Xuguang Yan

Mass Spectrometric Approaches Using Electrospray Ionization Charge States and Hydrogen-Deuterium Exchange for Determining Protein Structures and Their Conformational Changes

Electrospray ionization (ESI) mass spectrometry (MS) is a powerful analytical tool for elucidating structural details of proteins in solution especially when coupled with amide hydrogen/deuterium (H/D) exchange analysis. ESI charge-state distributions and the envelopes of charges they form from proteins can provide an abundance of information on solution conformations that is not readily available through other biophysical techniques such as near ultraviolet circular dichroism (CD) and tryptophan fluorescence. The most compelling reason for the use of ESI-MS over nuclear magnetic resonance (NMR) for measuring H/D after exchange is that larger proteins and lesser amounts of samples can be studied. In addition, MS can provide structural details on transient or folding intermediates that may not be accessible by CD, fluorescence, and NMR because these techniques measure the average properties of large populations of proteins in solution. Correlations between measured H/D and calculated parameters that are often available from crystallographic data can be used to extend the range of structural details obtained on proteins. Molecular dynamics and energy minimization by simulation techniques such as assisted model building with energy refinement (AMBER) force field can be very useful in providing structural models of proteins that rationalize the experimental H/D exchange results. Charge-state envelopes and H/D exchange information from ESI-MS data used complementarily with NMR and CD data provides the most powerful approach available to understanding the structures and dynamics of proteins in solution.

Hydrogen/deuterium exchange and mass spectrometric analysis of a protein containing multiple disulfide bonds: Solution structure of recombinant macrophage colony stimulating factor-beta (rhM-CSFβ)

Studies with the homodimeric recombinant human macrophage colony‐stimulating factor beta (rhM‐CSFβ), 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‐CSFβ) has fast and moderately fast exchangeable sections of amide hydrogens in the αA helix, and mostly slow exchanging sections of amide hydrogens in the αB, αC, and αD helices. Most of the amide hydrogens in the loop between the β1 and β4 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‐CSFα than with the average B‐factor. The rates of H/D exchange in rhM‐CSFβ appear to correlate well with the exposed surface calculated for each amino acid residue in the crystal structure except for the αD helix. Fast hydrogen isotope exchange throughout the segment amino acids 150–221 present in rhM‐CSFβ, but not rhM‐CSFα, provides evidence that the carboxy‐terminal region is unstructured. It is, therefore, proposed that the anomalous behavior of the αD helix is due to interaction of the carboxy‐terminal tail with this helical segment.

Hydrogen/Deuterium Exchange Mass Spectrometry

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.

Deuterium exchange and mass spectrometry reveal the interaction differences of two synthetic modulators of RXRα LBD

Protein amide hydrogen/deuterium (H/D) exchange was used to compare the interactions of two antagonists, UVI 2112 and UVI 3003, with that of the agonist, 9‐cis‐retinoic acid, upon binding to the human retinoid X receptor alpha ligand‐binding domain (hRXRα LBD) homodimer. Analysis of the H/D content by mass spectrometry showed that in comparison to 9‐cis‐retinoic acid, the antagonists provide much greater protection toward deuterium exchange‐in throughout the protein, suggesting that the protein–antagonist complex adopts a more restricted conformation or ensemble of conformations in which solvent accesses to amide protons are reduced. A comparison between the two antagonists shows that UVI 3003 is more protective in the C‐terminal region due to the extra hydrophobic interactions derived from the atoms in the benzene ring of the carboxylic acid chain. It was less protective within regions comprising peptides 271–278 and 326–330 due to differences in conformational orientation, and/or shorter carboxylic acid chain length. Decreased deuterium exchange‐in in the segment 234–239 where the residues do not involve interactions with the ligand was observed with the two antagonists, but not with 9‐cis‐RA. The amide protons of helix 12 of the agonist‐ or antagonist‐occupied protein in solution have the same deuterium exchange rates as the unliganded protein, supporting a suggestion made previously that helix 12 can cover the occupied binding cavity only with the cofactor present to adjust its location.

Structural comparison of recombinant human macrophage colony stimulating factor β and a partially reduced derivative using hydrogen deuterium exchange and electrospray ionization mass spectrometry

Hydrogen deuterium exchange, monitored by electrospray ionization mass spectrometry, has been employed to characterize structural features of a derivative of recombinant human macrophage colony stimulating factor beta (rhm‐CSFβ) in which two of the nine disulfide bridges (Cys157/Cys159–Cys′157/Cys′159) were selectively reduced and alkylated. Removal of these two disulfide bridges did not affect the biological activity of the protein. Similarities between CD and fluorescence spectra for rhm‐CSFβ and its derivative indicate that removing the disulfide bonds did not strongly alter the overall three‐dimensional structure of rhm‐CSFβ. However, differences between deuterium exchange data of the intact proteins indicate that more NHs underwent fast deuterium exchange in the derivative than in rhm‐CSFβ. Regions located near the disulfide bond removal site were shown to exhibit faster deuterium exchange behavior in the derivative than in rhm‐CSFβ.

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometric analysis of the recombinant human macrophage colony stimulating factor β and derivatives

The potential of electrospray ionization (ESI) Fourier transform ion cyclotron mass spectrometry (FTICR-MS) to assist in the structural characterization of monomeric and dimeric derivatives of the macrophage colony stimulating factor β (rhM-CSFβ) was assessed. Mass spectrometric analysis of the 49 kDa protein required the use of sustained off-resonance irradiation (SORI) in-trap cleanup to reduce adduction. High resolution mass spectra were acquired for a fully reduced and a fully S-cyanylated monomeric derivative (∼25 kDa). Mass accuracy for monomeric derivatives was better than 5 ppm, after applying a new calibration method (i.e., DeCAL) which eliminates space charge effects upon high accuracy mass measurements. This high mass accuracy allowed the direct determination of the exact number of incorporated cyanyl groups. Collisionally induced dissociation using SORI yielded b- and y-fragment ions within the N- and C-terminal regions for the monomeric derivatives, but obtaining information on other regions required proteolytic digestion, or potentially the use of alternative dissociation methods.

EQUILIBRIUM UNFOLDING OF THE RETINOID X RECEPTOR LIGAND BINDING DOMAIN AND CHARACTERIZATION OF AN UNFOLDING INTERMEDIATE

The retinoid X receptor (RXR) is a ligand-activated transcription factor that plays an important role in growth and development and the maintenance of cellular homeostasis. A thermodynamic ultraviolet circular dichroism, tryptophan fluorescence and ligand binding activity with guanidine as a chemical denaturant are consistent with a two step mechanism. The dimeric LBD equilibrates with a monomeric intermediate (DeltaG(0)(H(2)O) equal to 8.3 kcal/mol) that is in equilibrium with the unfolded state (DeltaG(0)(H(2)O) equal to 2.8 kcal/mol). The intermediate was characterized by analytical ultracentrifugation, spectroscopy, and collisional fluorescence quenching, which imply that the monomeric intermediate maintains a high degree, but not all, of native secondary structure. Although intrinsic fluorescence from native and intermediate suggests little change in tryptophan environments, fluorescence intensities from fluorescein reporter groups differ significantly between the two structures. Analysis of the collisional quenching results imply that the intermediate is characterized by tryptophans with increased accessibility to small solutes and less overall compactness than the native protein.