Aggregated CsgA Does Not Form In‐Register Parallel Structures

Abstract

Curli are hair‐like fibers used by the bacteria for adhesion and invasion, implicating them in the pathogenesis of bacterial disease. The main protein component of Curli is CsgA, and the main focus of our work is to characterize the structure of oligomerized CsgA. We expressed six C‐terminally histidine tagged single cysteine mutants of CsgA and used circular dichroism (CD) spectroscopy and pyrene fluorescence spectroscopic techniques to determine structural changes that occur during protein aggregation. CD spectra obtained immediately after addition of CsgA to buffer exhibit minima at approximately 200 nm, characteristic of a random coil conformation, indicating that CsgA is predominantly unstructured in solution. In contrast, CD spectra obtained after incubating CsgA in buffer exhibit minima at approximately 218 nm. These spectra are characteristic of β‐sheet conformation, indicating that CsgA undergoes a random coil to β‐sheet conformational change upon incubation. We also observed an increase in Thioflavin T fluorescence intensity upon incubation of CsgA in buffer indicating the formation of protein aggregates. Fluorescence spectra obtained of CsgA labeled at positions 24, 41, and 55 with pyrene did not contain a peak at 475 nm due to pyrene excimer formation, indicating that aggregated proteins are not arranged in an in‐register parallel fashion.Support or Funding Information1. This work is supported by NIH grant 1R15GM123430‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.