Current Research

Overview

  Caulobacter crescentus is a harmless aquatic bacterium with biotechnology potential. C. crescentus has a unique protein surface layer (S-layer) that can be engineered to display thousands of copies of any foreign peptide on its surface. My thesis will focus on how the S-layer attaches to the cell’s O-antigen. The mechanism of anchoring is likely a novel lectin-like domain. I aim to shed light on the unknown factors in this unique gram-negative S-layer system and explore and enhance the applications of surface peptide display.

Protein surface layers (S-layer) are often associated with Gram-positive bacteria and less commonly found on Gram-negative bacteria; C. cresentus is one such Gram-negative bacterium with an S-layer. C. crescentus’s S-layer is composed of a 98 kDa protein, RsaA, arranged in regular hexagonal paracrystalline lattice on the surface of the cell. The protein is secreted from the cell via a type I secretion system and then self organizes into a regular array non-covalently bound to the cell surface. Two things are required for S-layer anchoring in Caulobacter, the N-terminus of RsaA and the surface antigen, the O-antigen (SAO). Early NMR studies of the SAO have shown that the O-antigen is likely a polymer of N-acetylperosamine (N-acetyl-4,6-dideoxy-4-aminomannose), an unusual sugar in Gram-negative bacteria. The N-terminus of RsaA (first third of the protein) is the SAO binding region. The N-terminus alone can achieve binding both in vivo and in vitro. This activity, an N-acetylperosamine binding domain, has never been previously described. While we know the SAO is made of N-acetylperosamine and it is bound by the N-terminus of RsaA, we do not know the specific structure of either or how they interact with one another. I am working towards solving the mechanism of SAO binding, the structure of the N-terminus of RsaA, and the structure of SAO. Our lab has a large collection of Caulobacter species; by comparing the sequence of their S-layer proteins I will be able to identify regions of conserved homology that are important in SAO binding activity. For structure determination by X-ray crystallography, I am producing pure RsaA from Caulobacter and E. coli expression systems in parallel. My protein constructs are based on the predicted domain boundary and activity in an SAO binding assay. I am fortunate to have the support and expertise in crystallography of one of my supervisory committee members. I plan on crystallizing the N-terminal domain of RsaA alone and co-crystallizing with SAO. To date, there is no published structure of a paracrystalline S-layer protein. The O-antigen’s structure will be tackled by classical polysaccharide chemistry techniques, mass spectroscopy, and NMR analysis.