Determining the Regions Involved in CsgE Interaction with Human IAPP

Curli are proteinaceous fibers assembled by enteric bacteria (E. coli, Salmonella). These curli fibers are thought to be involved in cell adhesion, aggregation, and the formation of biofilms. The formation of curli fibers is thought to involve six proteins: CsgA, CagB, CsgC, CsgE, CsgF, and CsgG. The major and minor structural protein of curli fibers are CsgA and CsgB respectively provide a supporting role. CsgE and CsgF are thought to be periplasmic chaperone proteins that help export CsgA/CsgB through the periplasm. It has been shown that CsgE can prevent the aggregation of CsgA in vitro, supporting the hypothesis that CsgE acts as a chaperone to prevent improper CsgA aggregation in vivo. It has also been shown that CsgE can prevent the aggregation of hIAPP, another amyloid protein unrelated to curli formation. These observations suggest that CsgE may be able to prevent the aggregation of amyloidogenic protein in general. The main goal of this project was to determine the regions of CsgE involved in interacting with Human IAPP (hIAPP). Four single cysteine mutants of CsgE (H34C, N58C, V99C, L119C) were expressed in E. Coli, purified and labeled with the environment-sensitive fluorophore IAEDANS. The interaction between CsgE and hIAPP was investigated using fluorescence quenching in the absence and presence of hIAPP. Regions of CsgE that interact with hIAPP are expected to exhibit changes in the exposure of the fluorophore to the quencher which was measured using the Stern-Volmner quenching constant (Ksv). In absence of hIAPP, Ksv values for CsgE H34C, CsgE V99C, and CsgE L119C were 14.7, 8,3 and 11.6, respectively. In the presence of hIAPP, KsV values were 6.2, 5.6 and 7.6 respectively. A significant change in quenching is observed for the fluorophore at positions CsgE H34C and CsgE L119C. This suggests that residue CsgE H34C and L119C may be involved in the association of CsgE with hIAPP.