Josh Hoerner

Hydrogen Scrambling in the Gas Phase

Protein H/D exchange in solution (HDX) followed by protein ion desorption to the gas phase (by means of ESI) and subsequent fragmentation has been gaining prominence in recent years as a tool to study protein dynamics both in real time and in a site-specific fashion (J. Mass Spectrom., 2002, 37 557). However, the validity of the results obtained with this technique can easily be challenged due to the possibility of hydrogen scrambling within the protein ion prior to fragmentation. Such scrambling may potentially alter the deuterium content of the protein, thus rendering the results of the HDX/ESI CAD MS measurement meaningless. The goal of this work is to investigate presence and extent of hydrogen scrambling within protein ions under a variety of conditions typically used in HDX/ESI CAD MS experiments.

Relevent Publication:
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 126 (24): 7709-7717 JUN 23 2004

Structural Characterization of a Molten Globule State

Hydrogen-deuterium exchange (HDX) in the semi-correlated exchange regime (pH 7, 60% MeOH) was used in order to simultaneously probe the molten globule conformation of ubiquitin and make a comparison to its native structure. HDX simulations were used to aid in spectral interpretation of the observed HDX patterns under a variety of conditions. Local dynamics molten globule and native state were investigated using amide hydrogen exchange (HDX) electrospray ionization collision assisted dissociation mass spectrometry (ESI CAD MS) in the nozzle skimmer region of source and the extent of exchange with the existing HDX nuclear magnetic resonance (NMR) spectroscopic data. These results indicate the presence of a non-native conformation of the protein in which the two N- and C-terminal ?-sheets are the most dynamic regions of the protein. HDX CAD MS under native conditions (pH 6.8) indicates a similar phenomenon. The less dynamic regions of ubiquitin contrast with NMR protection, which may be a result of the detection of residual native structure in NMR measurements, as the detected intermediate conformer cannot be isolated from the native state.

Relevent Publication:
BIOCHEMISTRY 44 (33): 11286-11294 AUG 23 2005

Structure and Dynamics of Cellular Retinoic Acid Binding Proteins

Retinoic acid (RA), a metabolite of Vitamin A or retinol is vital for growth, maintenance, and repair of mammalian cells. The hydrophobic molecule is solubilized in the cytosol by two highly homologous proteins cellular retinoic acid binding proteins (CRABP) I and II. Despite the high degree of sequence homology and nearly identical tertiary folds, these proteins appear to have distinct function and remarkably different RA affinity. We have previously determined that the backbone dynamics is an important determinant of the ligand-binding properties of CRABP I. We are now expanding this study to include CRABP II, as well as the nuclear receptor of RA, retinoic acid receptor (RAR) to determine the mechanism of physiological RA delivery.