Deuterium Exchange Research Articles

Dissecting the molecular determinants of clinical PARP1 inhibitor selectivity for tankyrase1

Poly-ADP-ribosyltransferases play a critical role in DNA repair and cell death, and poly(ADP-ribosyl) polymerase 1 (PARP1) is a particularly important therapeutic target for the treatment of breast cancer because of its synthetic lethal relationship with breast cancer susceptibility proteins 1 and 2. Numerous PARP1 inhibitors have been developed, and their efficacy in cancer treatment is attributed to both the inhibition of enzymatic activity and their ability to trap PARP1 on to the damaged DNA, which is cytotoxic. Of the clinical PARP inhibitors, talazoparib is the most effective at trapping PARP1 on damaged DNA. Biochemically, talazoparib is also suspected to be a potent inhibitor of PARP5a/b (tankyrase1/2 [TNKS1/2]), which is an important regulator of Wnt/β-catenin pathway. Here we show using competition experiments in cell lysate that, at a clinically relevant concentration, talazoparib can potentially bind and engage TNKS1. Using surface plasmon resonance, we measured the dissociation constants of talazoparib, olaparib, niraparib, and veliparib for their interaction with PARP1 and TNKS1. The results show that talazoparib has strong affinity for PARP1 as well as uniquely strong affinity for TNKS1. Finally, we used crystallography and hydrogen deuterium exchange mass spectroscopy to dissect the molecular mechanism of differential selectivity of these PARP1 inhibitors. From these data, we conclude that subtle differences between the ligand-binding sites of PARP1 and TNKS1, differences in the electrostatic nature of the ligands, protein dynamics, and ligand conformational energetics contribute to the different pharmacology of these PARP1 inhibitors. These results will help in the design of drugs to treat Wnt/β-catenin pathway–related cancers, such as colorectal cancers.

Selective hydrogen–deuterium exchange in graphitic carbon nitrides: probing the active sites for photocatalytic water splitting by solid-state NMR

By selective deuteration, the exchangeable and unexchangeable protons of the H-containing groups of g-C3N4 were differentiated, providing a way to distinguish the hydrophilic and relatively non-hydrophilic H-containing groups of g-C3N4.

Biosimilars: An Update

Biologics are medicines primarily derived from living systems and produced through recombinant DNA (rDNA) and monoclonal technologies. Generic version of biologics with improved efficacy and safety is called biosimilar. Patent and copyright expiration of biological products permits the entry of biosimilars. Synthesis of biosimilars involves two main processes, such as monoclonal antibodies and rDNA technology, and characterized by various methods such as posttranslational modification, mass spectrometry, peptide mapping, three-dimensional (high-order) structure, X-ray crystallography, ion mobility spectrometry, and hydrogen deuterium exchange mass spectrometry. Though both generic and biosimilar products follow the same regulatory approval, the requirements are not the same due to the variability in composition and instability. Hence, it is essential to develop pharmacokinetic and pharmacodynamic data to support the efficacy and safety data on biosimilars. This review summarizes the recent updates on biosimilars, synthesis, characterization, and current market status. Brief information on the role of biosimilars in multiple sclerosis is also provided in the review.

Structural basis of specific interactions of Lp-PLA2 with HDL revealed by hydrogen deuterium exchange mass spectrometry (vol 54, pg 127, 2013)

Structural basis of specific interactions of Lp-PLA2 with HDL revealed by hydrogen deuterium exchange mass spectrometry (vol 54, pg 127, 2013)

Linkage Memory in Underivatized Protonated Carbohydrates.

Carbohydrates are among the most complex class of biomolecules, and even subtle variations in their structures are attributed to diverse biological functions. Mass spectrometry has been essential for large scale glycomics and glycoproteomics studies, but the gas-phase structures and sometimes anomalous fragmentation properties of carbohydrates present long-standing challenges. Here we investigate the gas-phase properties of a panel of isomeric protonated disaccharides differing in their linkage configurations. Multiple conformations were evident for most of the structures based on their fragment ion abundances by tandem mass spectrometry, their ion mobilities in several gases, and their deuterium uptake kinetics by gas-phase hydrogen-deuterium exchange. Most notably, we find that the properties of the Y-ion fragments are characteristically influenced by the precursor carbohydrate's linkage configuration. This study reveals how protonated carbohydrate fragment ions can retain "linkage memory" that provides structural insight into their intact precursor.

