Receptors for retinoic acid act as ligand activated transcription factors. The three-dimensional structure of the retinoid X receptor (RXR) ligand binding domain has been determined, but little information is available concerning the properties of the protein in solution. Hydrogen/deuterium exchange followed by electrospray ionization mass spectrometry was used to probe the solution conformation of the recombinant human RXRalpha homodimer ligand binding domain in the presence and absence of 9-cis-retinoic acid (9-cis-RA). Within the experimental time domain (0.25-180 min), about 20 amide hydrogens showed decreased exchange rates in the presence of saturating concentrations of 9-cis-RA as compared to those found for the homodimer in the absence of ligand. Most of the amides were located in peptides derived from regions of the protein shown by the X-ray structure to interact with the bound ligand: the amino termini of helices 3 and 9, the two beta sheets, helix 8, the H8-H9 loop, and the carboxyl terminus of helix 11. Unexpectedly, protection was also observed in peptides derived from helices 7, 10, 11, and the H7-H8 and H10-H11 loops, regions that are not directly in contact with bound 9-cis-RA. These results suggest that the binding of ligand results in additional effects on the conformation or dynamics of the homodimer in solution as compared to those observed for the X-ray structure. Overall, the change in deuterium exchange induced by the binding of 9-cis-RA correlated reasonably well with changes in hydrogen bonding, residue depth, and/or solvent accessibility predicted from the crystal structure.

Electrospray ionization (ESI) mass spectrometry (MS) isThe globular or braided topology with attendant surface crevices and interior cavities creates a unique three-dimensional structure for proteins and, together with their dynamic properties mediated through local fluctuations or large-scale conformational changes, the singular functions of these lifesustaining biomolecules are executed. Conformations and conformational dynamics are key to the protein's functional integrity and these characteristic properties are highly dependent on environmental conditions. Thus, varying physiological conditions may play an important role in protein conformational changes. For example, pH adjustments in organisms can drive ligand-receptor dissociation and receptor recycling (1). Protein conformational changes are also manifest in the protein life cycles from expression to final post-activity phase, i.e. birth, function, and death (2), and improper protein conformations may be responsible for a number of diseases (3). Detailed information is required on protein higher-order structures and dynamics to fully understand how proteins perform their biological functions at the molecular level.Protein conformational changes can be monitored by conventional biophysical and analytical methods, including circular dichroism (CD) 1 (4), tryptophan fluorescence (5), and infrared spectroscopy (6). Far-ultraviolet (UV) CD spectroscopy, for instance, provides information mainly on secondary structural elements of the polypeptide chains; near-UV-CD detects changes in the tertiary structure around aromatic amino acid side chains, and fluorescence techniques may be another more sensitive technique for this purpose. The low-resolving power inherent in these methods, however, limits the investigator's ability to focus on regions where conformational changes might occur. Detailed structural information at individual residues of a protein can, of course, be obtained from From the

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Studies with the homodimeric recombinant human macrophage colony-stimulating factor beta (rhM-CSF␤), show for the first time that a large number (9) of disulfide linkages can be reduced after amide hydrogen/ deuterium (H/D) exchange, and the protein digested and analyzed successfully for the isotopic composition by electrospray mass spectrometry. Analysis of amide H/D after exchange-in shows that in solution the conserved four-helix bundle of (rhM-CSF␤) has fast and moderately fast exchangeable sections of amide hydrogens in the ␣A helix, and mostly slow exchanging sections of amide hydrogens in the ␣B, ␣C, and ␣D helices. Most of the amide hydrogens in the loop between the ␤1 and ␤4 sheets exhibited fast or moderately fast exchange, whereas in the amino acid 63-67 loop, located at the interface of the two subunits, the exchange was slow. Solvent accessibility as measured by H/D exchange showed a better correlation with the average depth of amide residues calculated from reported X-ray crystallographic data for rhM-CSF␣ than with the average B-factor. The rates of H/D exchange in rhM-CSF␤ appear to correlate well with the exposed surface calculated for each amino acid residue in the crystal structure except for the ␣D helix. Fast hydrogen isotope exchange throughout the segment amino acids 150-221 present in rhM-CSF␤, but not rhM-CSF␣, provides evidence that the carboxy-terminal region is unstructured. It is, therefore, proposed that the anomalous behavior of the ␣D helix is due to interaction of the carboxy-terminal tail with this helical segment.

Amide hydrogen/deuterium (H/D) exchange of proteins monitored by mass spectrometry has established itself as a powerful method for probing protein conformational dynamics and protein interactions. The method uses isotope labeling to probe the rate at which protein backbone amide hydrogens undergo exchange. Backbone amide hydrogen exchange rates are particularly sensitive to hydrogen bonding; hydrogen bonding slows the exchange rates dramatically. Exchange rates reflect on the conformational mobility, hydrogen bonding strength, and solvent accessibility in protein structure. Mass spectrometric techniques are used to monitor the exchange events as mass shifts that arise through the incorporation of deuterium into the protein. Global conformational information can be deduced by monitoring the exchange profiles over time. Combining the labeling experiment with proteolysis under conditions that preserve the exchange information allows for localizing exchange events to distinct regions of the protein backbone and thus, the study of protein conformation with medium spatial resolution. Over the past decade, H/D exchange mass spectrometry has evolved into a versatile technique for investigating conformational dynamics and interactions in proteins, protein-ligand and protein-protein complexes.