Native State Hydrogen Exchange-Mass Spectrometry Methods to Probe Protein Folding and Unfolding.
|Title||Native State Hydrogen Exchange-Mass Spectrometry Methods to Probe Protein Folding and Unfolding.|
|Publication Type||Journal Article|
|Year of Publication||2022|
|Authors||Malhotra P, Udgaonkar JB|
|Journal||Methods Mol Biol|
Native state hydrogen exchange (HX) methods provide high-resolution structural data on the rare and transient opening motions in proteins under native conditions. Mass spectrometry-based HX methods (HX-MS) have gained popularity because of their ability to delineate population distributions, which allow a direct determination of the mechanism of inter conversion of the partially folded states under native conditions. Various technological advancements have provided further impetus to the development of HX-MS-based experiments to study protein folding. Classical HX-MS studies use proteolytic digestion to produce fragments of the protein subsequent to HX in solution, in order to obtain structural data. New chemical fragmentation methods, which achieve the same result as proteolysis and cause minimal change to the HX pattern in the protein, provide an attractive alternative to proteolysis. Moreover, when used in conjunction with proteolysis, chemical fragmentation methods have significantly increased the structural resolution afforded by HX-MS studies, even bringing them at par with the single amino acid resolution observed in NMR-based measurements. Experiments based on one such chemical fragmentation method, electron transfer dissociation (ETD), are described in this chapter. The ETD HX-MS method is introduced using data from a protein which is inherently resistant to proteolytic digestion as example of how such an experiment can provide high-resolution structural data on the folding-unfolding transitions of the protein under native conditions.
|Alternate Journal||Methods Mol Biol|