Effects of drying method and excipient on the structure and physical stability of protein solids: Freeze drying vs. spray freeze drying

This study aims to determine the impacts of drying method and excipient on changes in protein structure and physical stability of model protein solids. Protein solids containing one of two model proteins (lysozyme or myoglobin) were produced with or without excipients (sucrose or mannitol) using freeze drying or spray freeze drying (SFD). The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), circular dichroism spectrometry (CD), size exclusion chromatography (SEC), BET surface area measurements and solid-state hydrogen deuterium exchange with mass spectrometry (ssHDX-MS). ssFTIR and CD could identify little to no difference in structure of the proteins in the formulation. ssHDX-MS was able to identify the population heterogeneity, which was undetectable by conventional characterization techniques of ssFTIR and CD. ssHDX-MS metrics such as Dmax and peak area showed a good correlation with the protein physical instability (loss of the monomeric peak area by size exclusion chromatography) in 90-day stability studies conducted at 40 °C for lysozyme. Higher specific surface area was associated with greater loss in monomer content for myoglobin-mannitol formulations as compared to myoglobin-only formulations. Spray freeze drying seems a viable manufacturing technique for protein solids with appropriate optimization of formulations. The differences observed within the formulations and between the processes using ssHDX-MS, BET surface area measurements and SEC in this study provide an insight into the influence of drying methods and excipients on protein physical stability.

Hydrogen-deuterium exchange mass spectrometry identifies spatially distinct antibody epitopes on domain III of the Zika virus envelope protein.

Zika Virus (ZIKV) has become a global public health concern because it causes fetal microcephaly and other neurological complications in humans. Currently, there are no approved treatments or vaccines for ZIKV infection. We describe here the detailed epitopes for six monoclonal antibodies (mAbs) that bind to domain III of the envelope protein of ZIKV, some of which have therapeutic potential. We show that by using hydrogen-deuterium exchange mass spectrometry (HDX-MS), we can identify three spatially distinct epitopes for the six mAbs investigated. The HDX-MS approach identified epitopes for three mAbs that agreed well with recently reported X-ray crystallography data. The HDX-MS determined epitopes for the other three anti-ZIKV mAbs for which there were no crystal structures, and the epitopes were confirmed by structure-guided mutagenesis and biolayer interferometry (BLI) competition binding assay. Our results have implications for the design of vaccine and antibody therapeutics against ZIKV and demonstrate the use of HDX-MS as a rapid and valid approach for epitope mapping.

Structure and dynamics of an \u03b1-fucosidase reveal \ta mechanism for highly efficient IgG transfucosylation

Fucosylation is important for the function of many proteins with biotechnical and medical applications. Alpha-fucosidases comprise a large enzyme family that recognizes fucosylated substrates with diverse α-linkages on these proteins. Lactobacillus casei produces an α-fucosidase, called AlfC, with specificity towards α(1,6)-fucose, the only linkage found in human N-glycan core fucosylation. AlfC and certain point mutants thereof have been used to add and remove fucose from monoclonal antibody N-glycans, with significant impacts on their effector functions. Despite the potential uses for AlfC, little is known about its mechanism. Here, we present crystal structures of AlfC, combined with mutational and kinetic analyses, hydrogen–deuterium exchange mass spectrometry, molecular dynamic simulations, and transfucosylation experiments to define the molecular mechanisms of the activities of AlfC and its transfucosidase mutants. Our results indicate that AlfC creates an aromatic subsite adjacent to the active site that specifically accommodates GlcNAc in α(1,6)-linkages, suggest that enzymatic activity is controlled by distinct open and closed conformations of an active-site loop, with certain mutations shifting the equilibrium towards open conformations to promote transfucosylation over hydrolysis, and provide a potentially generalizable framework for the rational creation of AlfC transfucosidase mutants.

The substrate specificity of the human TRAPPII complex\u2019s Rab-guanine nucleotide exchange factor activity

The TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.

