TY - JOUR
T1 - Deamidation Slows Curli Amyloid-Protein Aggregation
AU - Wang, Hanliu
AU - Shu, Qin
AU - Frieden, Carl
AU - Gross, Michael L.
N1 - Funding Information:
We thank Dr. Chris Becker from Protein Metrics for his help with the deamidation quantification feature in Byologics. This work was supported by NIH National Institutes of General Medical Sciences: 2P41GM103422 to M.L.G. and 1R01AI099099 to C.F. Instrumentation was provided by the NIH 1S10OD016298.
Funding Information:
#Analytical Research and Development, Pfizer Inc., Chesterfield, MO 63017, United States Notes The authors declare no competing financial interest.
Publisher Copyright:
© 2017 American Chemical Society.
PY - 2017/6/13
Y1 - 2017/6/13
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85020743805&partnerID=8YFLogxK
U2 - 10.1021/acs.biochem.7b00241
DO - 10.1021/acs.biochem.7b00241
M3 - Article
C2 - 28497950
AN - SCOPUS:85020743805
SN - 0006-2960
VL - 56
SP - 2865
EP - 2872
JO - Biochemistry
JF - Biochemistry
IS - 23
ER -