Infectious diseases cause widespread sickness throughout the world each year and are the second leading cause of death, particularly in underdeveloped countries. And with the emergence of multi-drug resistance strains, the necessity for new, more effective, and more sustainable therapies is immediate and vital to protect against any future pandemics. My studies will provide crucial insight into the biogenesis of surface receptors and proteins that assist pathogenic bacteria in their virulence. This knowledge will significantly assist in the development of better therapies against these infectious agents. Gram-negative bacteria contain an inner and outer membrane. The outer membrane contains a host of beta-barrel proteins commonly called outer membrane proteins (OMPs), which serve essential functions in cargo transport, signaling, and bacterial virulence. In Gram-negative bacteria, it is known that OMPs are synthesized in the cytoplasm and then transported across the inner membrane into the periplasm via a Sec translocon. Once in the periplasm, chaperones guide the nascent OMPs across the periplasm and peptidoglycan to the inner surface of the outer membrane. Here, the nascent OMPs are recognized by a complex known as the BAM complex which folds and inserts the new OMPs into the outer membrane. Exactly how the BAM complex is able to accomplish its function remains unknown. However, we do know that the BAM complex consists of five subunits named BamA (an OMP itself), BamB, BamC, BamD, and BamE, which are all lipoproteins. Studies have shown that BamA and BamD are absolutely essential for cell viability and OMP biogenesis. Our lab and others have reported the structures of BamB, BamC, BamD, BamE and a large portion of the periplasmic domain of BamA, providing initial insight into how the BAM complex may function. However, even with these structures being known, the mechanism for how the BAM complex recognizes, folds, and inserts nascent OMPs into the outer membrane remains elusive, largely due to the lack of a full length BamA structure and complexes with BamA. Recently, I determined the crystal structures of a truncated BamA construct from Haemophilus ducreyi to 2.9 ? and of a full length BamA construct from Neisseria gonorrhea to 3.2 ?. In my proposed studies, I aim to build on this recent success to use X-ray crystallography to determine structures of BamA in complex with BamB-E, to use DEER spectroscopy and crosslinking to characterize the conformational dynamics of BamA, and to use crosslinking to explore the interactions between nascent OMPs and BamA and the other BAM components, with my goal being understand the functional role of BamA within the BAM complex. I have a strong background in X-ray crystallography and during my postdoctoral studies, have added a strong background in working with and crystallizing membrane proteins using the latest technologies such as new stabilizing detergents, bicelles, and lipidic cubic phase (LCP) methods. Recently, I solved the structures of two important surface proteins from pathogenic Neisseria and this work was published in Nature as a full research article (March 2012). For this manuscript, I also solved the structure of diferric human transferrin which research groups have been trying to solve for decades without success. More recently, I also solved the crystal structure of the agonist bound neurotensin receptor NTSR1, a GPCR responsible for binding neurotensin and other neurotransmitters. This work was published in Nature as well in October 2012 (Research Article). My current efforts are focused on studying BamA of the BAM complex and I will take this project with me as a tenure-track faculty member in academia. And with this, my most recent results, the crystal structures of BamA (H. ducreyi and N. gonorrhea), were also recently published in Nature as a full research article as well (Sept 2013). My long-term goal is to have my own research lab as a faculty member at a Research I academic institution where I can continue my research interests. This has been my lifelong ambition and while funding for academic research is more competitive now than ever, I remain dedicated to a career in research with aspirations that I will be able to establish a research group that significantly advances our current understanding of beta-barrel membrane proteins in both bacteria and humans.

Public Health Relevance

Infectious diseases cause widespread sickness and death throughout the world each year, particularly in underdeveloped countries. And with the emergence of multi-drug resistance strains, the necessity for new, more effective, and more sustainable therapies is immediate. Gram-negative bacteria have an outer membrane which provides nutrients and added protection for the cell, yet the outer membrane also contains proteins that can enable pathogenic bacteria to evade host immune responses and cause a number of serious infectious diseases. Despite the importance of these outer membrane proteins for cell survival and virulence, little is known about how these proteins are folded and inserted into the outer membrane. However, we know that a complex called the BAM complex mediates the biogenesis of these membrane proteins and it is my goal is to use X-ray crystallography, electron microscopy, DEER spectroscopy, and crosslinking to characterize the role of BamA, the central and essential component of the BAM complex, to determine the mechanism for the biogenesis of beta-barrel membrane proteins in Gram-negative bacteria.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Career Transition Award (K22)
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Study Section
Microbiology and Infectious Diseases B Subcommittee (MID)
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Hiltke, Thomas J
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Purdue University
Schools of Arts and Sciences
West Lafayette
United States
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Lundquist, Karl; Bakelar, Jeremy; Noinaj, Nicholas et al. (2018) C-terminal kink formation is required for lateral gating in BamA. Proc Natl Acad Sci U S A 115:E7942-E7949
Sikora, Aleksandra E; Wierzbicki, Igor H; Zielke, Ryszard A et al. (2018) Structural and functional insights into the role of BamD and BamE within the ?-barrel assembly machinery in Neisseria gonorrhoeae. J Biol Chem 293:1106-1119
O'Neil, Patrick K; Richardson, Lynn G L; Paila, Yamuna D et al. (2017) The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts. Proc Natl Acad Sci U S A 114:E4868-E4876
Bakelar, Jeremy; Buchanan, Susan K; Noinaj, Nicholas (2017) Structural snapshots of the ?-barrel assembly machinery. FEBS J 284:1778-1786
Noinaj, Nicholas; Gumbart, James C; Buchanan, Susan K (2017) The ?-barrel assembly machinery in motion. Nat Rev Microbiol 15:197-204
Botos, Istvan; Noinaj, Nicholas; Buchanan, Susan K (2017) Insertion of proteins and lipopolysaccharide into the bacterial outer membrane. Philos Trans R Soc Lond B Biol Sci 372:
Abeykoon, Amila H; Noinaj, Nicholas; Choi, Bok-Eum et al. (2016) Structural Insights into Substrate Recognition and Catalysis in Outer Membrane Protein B (OmpB) by Protein-lysine Methyltransferases from Rickettsia. J Biol Chem 291:19962-74
Celia, Hervé; Noinaj, Nicholas; Zakharov, Stanislav D et al. (2016) Structural insight into the role of the Ton complex in energy transduction. Nature 538:60-65
Noinaj, Nicholas; Mayclin, Stephen; Stanley, Ann M et al. (2016) From Constructs to Crystals - Towards Structure Determination of ?-barrel Outer Membrane Proteins. J Vis Exp :
Bakelar, Jeremy; Buchanan, Susan K; Noinaj, Nicholas (2016) The structure of the ?-barrel assembly machinery complex. Science 351:180-6

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