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The exchange of water hydroxyl hydrogen bonds between anions and water oxygens is observed directly with ultrafast 2D IR vibrational echo chemical exchange spectroscopy (CES). The OD hydroxyl stretch of dilute HOD in H₂O in concentrated (5.5 M) aqueous solutions of sodium tetrafluoroborate (NaBF₄) displays a spectrum with a broad water-like band (hydroxyl bound to water oxygen) and a resolved, blue shifted band (hydroxyl bound to BF[Formula: see text]). At short time (200 fs), the 2D IR vibrational echo spectrum has 4 peaks, 2 on the diagonal and 2 off-diagonal. The 2 diagonal peaks are the 0-1 transitions of the water-like band and the hydroxyl-anion band. Vibrational echo emissions at the 1-2 transition frequencies give rise to 2 off-diagonal peaks. On a picosecond time scale, additional off-diagonal peaks grow in. These new peaks arise from chemical exchange between water hydroxyls bound to anions and hydroxyls bound to water oxygens. The growth of the chemical exchange peaks yields the time dependence of anion-water hydroxyl hydrogen bond switching under thermal equilibrium conditions as Taw = 7 ± 1 ps. Pump-probe measurements of the orientational relaxation rates and vibrational lifetimes are used in the CES data analysis. The pump-probe measurements are shown to have the correct functional form for a system undergoing exchange.

Protein molecules naturally emit streams of information-rich signals in the language of hydrogen exchange concerning the intimate details of their stability, dynamics, function, changes therein, and effects thereon, all resolved to the level of their individual amino acids. The effort to measure protein hydrogen exchange behavior, understand the underlying chemistry and structural physics of hydrogen exchange processes, and use this information to learn about protein properties and function has continued for 50 years. Recent work uses mass spectrometric analysis together with an earlier proteolytic fragmentation method to extend the hydrogen exchange capability to large biologically interesting proteins. This article briefly reviews the advances that have led us to this point and the understanding that has so far been achieved.

Gas-phase hydrogen/deuterium exchange measured by mass spectrometry (gas-phase HDX-MS) is a fast method to probe the conformation of protein ions. The use of gas-phase HDX-MS to investigate the structure and interactions of protein complexes is however mostly unharnessed. Ionizing proteins under conditions that maximize preservation of their native structure (native MS) enables the study of solution-like conformation for milliseconds after electrospray ionization (ESI), which enables the use of ND 3 -gas inside the mass spectrometer to rapidly deuterate heteroatom-bound non-amide hydrogens. Here, we explored the utility of gas-phase HDX-MS to examine protein-protein complexes and inform on their binding surface and the structural consequences of gas-phase dissociation. Protein complexes ranging from 24 kDa dimers to 395 kDa 24mers were analyzed by gas-phase HDX-MS with subsequent collision-induced dissociation (CID). The number of exchangeable sites involved in complex formation could, therefore, be estimated. For instance, dimers of cytochrome c or α-lactalbumin incorporated less deuterium/subunit than their unbound monomer counterparts, providing a measure of the number of heteroatom-bound side-chain hydrogens involved in complex formation. We furthermore studied if asymmetric charge-partitioning upon dissociation of protein complexes caused intermolecular H/D migration. In larger multimeric protein complexes, the dissociated monomer showed a significant increase in deuterium. This indicates that intermolecular H/D migration occurs as part of the asymmetric partitioning of charge during CID. We discuss several models that may explain this increase deuterium content and find that a model where only deuterium involved in migrating charge can account for most of the deuterium enrichment observed on the ejected monomer. In summary, the deuterium content of the ejected subunit can be used to estimate that of the intact complex with deviations observed for large complexes accounted for by charge migration. Graphical abstract ᅟ

