title: Comparison of continuous and pulsed labeling amide hydrogen exchange/mass spectrometry for studies of protein dynamics.
abstract: In contrast to the rigid structures portrayed by X-ray diffraction, proteins in solution display constant motion which leads to populations that are momentarily unfolded. To begin to understand protein dynamics, we must have experimental methods for determining rates of folding and unfolding, as well as for identifying structures of folding and unfolding intermediates. Amide hydrogen exchange has become an important tool for such measurements. When urea is used to stabilize unfolded forms of proteins, the refolding rates may become slower than the rates of isotope exchange. In such cases, the intermolecular distribution of deuterium among the entire population of molecules may become bimodal, giving rise to a bimodal distribution of isotope peaks in mass spectra of the protein or its peptic fragments. When the protein is exposed continuously to D2O, the relative intensities of the two envelopes of isotope peaks give an integrated account of populations participating in the folding/unfolding process. However, when the protein is exposed only briefly to D2O, the relative intensities of the two envelopes of isotope peaks give an instantaneous measure of the folded/unfolded populations. Application of these two labeling methods to a large protein, aldolase, is described along with a discussion of specific parameters required to optimize these experiments.
authors.affiliation: Department of Chemistry, University of Nebraska, Lincoln 68588-0304, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Deng
authors.forename: Z
authors.initials: Z
authors.lastname: Zhang
authors.forename: D L
authors.initials: DL
authors.lastname: Smith
mesh_terms: D000577:Amides / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
chemical_list: D000577:Amides; D010446:Peptide Fragments; D011506:Proteins; D006859:Hydrogen