Hydrogen exchange mass spectrometry: what is it and what can it tell us?

Marcsisin, Sean R.; Engen, John R.

doi:10.1007/s00216-010-3556-4

Cited by 170 publications

(157 citation statements)

References 30 publications

Supporting

Mentioning

156

Contrasting

Unclassified

Order By: Relevance

“…2A). (14,15). Amides involved in hydrogen bonds through stable secondary structure, or that are substantially solvent-occluded, exchange much slower than accessible ones.…”

Section: Resultsmentioning

confidence: 99%

“…with mass spectrometry (XF-MS) and hydrogen/deuterium exchange with mass spectrometry (HDX-MS), which detect residuespecific changes (13)(14)(15), to investigate the structural changes that occur during OCP photoactivation. In conjunction with small angle X-ray scattering (SAXS), which enables characterization of global conformational changes in the solution state (16), we show that dissociation of the NTD and CTD is complete in photoactivated OCP.…”

Section: Significancementioning

confidence: 99%

See 1 more Smart Citation

Local and global structural drivers for the photoactivation of the orange carotenoid protein

Gupta

Guttman

Leverenz

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

136

206

View full text Add to dashboard Cite

Photoprotective mechanisms are of fundamental importance for the survival of photosynthetic organisms. In cyanobacteria, the orange carotenoid protein (OCP), when activated by intense blue light, binds to the light-harvesting antenna and triggers the dissipation of excess captured light energy. Using a combination of small angle X-ray scattering (SAXS), X-ray hydroxyl radical footprinting, circular dichroism, and H/D exchange mass spectrometry, we identified both the local and global structural changes in the OCP upon photoactivation. SAXS and H/D exchange data showed that global tertiary structural changes, including complete domain dissociation, occur upon photoactivation, but with alteration of secondary structure confined to only the N terminus of the OCP. Microsecond radiolytic labeling identified rearrangement of the H-bonding network associated with conserved residues and structural water molecules. Collectively, these data provide experimental evidence for an ensemble of local and global structural changes, upon activation of the OCP, that are essential for photoprotection.orange carotenoid protein | photoprotection | X-ray footprinting | hydrogen deuterium exchange | SAXS P hotosynthetic organisms have evolved a protective mechanism known as nonphotochemical quenching (NPQ) to dissipate excess energy, thereby preventing oxidative damage under high light conditions (1). In plants and algae, NPQ involves pHinduced conformation changes in membrane-embedded protein complexes and enzymatic interconversion of carotenoids (2, 3). Cyanobacteria, in contrast, use a relatively simple NPQ mechanism governed by the water soluble orange carotenoid protein (OCP). The OCP is composed of an all α-helical N-terminal domain (NTD) consisting of two discontinuous four-helix bundles and a mixed α/β C-terminal domain (CTD), which is a member of the widely distributed nuclear transport factor 2-like superfamily (Fig. S1A) (4, 5). There are two regions of interaction between the NTD and CTD (4, 5): the major interface, which buries 1,722 Å of surface area, and the interaction between the N-terminal alpha-helix (αA) and the CTD (minor interface) (Fig. S1A). A single noncovalently bound keto-carotenoid [e.g., echinenone (ECN)] spans both domains in the structure of the resting (inactive) form of the protein (OCP O ).The NTD and CTD of the OCP have discrete functions. The isolated NTD acts as an effector domain that binds to the antenna whereas the CTD has been proposed to play a sensory/regulatory role in controlling the OCP's photoprotective function (6). Exposure to blue light converts OCP O to the active (red) form, OCP R (7). OCP R is involved in protein-protein interactions with the phycobilisome (PB) (5) and the fluorescence recovery protein (FRP), which converts OCP R back to OCP O (8). The OCP R form is therefore central to the photoprotective mechanism, and determining the exact structural changes that accompany its formation are critical for a complete mechanistic understanding of the reversible quenching process in cy...

show abstract

“…2A). (14,15). Amides involved in hydrogen bonds through stable secondary structure, or that are substantially solvent-occluded, exchange much slower than accessible ones.…”

Section: Resultsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Local and global structural drivers for the photoactivation of the orange carotenoid protein

Gupta

Guttman

Leverenz

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

136

206

View full text Add to dashboard Cite

show abstract

“…HDX-MS reports on the solvent accessibility and local environment of residues by measuring the exchange rate of backbone amide protons with the deuterons of a D 2 O buffer (12,13). Decreases in exchange rates are diagnostic of buried surfaces or decreased local backbone dynamics.…”

Section: Significancementioning

confidence: 99%

Nitric oxide synthase domain interfaces regulate electron transfer and calmodulin activation

