Structural Characterization of Proteins from the E. coli β-barrel Assembly Machine using NMR Spectroscopy

Warner, Lisa Rose

Graduate Thesis Or Dissertation

Structural Characterization of Proteins from the E. coli β-barrel Assembly Machine using NMR Spectroscopy Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/cc08hf78d

Abstract

The cytosol of Gram-negative bacteria is fortified by an inner membrane and an outer membrane that are separated by ~140 Å of periplasmic space. Spanning the outer membrane are β-barrel proteins that are integral for basic physiological functions, virulence and multidrug resistance. The E. coli β-barrel assembly machine is a five protein complex responsible for the folding and insertion of β-barrel proteins into the outer membrane. How this molecular machine works in the absence of ATP or a proton gradient has remained elusive. The prevailing theory is that interactions between proteins in the complex, protein chaperones in the periplasm, and outer membrane protein precursors drive this molecular machine. The work presented here contributes to understanding the mechanics of the β-barrel assembly machine by providing a structural description of two of the components: BamC and BamA.

NMR is a powerful tool to investigate the structure and dynamics of proteins in solution. Here, a novel approach was used to determine the solutions structure of the 27 kDa BamC by combining a limited NMR dataset of chemical shifts, residual dipolar couplings (RDCs) and nuclear Overhauser effect (NOE) distance restraints with the protein fold prediction program, Rosetta. The structure of BamC was determined to consist of two helix-grip type domains connected by an ∼18-residue flexible linker. The structure was validated with a supplementary NOE NMR dataset including amide-amide ¹H-¹H and isoleucine, leucine and valine methyl methyl ¹H-¹H NOEs. Interestingly, regions of the structural ensemble that did not converge to a unique conformation also showed increased ¹⁵N backbone amide dynamics.

The domain orientation and flexibility of the periplasmic POlypeptide TRansport Associated (POTRA) domains of BamA were investigated. Solution domain orientation using RDCs validated the orientation of POTRA4–5 in a spliced crystallographic model of POTRA1–5. The flexibility of POTRA1–5 in solution was assessed with ¹⁵N amide backbone dynamic, paramagnetic relaxation enhancement and analysis of RDCs. Previous reports suggested that POTRA1–5 was comprised of two rigid units, POTRA1–2 and POTRA3–5. The results presented here indicate that POTRA1–5 may be more flexible than previously thought.

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