Inhibiting Functional Amyloid Formation Using Protein Engineering

Amyloids are a class of protein assembly known for their extreme stability and ordered, fibrous structure. Functional amyloids are a subclass of amyloids that can be found in all domains of cellular life. Functional amyloids fulfill key biological roles that help the organism that produce them grow and thrive in their environment. Functional amyloids can act as structural scaffolds, storage mechanisms, antibacterial agents, and major components of microbial biofilms. The most well studied functional amyloid is curli, the predominant protein component of the E. coli biofilm. The major protein subunit of curli is a fast-aggregating amyloid protein called CsgA. E. coli secretes intrinsically disordered CsgA to the outside of the cell where it can then fold into an amyloid-competent state and form fibers. Though CsgA has a high propensity to form amyloid fibers its polymerization can be controlled through different biochemical mechanisms. In this dissertation I focused on two ways that functional amyloid formation is controlled, both in vivo and in vitro. E. coli uses an amyloid inhibitor protein called CsgC to control CsgA aggregation within the cell. CsgC is a periplasmic chaperone-like protein that maintains CsgA in in an intrinsically disordered state. In collaboration with the Olofsson (Umeå University, Sweden) and Ruotolo (University of Michigan) labs, I investigated the interaction between CsgC and CsgA. We observed a 1:1 heterodimeric complex of CsgC:CsgA that suggested that CsgC interacts with monomers to keep CsgA unfolded and non-amyloidogenic. We found that after interacting transiently with CsgC, CsgA monomers aggregate much more slowly than compared to a negative control. To learn more about how CsgC interacts with CsgA, I developed an in vivo screening assay that was able to identify CsgC residues that are important for structural stability and amyloid inhibition activity. Controlling CsgA aggregation in vitro is also a useful research endeavor. Upon purification, intrinsically disordered CsgA proteins immediately begin oligomerizing and within a few hours stable, mature amyloid fibers will have formed. To aid the study of amyloid proteins, I created a CsgA variant called CsgACC that remains monomeric and unfolded while oxidized. When CsgACC is incubated under reducing conditions, an intramolecular disulfide bond is broken, and the protein adopts the amyloid conformation. Therefore, the aggregation propensity of CsgACC can be manipulated by the addition of a reducing agent. CsgACC will enable scientists to control amyloid formation in ways that were never possible before, hopefully leading to insights into how aggregation occurs. In addition, an amyloid protein that responds to a reducing agent to begin polymerization could prove to be a useful building block for constructing complex structures using functional amyloid fibers as the core structural element.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/175607/1/abalist_1.pd