The research proposal outlined in this application is consisted of two components designed to facilitate the transition of the principal investigator (PI) to an independent research career, a career development plan and a research plan. The PI has a multidisciplinary background, and through this proposal, seeks to finalize his training in Structural Biology of Membrane Proteins. The career development plan comprises a structured educational experience for the first two years of the award (K99 phase) that includes training in Electron Microscopy and Electron Paramagnetic Resonance (EPR) Spectroscopy for the study of Membrane Proteins, and enhancement of career skills, such as grant writing, laboratory management, teaching and responsible conduct of research. It includes a clear and actionable plan for identifying and successfully competing for an independent tenure-track faculty position by the end of the K99 phase. The PI has assembled a multi- disciplinary team of mentors, advisors and collaborators that will oversee and guide his training, research program and transition to independence. The research plan spans both the mentored (K99) and independent (R00) phases of the award. It involves mechanistic studies of the aminoarabinose sugar biosynthetic pathway in Gram-negative bacteria that is linked to resistance to polymyxin-class antibiotics, our last line of defense against multi-drug resistant infections. The research program for the K99 phase aims to build on knowledge gained from a recent solution structure of an enzyme in the aminoarabinose pathway, named ArnT, by the PI and his colleagues. The independent (R00) research program then aims to extend the structural studies to other transmembrane enzymes of the aminoarabinose pathway, working towards a complete structural description and functional characterization of the pathway. The core questions that the research program aims to address are: (1) How does the enzyme ArnT accommodate its two lipidic substrates within the context of its fold? (2) What are the molecular determinants that impart stereospecificity to some members of the ArnT enzyme family but not others? (3) How is the ArnT enzyme changing during substrate binding and catalysis? (4) How do the other transmembrane enzymes in the pathway look and how do they accomplish their functions? (5) How do other transmembrane enzymes in the pathway accommodate the lipidic sugar carrier undecaprenyl phosphate? To address these questions, the PI has put together a comprehensive research plan that utilizes state-of-the-art methodologies. Moreover, the plan includes some technology development aimed at extending the capabilities of electron microscopy within the context of the research program, and which, upon completion of the program, may be applicable to other research programs. The proposed studies for the K99 phase will largely take place in the prominent environment of Columbia University, which harbors a vibrant structural biology community. This environment is certain to facilitate the PI in the successful completion of the proposed research program and achieving his goal of transitioning into an independent research career.
The World Health Organization has identified antibiotic resistance as one the three greatest threats to human health and emphasized that it is no longer a concern for the future, but a present and rapidly growing global health challenge. Our last line of defense antibiotics, polymyxins, are used to treat multi-drug resistant infections, however, incidence of resistance to polymyxins is growing and the modifications that cause it are poorly understood in mechanistic terms. This project aims to investigate the molecular mechanisms responsible for polymyxin resistance and its impact lies within its potential to inform the design of therapeutic compounds capable of countering resistance to polymyxins.
