Question

Curli are cell surface filaments that are found on pathogenic bacteria such as E. Coli and Salmonella. These filaments are thought the help bacterial adhere to surfaces, to facilitate bacterial interaction with cells, and to form biofilms. In a general sense, Curli are thought to help bacterial pathogenicity. Curli filaments are composed of the protein CsgA (Curli Specific Gene A). CsgA is synthesized in the cytosol (as all proteins are) and then transported (though the inner membrane, the periplasm and finally though the outer membrane) to the cell exterior where they aggregate (associate together) to form filaments. a. Aggregated CsgA exhibit characteristics similar to amyloid fibrils found in a number of human diseases (such as Alzheimers, Parkinson’s, and Huntignton’s). Almost all of the amyloid fibrils found in human diseases exhibit β-sheet secondary structure, and the proteins involved aggregate to form in-register parallel structures (i.e. structures in which each single polypeptide chain associates with another chain in a parallel fashion, and where the same residues from each chain is next to each other and where each polypeptide chain forms a strand in a large β-sheet). The structure of CsgA in its aggregated form is not known. Imagine that you are writing a proposal to NIH to determine the structure of aggregated CsgA, and to determine if CsgA forms in-register parallel structures. Propose a set of experiments to determine if CsgA form fibers with in-register parallel structures (keep in mind that amyloid do not crystalize well and that the structures are typically too large to obtain good NMR spectra). In your answer discuss : i. how you would obtain sufficient quantities of protein to carry out your studies. ii. the types of experiments you would carry out, the types of techniques you might use, as well as the type of results you would expect. b. The transport of CsgA through the periplasm is thought to be aided by a chaperon protein CsgE (since, in the absence of CsgE CsgA would aggregate inside the cell). In vitro studies have shown that CsgE is able to inhibit the aggregation of CsgA. The region of CsgE that interacts with CsgA is not known, however it has been proposed that the last 20 residues of CsgE (the C-terminal 20 residues of CsgE) may be involved in its interaction with CsgA. Propose a set of experiments to determine if the C-terminus of CsgE is involved in interacting with CsgA.

Answer 1