Hydrogen/deuterium exchange (HDX) methods generate useful information on protein structure and dynamics, ideally at the individual residue level. Most MS-based HDX methods involve a rapid proteolytic digestion followed by LC/MS analysis, with exchange kinetics monitored at the peptide level. Localizing specific sites of HDX is usually restricted to a resolution the size of the host peptide because gas-phase processes can scramble deuterium throughout the peptide. Subtractive methods may improve resolution, where deuterium levels of overlapping and nested peptides are used in a subtractive manner to localize exchange to smaller segments. In this study, we explore the underlying assumption of the subtractive method, namely, that the measured back exchange kinetics of a given residue is independent of its host peptide. Using a series of deuterated peptides, we show that secondary structure can be partially retained under quenched conditions, and that interactions between peptides and reversed-phase LC columns may both accelerate and decelerate residue HDX, depending upon peptide sequence and length. Secondary structure is induced through column interactions in peptides with a solution-phase propensity for structure, which has the effect of slowing HDX rates relative to predicted random coil values. Conversely, column interactions can orient random-coil peptide conformers to accelerate HDX, the degree to which correlates with peptide charge in solution, and which can be reversed by using stronger ion pairing reagents. The dependency of these effects on sequence and length suggest that subtractive methods for improving structural resolution in HDX-MS will not offer a straightforward solution for increasing exchange site resolution. Figure ᅟ

Chromatographic carry-over can severely distort measurements of amide H/D exchange in proteins analyzed by LC/MS. In this work, we explored the origin of carry-over in the online digestion of an IgG1 monoclonal antibody using an immobilized pepsin column under quenched H/D exchange conditions (pH 2.5, 0 °C). From a consensus list of 169 different peptides consistently detected during digestion of this large, ~150 kDa protein, approximately 30 % of the peptic peptides exhibited carry-over. The majority of carry-over originates from the online digestion. Carry-over can be substantially decreased by washing the online digestion flow-path and pepsin column with two wash cocktails: [acetonitrile (5 %)/ isopropanol (5 %)/ acetic acid (20 %) in water] and [2 M guanidine hydrochloride in 100 mM phosphate buffer pH 2.5]. Extended use of this two-step washing procedure does not adversely affect the specificity or activity of the immobilized pepsin column. The results suggest that although the mechanism of carry-over appears to be chemical in nature, and not hydrodynamic, carry-over cannot be attributed to a single factor such as mass, abundance, pI, or hydrophobicity of the peptides.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

Mutations in the E3 ubiquitin ligase parkin (PARK2, also known as PRKN) and the protein kinase PINK1 (also known as PARK6) are linked to autosomal-recessive juvenile parkinsonism (AR-JP) 1 , 2 ; at the cellular level, these mutations cause defects in mitophagy, the process that organizes the destruction of damaged mitochondria 3 , 4 . Parkin is autoinhibited, and requires activation by PINK1, which phosphorylates Ser65 in ubiquitin and in the parkin ubiquitin-like (Ubl) domain. Parkin binds phospho-ubiquitin, which enables efficient parkin phosphorylation; however, the enzyme remains autoinhibited with an inaccessible active site 5 , 6 . It is unclear how phosphorylation of parkin activates the molecule. Here we follow the activation of full-length human parkin by hydrogen–deuterium exchange mass spectrometry, and reveal large-scale domain rearrangement in the activation process, during which the phospho-Ubl rebinds to the parkin core and releases the catalytic RING2 domain. A 1.8 Å crystal structure of phosphorylated human parkin reveals the binding site of the phospho-Ubl on the unique parkin domain (UPD), involving a phosphate-binding pocket lined by AR-JP mutations. Notably, a conserved linker region between Ubl and the UPD acts as an activating element (ACT) that contributes to RING2 release by mimicking RING2 interactions on the UPD, explaining further AR-JP mutations. Our data show how autoinhibition in parkin is resolved, and suggest a mechanism for how parkin ubiquitinates its substrates via an untethered RING2 domain. These findings open new avenues for the design of parkin activators for clinical use. Structural mass spectrometry of full-length human parkin and a structure of the activated parkin core reveal large-scale domain rearrangements involved in activation of parkin by PINK1.

