Probing the non-covalent structure of proteins by amide hydrogen exchange and mass spectrometry.
The rates at which hydrogens located at peptide amide linkages in proteins undergo isotopic exchange when a protein is exposed to D2O depend on whether these amide hydrogens are hydrogen bonded and whether they are accessible to the aqueous solvent. Hence, amide hydrogen exchange rates are a sensitive probe for detecting changes in protein conformation and dynamics. Hydrogen exchange rates in proteins are most often measured by NMR or Fourier transform IR spectroscopy. After a brief introduction to model kinetics used to relate amide hydrogen exchange rates to protein structure and dynamics, information required to understand and implement a new method that uses acid proteases and mass spectrometry to determine amide hydrogen exchange rates in proteins is presented. Structural and dynamic features affecting isotopic exchange rates can be detected and localized from the deuterium levels detected by mass spectrometry in proteolytic fragments of the protein. Procedures used to adjust for isotopic exchange occurring during the analysis, to extract isotope exchange rate constants from mass spectra and to link bimodal isotope patterns to protein unfolding and structural heterogeneity are also discussed. In addition, the relative merits of using mass spectrometry or NMR combined with amide hydrogen exchange to study protein structure and dynamics are discussed. The spatial resolution of hydrogen exchange results obtained by this method is typically in the range of 1-10 residues, which is substantially less than that obtained by high-resolution NMR, but sufficient to detect many functionally significant structural changes. Advantages in the areas of sensitivity, protein solubility, detection of correlated exchange and high molecular mass proteins make this approach particularly attractive for a wide range of studies.
Smith
D L
DL
Department of Chemistry, University of Nebraska-Lincoln 68588-0304, USA.
Deng
Y
Y
Zhang
Z
Z
D000577:Amides / Q000737:chemistry*; D017666:Deuterium Oxide / Q000737:chemistry; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D019061:src-Family Kinases / Q000737:chemistry
D000577:Amides; D010455:Peptides; D011506:Proteins; D006859:Hydrogen; D019061:src-Family Kinases; D005634:Fructose-Bisphosphate Aldolase; D017666:Deuterium Oxide