Integration of hydrogen/deuterium exchange and cyanylation-based methodology for conformational studies of cystinyl proteins

Analytical Biochemistry
Xue LiJ Throck Watson

Abstract

This report documents the feasibility and advantages of integrating hydrogen/deuterium exchange (HDX) methodology with cyanylation (CN)-based methodology to determine the conformation of cystinyl proteins and intermediates during refolding. The CN-based methodology can be used to trap, identify, and preserve the disulfide structure of a given cystinyl protein folding intermediate, while the HDX methodology can be used to assess other conformational features of the intermediate. Specifically, in this study, CN-based methodology was used to trap a 1-disulfide bond and a 2-disulfide intermediate of long Arg(3) insulin-like growth factor-I (LR(3)IGF-I), which was then exposed to HDX using D(2)O at pD 6.8 and subsequently digested with pepsin before analysis by matrix-assisted laser desorption/ionization mass spectrometry. The HDX results show an increasing degree of secondary and tertiary structure as a function of disulfide bond formation. In addition, the HDX results for two overlapping peptic fragments suggest that a segment of the polypeptide exists in two conformations, which can be distinguished by HDX and pepsin. These results from HDX mass spectrometry are in reasonably good agreement with those from nuclear magnetic resona...Continue Reading

References

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