assessments, and questionnaires will aid in fulfilling these educational goals. Overall, implementing this educational plan will increase high school students interest in STEM careers, promote interest in computational biology, and train the next generation of science and engineering researchers.This project is jointly supported by the Cellular Processes Cluster in the Division of Molecular and Cellular Biosciences and the Chemical Theory, Models and Computational Methods program in the Chemistry Division.