Hydrogen-exchange mass spectrometry for the study of intrinsic disorder in proteins

Balasubramaniam, Deepa; Komives, Elizabeth A.

doi:10.1016/j.bbapap.2012.10.009

Cited by 80 publications

(67 citation statements)

References 70 publications

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“…32 Some such proteins are only unfolded until they find their binding partner, and then fold upon binding, which is reflected by a change in the protein’s deuterium exchange profile. 33–35 Many other intrinsically disordered proteins tend to aggregate or undergo oligomer formation, which is accompanied by a shift to a more ordered structure.…”

Section: Discussionmentioning

confidence: 99%

Structural Insights into Fibrinogen Dynamics Using Amide Hydrogen/Deuterium Exchange Mass Spectrometry

Marsh

Guan

et al. 2013

Biochemistry

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We determined the amide hydrogen/deuterium exchange profile of native human fibrinogen under physiologic conditions. After optimization of the quench and proteolysis conditions, more than 1,200 peptides were identified by mass spectrometry, spanning more than 90% of the constituent Aα, Bβ, and γ chain amino acid sequences. The compact central and distal globular regions of fibrinogen were well protected from deuterium exchange, with the exception of the unfolded amino-terminal segments of the Aα and Bβ chains extending from the central region, and the short γ chain “tail” extending from each distal globular region. The triple-helical coiled-coil regions, which bridge the central region to each distal region, were also well protected with the exception of a moderately fast-exchanging area in the middle of each coiled coil adjacent to the γ chain carbohydrate attachment site. These dynamic regions appear to provide flexibility to the fibrinogen molecule. The γ chain “out loop” contained within each coiled-coil also exchanged rapidly. The αC domain (Aα 392–610) exchanged rapidly, with the exception of a short segment sandwiched between a conserved disulfide linkage in the N-terminal αC subdomain. This latter finding is consistent with a mostly disordered structure for the αC domain in native fibrinogen. Analysis of the dysfibrinogen Bβ 235 Pro/Leu, which exhibits abnormal fibrin structure, revealed enhanced deuterium exchange surrounding the Pro/Leu substitution site as well as in the vicinity of the high affinity calcium binding site and the A knob polymerization pocket within the γC domain. The implication of these changes with respect to fibrin structure is discussed.

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Section: Discussionmentioning

confidence: 99%

Structural Insights into Fibrinogen Dynamics Using Amide Hydrogen/Deuterium Exchange Mass Spectrometry

Marsh

Guan

et al. 2013

Biochemistry

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“…Although mass spectrometry is primarily applicable to measuring hydrogen exchange on slower timescales (Hitchens & Bryant, 1998; Konermann et al . 2008; Zhang & Smith, 1993), fiow-quench methods have made timescales ≪1 s accessible (Balasubramaniam & Komives, 2013; Dharmasiri & Smith, 1996). Methods for measuring hydrogen exchange come in two fiavors: monitoring of hydrogen–deuterium exchange (HDX) with the solvent by either NMR or mass spectrometry (Hitchens & Bryant, 1998; Konermann et al .…”

Section: Survey Of Nmr Methods For Studying Invisible Statesmentioning

confidence: 99%

Visualizing transient dark states by NMR spectroscopy

Anthis

Clore

2015

Quart. Rev. Biophys.

206

230

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Myriad biological processes proceed through states that defy characterization by conventional atomic-resolution structural biological methods. The invisibility of these ‘dark’ states can arise from their transient nature, low equilibrium population, large molecular weight, and/or heterogeneity. Although they are invisible, these dark states underlie a range of processes, acting as encounter complexes between proteins and as intermediates in protein folding and aggregation. New methods have made these states accessible to high-resolution analysis by nuclear magnetic resonance (NMR) spectroscopy, as long as the dark state is in dynamic equilibrium with an NMR-visible species. These methods – paramagnetic NMR, relaxation dispersion, saturation transfer, lifetime line broadening, and hydrogen exchange – allow the exploration of otherwise invisible states in exchange with a visible species over a range of timescales, each taking advantage of some unique property of the dark state to amplify its effect on a particular NMR observable. In this review, we introduce these methods and explore two specific techniques – paramagnetic relaxation enhancement and dark state exchange saturation transfer – in greater detail.

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“…HDX-MS has been successfully used to probe small molecule binding to nuclear receptors [26–28]. HDX-MS reports on changes of backbone amide hydrogen exchange rates which are highly sensitive to the structural changes, conformational flexibility, hydrogen bonding strength, and solvent accessibility of protein surfaces [29].…”

Section: Introductionmentioning

confidence: 99%

Conformational modulation of the farnesoid X receptor by prenylflavonoids: Insights from hydrogen deuterium exchange mass spectrometry (HDX-MS), fluorescence titration and molecular docking studies

Yang

Broderick

Campbell

et al. 2016

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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We report on the molecular interactions of the Farnesoid X Receptor (FXR) with prenylflavonoids, an emerging class of FXR modulators. FXR is an attractive therapeutic target for mitigating metabolic syndromes (MetS) because FXR activates the inhibitory nuclear receptor, small heterodimer partner (SHP), thereby inhibiting both gluconeogenesis and de novo lipogenesis. We and others have shown that xanthohumol (XN), the principal prenylflavonoid of the hop plant (Humulus lupulus L.), is a FXR agonist based on its ability to affect lipid and glucose metabolism in vivo and to induces FXR target genes in biliary carcinoma cells and HEK293 cells. However, studies are currently lacking to rationalize the molecular mechanisms of FXR modulation by prenylflavonoids. We addressed this deficiency and report the first systematic study of FXR prenylflavonoid interactions. We combined Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) with computational studies for dissecting molecular recognition and conformational impact of prenylflavonoid interactions on the ligand binding domain (LBD) of human FXR. Four prenylflavonoids were tested: xanthohumol, a prenylated chalcone, two prenylated flavonones, namely isoxanthohumol (IX) and 8-prenylnaringenin (8PN), and a semisynthetic prenylflavonoid derivative, tetrahydroxanthohumol (TX). Enhancement of the HDX protection profile data by in silico predicted models of FXR prenylflavonoid complexes resulted in mapping of the prenylflavonoid interactions within the canonical ligand binding pocket. Our findings provide a foundation for the exploration of the chemical scaffolds of prenylated chalcones and flavanones as leads for future structure activity studies of this important nuclear receptor with potential relevance for ameliorating lipid metabolic disorders associated with obesity and MetS.

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Hydrogen-exchange mass spectrometry for the study of intrinsic disorder in proteins

Cited by 80 publications

References 70 publications

Structural Insights into Fibrinogen Dynamics Using Amide Hydrogen/Deuterium Exchange Mass Spectrometry

Structural Insights into Fibrinogen Dynamics Using Amide Hydrogen/Deuterium Exchange Mass Spectrometry

Visualizing transient dark states by NMR spectroscopy

Conformational modulation of the farnesoid X receptor by prenylflavonoids: Insights from hydrogen deuterium exchange mass spectrometry (HDX-MS), fluorescence titration and molecular docking studies

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