5 years ago

Deamidation Slows Curli Amyloid-Protein Aggregation

Deamidation Slows Curli Amyloid-Protein Aggregation
Michael L. Gross, Qin Shu, Carl Frieden, Hanliu Wang
Nonenzymatic deamidation of asparagine and glutamine in peptides and proteins is a frequent modification both in vivo and in vitro. The biological effect is not completely understood, but it is often associated with protein degradation and loss of biological function. Here we describe the deamidation of CsgA, the major protein subunit of curli, which are important proteinaceous components of biofilms. CsgA has a high content of Asn and Gln, a feature seen in a few proteins that self-aggregate. We have implemented an approach to monitor deamidation rapidly by following the globally centroid mass shift, providing guidance for studies at the residue level. From the global mass measurement, we identified, using LC-MS/MS, extensive deamidation of several Asn residues and discovered three “Asn–Gly” sites to be the hottest spots for deamidation. The fibrillization of deamidated CsgA was measured using thioflavin T (ThT) fluorescence, circular dichroism (CD), and a previously reported hydrogen–deuterium exchange (HDX) platform. Deamidated proteins exhibit a longer lag phase and lower final ThT fluorescence, strongly suggesting slower and less amyloid fibril formation. CD spectra show that extensively deamidated CsgA remains unstructured and loses its ability to form amyloids. Mass-spectrometry-based HDX also shows that deamidated CsgA aggregates more slowly than wild-type CsgA. Taken together, the results show that deamidation of CsgA slows its fibrillization and disrupts its function, suggesting an opportunity to modulate CsgA fibrillization and affect curli and biofilm formation.

Publisher URL: http://dx.doi.org/10.1021/acs.biochem.7b00241

DOI: 10.1021/acs.biochem.7b00241

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