The human glucagon receptor, GCGR, belongs to the class B G-protein-coupled receptor family and plays a key role in glucose homeostasis and the pathophysiology of type 2 diabetes. Here we report the 3.0 Å crystal structure of full-length GCGR containing both the extracellular domain and transmembrane domain in an inactive conformation. The two domains are connected by a 12-residue segment termed the stalk, which adopts a β-strand conformation, instead of forming an α-helix as observed in the previously solved structure of the GCGR transmembrane domain. The first extracellular loop exhibits a β-hairpin conformation and interacts with the stalk to form a compact β-sheet structure. Hydrogen–deuterium exchange, disulfide crosslinking and molecular dynamics studies suggest that the stalk and the first extracellular loop have critical roles in modulating peptide ligand binding and receptor activation. These insights into the full-length GCGR structure deepen our understanding of the signalling mechanisms of class B G-protein-coupled receptors. The crystal structure of the full-length human glucagon receptor reveals the essential role of the 12-residue ‘stalk’ segment and an extracellular loop in the regulation of ligand binding and receptor activation. Full-length class B GPCR structures The glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR) belong to the class B G-protein-coupled receptor family and have opposing physiological roles in glucose homeostasis and insulin release. As such, they are important in regulating metabolism and appetite and offer significant treatment possibilities for type 2 diabetes. However, as yet, no full-length structures of these receptors have been solved. Three papers in this issue of Nature report the structure of GLP-1R. Ray Stevens and colleagues describe the crystal structure of the human GLP-1R transmembrane domain in an inactive state in complex with negative allosteric modulators. Fiona Marshall and colleagues describe the active-state full-length receptor in complex with truncated peptide agonists, which have potent activity in mice on oral administration. Georgios Skiniotis, Brian Kobilka and colleagues describe the cryo-electron microscopy structure of an unmodified GLP-1R in complex with its endogenous peptide ligand, GLP-1, and the heterotrimeric G protein. Finally, in a fourth paper in this week's issue of Nature , Beili Wu and colleagues report the crystal structure of the full-length GCGR in an inactive conformation. Taken together, these studies provide key insights into the activation and signalling mechanisms of class B receptors and provide therapeutic opportunities for targeting this receptor family.

G Protein Coupled Receptors (GPCRs) constitute the largest family of signalling proteins responsible for translating extracellular stimuli into intracellular functions. They play crucial roles in numerous physiological processes and are major targets for drug discovery. Dysregulation of GPCRs is implicated in various diseases, making understanding their structural dynamics critical for therapeutic development. Here, we use Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) to explore the structural dynamics of the turkey β1-adrenergic receptor (tβ1AR) bound with nine different ligands, including agonists, partial agonists, and antagonists. We find that these ligands induce distinct dynamic patterns across the receptor, which can be grouped by compound modality. Notably, full agonist binding destabilises the intracellular loop 1 (ICL1), while antagonist binding stabilises it, highlighting ICL1’s role in G protein recruitment. Our findings indicate that the conserved L72 residue in ICL1 is crucial for maintaining receptor structural integrity and stabilising the GDP-bound state. Overall, our results provide a platform for determining drug modality and highlight how HDX-MS can be used to dissect receptor ligand interaction properties and GPCR mechanism. GPCRs are vital drug targets. Here, authors use HDX-MS to gain insight into structural dynamics of β1AR upon ligand binding, revealing agonist-induced destabilisation and antagonist stabilisation of ICL1.

Secondary transporters undergo structural rearrangements to catalyze substrate translocation across the cell membrane – yet how such conformational changes happen within a lipid environment remains poorly understood. Here, we combine hydrogen-deuterium exchange mass spectrometry (HDX-MS) with molecular dynamics (MD) simulations to understand how lipids regulate the conformational dynamics of secondary transporters at the molecular level. Using the homologous transporters XylE, LacY and GlpT from Escherichia coli as model systems, we discover that conserved networks of charged residues act as molecular switches that drive the conformational transition between different states. We reveal that these molecular switches are regulated by interactions with surrounding phospholipids and show that phosphatidylethanolamine interferes with the formation of the conserved networks and favors an inward-facing state. Overall, this work provides insights into the importance of lipids in shaping the conformational landscape of an important class of transporters. Secondary transporters catalyse substrate translocation across the cell membrane but the role of lipids during the transport cycle remains unclear. Here authors used hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations to understand how lipids regulate the conformational dynamics of secondary transporters.