Smith¹,

Underbakke²,

Kulp

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

136

View full text Add to dashboard Cite

Nitric oxide (NO) produced by NO synthase (NOS) participates in diverse physiological processes such as vasodilation, neurotransmission, and the innate immune response. Mammalian NOS isoforms are homodimers composed of two domains connected by an intervening calmodulin-binding region. The N-terminal oxidase domain binds heme and tetrahydrobiopterin and the arginine substrate. The C-terminal reductase domain binds FAD and FMN and the cosubstrate NADPH. Although several highresolution structures of individual NOS domains have been reported, a structure of a NOS holoenzyme has remained elusive. Determination of the higher-order domain architecture of NOS is essential to elucidate the molecular underpinnings of NO formation. In particular, the pathway of electron transfer from FMN to heme, and the mechanism through which calmodulin activates this electron transfer, are largely unknown. In this report, hydrogendeuterium exchange mass spectrometry was used to map critical NOS interaction surfaces. Direct interactions between the heme domain, the FMN subdomain, and calmodulin were observed. These interaction surfaces were confirmed by kinetic studies of site-specific interface mutants. Integration of the hydrogen-deuterium exchange mass spectrometry results with computational docking resulted in models of the NOS heme and FMN subdomain bound to calmodulin. These models suggest a pathway for electron transfer from FMN to heme and a mechanism for calmodulin activation of this critical step.iNOS | NO signaling | flavin | hemoprotein N itric oxide (NO) has several essential functions in mammalian physiology. NO produced by the neuronal and endothelial nitric oxide synthase isoforms (nNOS and eNOS, respectively) initiates diverse signaling processes including vasodilation, myocardial function, and neurotransmission (1). The eNOS and nNOS isoforms are constitutively expressed and their activity responds to intracellular calcium concentrations. The inducible NOS isoform (iNOS) is transcriptionally controlled and produces NO as a cytotoxin at sites of inflammation or infection. Aberrant NO signaling contributes to a variety of diseases including stroke, hypertension, and neurodegeneration (2).Mammalian NOS isoforms are homodimeric and composed of two principal domains: the N-terminal oxidase domain and C-terminal reductase domain, which are connected by an intervening calmodulin (CaM) binding region (Fig. 1A). The N-terminal oxidase domain contains the heme and tetrahydrobiopterin cofactors and the binding site for the substrate arginine. The reductase domain is further divided into the FMN-binding subdomain and the FAD/NADPH-binding subdomains. This array of cofactors works in concert to catalyze the conversion of arginine to the intermediate N-hydroxyarginine and, ultimately, citrulline and NO. NADPH and oxygen are consumed in the process. During catalysis, electrons are shuttled from the reductase domain of one monomer to the heme domain of the opposite monomer in the homodimer (Fig. 1B) (1, 3). Electron transfer is initiat...

show abstract

“…We thus began examining whether and how different peptides alter HLA-A2 flexibility by monitoring hydrogen/deuterium exchange via proteolysis followed by mass spectrometry (HDX-MS). In HDX-MS, protein prepared in aqueous solution is incubated in 2 H 2 O and digested into fragments and deuteron incorporation for each fragment is determined via mass spectrometry (22). Increased levels of deuteration indicate that backbone amides are more accessible to exchange through greater sampling of more open, solvent-exposed states, whereas lower levels of incorporation indicate that backbone amides are protected from exchange through less frequent excursions into solvent-exposed states.…”

Section: Peptides Alter the Hydrogen/deuterium Exchange Behavior Of Tmentioning

confidence: 99%

Peptide Modulation of Class I Major Histocompatibility Complex Protein Molecular Flexibility and the Implications for Immune Recognition*

Hawse¹,

Gloor²,

Ayres³

et al. 2013

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: Peptide modulation of MHC flexibility can affect recognition by immune receptors. Results: Different peptides alter the flexibility of the MHC protein at sites around the peptide-binding groove. Conclusion: Peptide modulation of MHC flexibility is not limited to specific peptides or isolated regions. Significance: Peptide modulation of MHC flexibility indicates an extension of antigenicity from the peptide to the MHC.

show abstract

Hydrogen exchange mass spectrometry: what is it and what can it tell us?

Cited by 170 publications

References 30 publications

Local and global structural drivers for the photoactivation of the orange carotenoid protein

Local and global structural drivers for the photoactivation of the orange carotenoid protein

Nitric oxide synthase domain interfaces regulate electron transfer and calmodulin activation

Peptide Modulation of Class I Major Histocompatibility Complex Protein Molecular Flexibility and the Implications for Immune Recognition*

Contact Info

Product

Resources

About