Developments towards an ELM-free pedestal radiative cooling scenario using noble gas seeding in ASDEX Upgrade

Integrated edge-localized-mode (ELM)-free or small ELM scenarios for the demonstration fusion power plant (DEMO) are investigated in ASDEX Upgrade using argon seeding for radiative power removal mainly in the pedestal region. An important aspect is the modification of the electron pressure in the pedestal by the additional radiative power losses. Full ELM suppression could be achieved in a no-ELM H-mode scenario featuring an edge electromagnetic quasicoherent mode up to a heating power of 5 MW, where argon radiation allowed the extension of the heating power operational space. At higher powers up to 12 MW (reaching the beta limit), ELMs of reduced size prevail and detachment is obtained by the argon seeding. Control of the position of a radiating zone localized inside the X-point was found to be favorable compared to the control of the separatrix power for low /. Integration of pedestal cooling with Ar and ELM suppression by resonant magnetic perturbations (RMP) allowed an increase of the core radiation and a partial recovery of normalized confinement to H98 = 1. This favorable behavior is finally limited by the loss of the RMP density pump-out effect, followed by an ELM-free H-mode phase and re-occurrence of ELMs. For an active tailoring of the pedestal pressure profile, precise knowledge of the radiation profile is required. Modeling of the argon radiation profile with the STRAHL code showed good agreement with bolometry only if charge exchange of neutral deuterium with argon ions was taken into account, highlighting the importance of the neutral density in the pedestal region. In view of best integration of a no-ELM scenario with divertor detachment and high heating power, the divertor compression and enrichment of argon and neon were compared using dynamic gas puff experiments. Argon shows more than a factor of 3 higher divertor enrichment compared to neon, but the absolute values decrease with higher neutral deuterium pressure.

Isotopic Exchange Extends Charge Carrier Lifetime in Metal Lead Perovskites by Quantum Dynamics Simulations.

One may expect that isotopic exchange has no influence on charge carrier lifetime and perovskite solar cell performance because isotopic effects do not affect the fundamental electronic structure of materials. Experiments defy this expectation. By performing nonadiabatic (NA) molecular dynamics simulations, we demonstrate that hydrogen and deuterium exchange significantly enhances the excited-state lifetime and stability of CH3NH3PbI3. Replacing lighter hydrogen with heavier deuterium suppresses the collective motions of organic and inorganic components, thus enhancing lattice stiffness and decreasing the NA coupling. Isotopic exchange further reduces NA coupling by localizing electron wave functions for separation of electrons and holes, which beats the extended coherence time, slowing down nonradiative electron-hole recombination from CH3ND3PbI3 to CD3ND3PbI3 with respect to the pristine system. The unchanged fundamental electronic structure together with the prolonged carrier lifetime and enhanced stability rationalize the improvement of the deuterated CH3NH3PbI3 solar cells. Our work provides valuable insights into isotope effects for the design of high-performance perovskite photovoltaic and optoelectronic devices.

High-throughput hydrogen deuterium exchange mass spectrometry (HDX-MS) coupled with subzero-temperature ultrahigh pressure liquid chromatography (UPLC) separation for complex sample analysis

Hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) is a powerful technique for the characterization of protein dynamics and protein interactions. Recent technological developments in the HDX-MS field, such as sub-zero LC separations, large-scale data analysis tools, and efficient protein digestion methods, have allowed for the application of HDX-MS to the analysis of multi protein systems in addition to pure protein analysis. Still, high-throughput HDX-MS analysis of complex samples is not widespread because the co-elution of peptides combined with increased peak complexity after labeling makes peak de-convolution extremely difficult. Here, for the first time, we evaluated and optimized long gradient subzero-temperature ultra-high-pressure liquid chromatography (UPLC) separation conditions for the HDX-MS analysis of complex protein samples such as E. coli cell lysate digest. Under the optimized conditions, we identified 1419 deuterated peptides from 320 proteins at −10 °C, which is about 3-fold more when compared with a 15-min gradient separation under the same conditions. Interestingly, our results suggested that the peptides eluted late in the gradient are well-protected by peptide-column interactions at −10 °C so that peptides eluted even at the end of the gradient maintain high levels of deuteration. Overall, our study suggests that the optimized, sub-zero, long-gradient UPLC separation is capable of characterizing thousands of peptides in a single HDX-MS analysis with low back-exchange rates. As a result, this technique holds great potential for characterizing complex samples such as cell lysates using HDX-MS.

