Characterizing Curli Accessory Proteins CsgE/CsgF

The term curli is used to define cell surface organelles that are present on Gram-negative bacteria such as E. coli and Salmonella. These organelles are hair-like filaments comprised of protein. The curli facilitate functions such as bacterial motility, cell aggregation, biofilm formation and host colonization. The assembly of curli involves six proteins CsgA, CsgB, CsgC, CsgE, CsgF and CsgG. CsgA and CsgB are the curli structure proteins while CsgC, CsgE, CsgF and CsgG help in proper curli assembly. It is thought that CsgE and CsgF are periplasmic chaperone proteins that help export CsgA/CsgB through the periplasm to the outer membrane. The main goal of this project was to characterize regions of CsgE/CsgF that maybe involved in the structure and/or function of curli fibers. A series of single cysteine mutants of CsgF (T23C, S44C, S66C, T97C T123C) were expressed in E.coli, purified and labeled with environment-sensitive fluorophore IAEDANS. The protein structure of the unlabeled and labeled mutants were investigated using Circular dichroism (CD) and compared with the CsgF wild type (WT). The CD spectra of CsgF WT contained a broad negative peak based around 218 nm while the unlabeled mutants S44C, S66C and T23C had negative peaks at 210, 216 and 208 nm respectively. The labeled mutants S44C, S66C and T23C contained negative peaks centered at approximately 218, 210 and 206 nm respectively. Structural differences were observed in the unlabeled T23C,S44C and labeled S66C, T23C compared to the WT. This could be due to protein denaturation or because these sites influenced the structure of the protein. Protein bioinformatics analysis was conducted to identify significant conserved regions that maybe important structure and/or function for CsgE. The residues that were absolutely conserved were found to be located at the positions 33,37,44,47,48,51,71,76,78,109 and 134 which may be used to elucidate the mechanistic details of curli assembly. Future direction - mutation of the residues at positions 33,51,71 and 76 to cysteines. Cysteine is the selected residue as it is known to have role in stabilizing protein structure.