Single molecule studies of the bacterial curli protein CsgA reveal a structurally dynamic monomeric structure
Foster, D. A. N.; dee, d. r.
Show abstract
Functional amyloid forms the scaffold that supports bacterial communities, making them an important antimicrobial target. In particular, the subunit of curli fibrils, CsgA, has recently had its fibril assembly deciphered by cryoEM. However, the folding of CsgA has yet to be careful examined. We report the first in vitro single molecule folding trajectories of CsgA, constituting two principal modes: discrete and non-piecewise unfolding reflective of the assembly of R domains, and a blend of metastable states or of a molten globule. Moreover, extending refolding time to many seconds does not obstruct the appearance of metastable states. We also present the refolding CsgA using constant trap separation, lengthening out the process to several minutes. In order to restrain the aggregation propensity of this protein, we developed an assay to trigger native folding of CsgA under controlled conditions in a microfluidic flow cell. From there we measured the individual folding trajectories of single molecules of CsgA using force spectroscopy by optical tweezers. This work exploits an engineered disulfide trap in the protein, where native folding becomes possible after reduction of the cysteine substitutions. When folding, CsgA does not get locked into a native {beta}-hairpin assembly, but can be found in a metastable state in about a quarter of the incidences in which it is probed.
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