A Disorder-to-Order Transition Activates an ATP-Independent Membrane Protein Chaperone

The 43 kDa subunit of the chloroplast signal recognition particle, cpSRP43, is an ATP-independent chaperone essential for the biogenesis of the light harvesting chlorophyll-binding proteins (LHCP), the most abundant membrane protein family on earth. cpSRP43 is activated by a stromal factor, cpSRP54, to more effectively capture and solubilize LHCPs. The molecular mechanism underlying this chaperone activation is unclear. Here, a combination of hydrogen–deuterium exchange, electron paramagnetic resonance, and NMR spectroscopy experiments reveal that a disorder-to-order transition of the ankyrin repeat motifs in the substrate binding domain of cpSRP43 drives its activation. An analogous coil-to-helix transition in the bridging helix, which connects the ankyrin repeat motifs to the cpSRP54 binding site in the second chromodomain, mediates long-range allosteric communication of cpSRP43 with its activating binding partner. Our results provide a molecular model to explain how the conformational dynamics of cpSRP43 enables regulation of its chaperone activity and suggest a general mechanism by which ATP-independent chaperones with cooperatively folding domains can be regulated.

Interaction interface in the C-terminal parts of centriole proteins Sas6 and Ana2.

The centriole is a ninefold symmetrical structure found at the core of centrosomes and, as a basal body, at the base of cilia, whose conserved duplication is regulated by Plk4 kinase. Plk4 phosphorylates a single serine residue at the N-terminus of Ana2 to promote Ana2's loading to the site of procentriole formation. Four conserved serines in Ana2's STAN motif are then phosphorylated by Plk4, enabling Sas6 recruitment. Crystallographic data indicate that the coiled–coil domain of Ana2 forms a tetramer but the structure of full-length Ana2 has not been solved. Here, we have employed hydrogen–deuterium exchange coupled with mass spectrometry (HDX-MS) to uncover the conformational dynamics of Ana2, revealing the high flexibility of this protein with one rigid region. To determine the elusive nature of the interaction surfaces between Ana2 and Sas6, we have confirmed complex formation between the phosphomimetic form of Ana2 (Ana2-4D) and Sas6 in vitro and in vivo. Analysis of this complex by HDX-MS identifies short critical regions required for this interaction, which lie in the C-terminal parts of both proteins. Mutational studies confirmed the relevance of these regions for the Ana2–Sas6 interaction. The Sas6 site required for Ana2 binding is distinct from the site required for Sas6 to bind Gorab and Sas6 is able to bind both these protein partners simultaneously.

Effects of Additives on Catalytic Arene C–H Activation: Study of Rh Catalysts Supported by Bis-phosphine Pincer Ligands

Hydrogen–deuterium exchange (H/D exchange) is a method commonly used for studying catalytic activation of C–H(D) bonds by transition metal complexes. In this study, a series of additives were studi...

HD-eXplosion: visualization of hydrogen-deuterium exchange data as chiclet and volcano plots with statistical filtering.

Hydrogen-Deuterium eXchange coupled to mass spectrometry is a powerful tool for the analysis of protein dynamics and interactions. Bottom-up experiments looking at deuterium uptake differences between various conditions are the most common. These produce multi-dimensional data that can be challenging to depict in a single visual format. Each user must also set significance thresholds to define meaningful differences and make these apparent in data presentation. To assist in this process, we have created HD-eXplosion, an open-source, web-based application for the generation of chiclet and volcano plots with statistical filters. HD-eXplosion fills a void in available software packages and produces customizable plots that are publication quality. The HD-eXplosion application is available at http://hd-explosion.utdallas.edu. The source code can be found at https://github.com/HD-Explosion.

A Review on Computational Approaches for Analyzing Hydrogen- Deuterium (H/D) Exchange of Proteins.

