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Understanding Curli Amyloid-Protein Aggregation by Hydrogen-Deuterium Exchange and Mass Spectrometry.

International journal of mass spectrometry (2017-10-24)
Hanliu Wang, Qin Shu, Don L Rempel, Carl Frieden, Michael L Gross
RESUMEN

Bacteria within Curli biofilms are protected from environmental pressures (e.g., disinfectants, antibiotics), and this is responsible for intractable infections. Understanding aggregation of the major protein component of Curli, CsgA, may uncover disease-associated amyloidogenesis mechanisms. Here, we report the application of pulsed hydrogen-deuterium exchange and mass spectrometry (HDX-MS) to study CsgA aggregation, thereby obtaining region-specific information. By following time-dependent peptide signal depletion, presumably a result of insoluble fibril formation, we acquired sigmoidal profiles that are specific for regions (region-specific) of the protein. These signal-depletion profiles not only provide an alternative aggregation measurement, but also give insight on soluble species in the aggregation. The HDX data present as bimodal isotopic distributions, one representing a highly disordered species whereas the other a well-structured one. Although the extents of deuterium uptake of the two species remain the same with time, the relative abundance of the lower mass, less-exchanged species increases in a region-specific manner. The same region-specific aggregation properties also pertain to different aggregation conditions. Although CsgA is an intrinsically disordered protein, within the fibril it is thought to consist of five imperfect β-strand repeating units (labeled R1-R5). We found that the exterior repeating units R1 and R5 have higher aggregation propensities than do the interior units R2, R3, and R4. We also employed TEM to obtain complementary information of the well-structured species. The results provide insight on aggregation and a new approach for further application of HDX-MS to unravel aggregation mechanisms of amyloid proteins.

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L-Aspartic acid β-hydroxamate, serine racemase inhibitor