Local structure and dynamics in proteins characterized by hydrogen exchange and mass spectrometry.
Amide hydrogen exchange rates, determined by NMR spectroscopy, have become an important tool that is often used to investigate structure and dynamics of small proteins. Recent developments in mass spectrometry and sample handling methods make possible measurement of deuterium levels at peptide amide linkages in polypeptides. The ability to make these measurements has led to development of the protein fragmentation/mass spectrometry approach for determining amide hydrogen exchange rates in short segments of intact proteins following their incubation in D2O. Partially deuterated proteins are proteolytically fragmented into peptides whose molecular weights are determined by on-line liquid chromatography/mass spectrometry. Deuterium levels, which are determined from the molecular weights of the peptic fragments, can be used to determine amide hydrogen exchange rates. Details of the protein fragmentation/mass spectrometry approach, along with a brief review of the theory of amide hydrogen exchange, are described. The ability to detect and locate minor structural differences in proteins by the protein fragmentation/mass spectrometry approach is illustrated using oxidized and reduced cytochrome c. These results show that oxidation of iron has little effect on the N- and C-terminal regions, but significantly destabilizes the interior regions of cytochrome c. The ability to detect localized unfolding in large proteins is illustrated with aldolase that was equilibrated in acid. Despite the success achieved by NMR spectroscopy for determining amide hydrogen exchange rates, mass spectrometry is advantageous because it permits studies of large proteins, requires only picomoles of protein, and provides a direct measure of structural heterogeneity.
Smith
D L
DL
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA. DLS@UNLINFO.UNL.EDU
D003903:Deuterium / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism*
D011506:Proteins; D003903:Deuterium