Native state Hydrogen-Deuterium (H/D) exchange method has been used to study the structures and the unfolding pathways for quite a number of proteins. The H/D exchange method is generally monitored using nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) techniques. NMR-assisted H/D exchange methods primarily monitor the residue level fluctuation of proteins, whereas MS-assisted H/D exchange methods analyze multifold ensemble conformations of proteins. In this connection, quite a large number of computational tools and algorithms have been developed for processing and analyzing huge amount of the H/D exchange data generated from these techniques. In this review, most of the freely available computational tools associated with the H/D exchange of proteins have been comprehensively reviewed and scopes to improve/ develop novel computational approaches for analyzing the H/D exchange data of proteins have also been brought into fore.

Regiodivergent Access to 2- or 3-Substituted Indanones: Catalyst-Controlled Carboacylation via C–C Bond Activation

Indanones are ubiquitous in biologically active compounds. Intramolecular hydroacylation of aldehydes and alkenes is an efficient and atom-economic route to indane rings. However, these reactions a...

Synergistic Allostery in Multiligand-Protein Interactions

Amide hydrogen-deuterium exchange mass spectrometry is powerful for describing combinatorial coupling effects of a cooperative ligand pair binding at noncontiguous sites: adenosine at the ATP-pocket and a docking peptide (PIFtide) at the PIF-pocket, on a model protein kinase PDK1. Binding of two ligands to PDK1 reveal multiple hotspots of synergistic allostery with cumulative effects greater than the sum of individual effects mediated by each ligand. We quantified this synergism and ranked these hotspots using a difference in deuteration-based approach, which showed that the strongest synergistic effects were observed at three of the critical catalytic loci of kinases: the αB-αC helices, and HRD-motif loop, and DFG-motif. Additionally, we observed weaker synergistic effects at a distal GHI-subdomain locus. Synergistic changes in deuterium exchange observed at a distal site but not at the intermediate sites of the large lobe of the kinase reveals allosteric propagation in proteins to operate through two modes. Direct electrostatic interactions between polar and charged amino acids that mediate targeted relay of allosteric signals, and diffused relay of allosteric signals through soft matter-like hydrophobic core amino acids. Furthermore, we provide evidence that the conserved β-3 strand lysine of protein kinases (Lys111 of PDK1) functions as an integrator node to coordinate allosteric coupling of the two ligand-binding sites. It maintains indirect interactions with the ATP-pocket and mediates a critical salt bridge with a glutamate (Glu130) of αC helix, which is conserved across all kinases. In summary, allosteric propagation in cooperative, dual-liganded enzyme targets is bidirectional and synergistic and offers a strategy for combinatorial drug development.

Voltage-gated potassium channel proteins and stereoselective S-nitroso-l-cysteine signaling.

S-nitroso-l-cysteine (L-CSNO) behaves as a ligand. Its soluble guanylate cyclase–independent (sGC-independent) effects are stereoselective — that is, not recapitulated by S-nitroso-d-cysteine (D-CSNO) — and are inhibited by chemical congeners. However, candidate L-CSNO receptors have not been identified. Here, we have used 2 complementary affinity chromatography assays — followed by unbiased proteomic analysis — to identify voltage-gated K+ channel (Kv) proteins as binding partners for L-CSNO. Stereoselective L-CSNO–Kv interaction was confirmed structurally and functionally using surface plasmon resonance spectroscopy; hydrogen deuterium exchange; and, in Kv1.1/Kv1.2/Kvβ2-overexpressing cells, patch clamp assays. Remarkably, these sGC-independent L-CSNO effects did not involve S-nitrosylation of Kv proteins. In isolated rat and mouse respiratory control (petrosyl) ganglia, L-CSNO stereoselectively inhibited Kv channel function. Genetic ablation of Kv1.1 prevented this effect. In intact animals, L-CSNO injection at the level of the carotid body dramatically and stereoselectively increased minute ventilation while having no effect on blood pressure; this effect was inhibited by the L-CSNO congener S-methyl-l-cysteine. Kv proteins are physiologically relevant targets of endogenous L-CSNO. This may be a signaling pathway of broad relevance.