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Keywords = hydrogen–deuterium exchange mass spectrometry

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28 pages, 5658 KiB  
Review
Mechanistic Insights into the Mutational Landscape of the Main Protease/3CLPro and Its Impact on Long-Term COVID-19/SARS-CoV-2 Management
by Aganze Gloire-Aimé Mushebenge, Samuel Chima Ugbaja, Nonjabulo Ntombikhona Magwaza, Nonkululeko Avril Mbatha, Tambwe Willy Muzumbukilwa, Mukanda Gedeon Kadima, Fave Yohanna Tata, Mthokosizi Bongani Nxumalo, Riziki Ghislain Manimani, Ntabaza Ndage, Bakari Salvius Amuri, Kahumba Byanga, Manimbulu Nlooto, Rene B. Khan and Hezekiel M. Kumalo
Future Pharmacol. 2024, 4(4), 825-852; https://doi.org/10.3390/futurepharmacol4040044 - 28 Nov 2024
Viewed by 982
Abstract
The main proteinase (Mpro), or 3CLpro, is a critical enzyme in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lifecycle and is responsible for breaking down and releasing vital functional viral proteins crucial for virus development and transmission. As a catalytically active dimer, [...] Read more.
The main proteinase (Mpro), or 3CLpro, is a critical enzyme in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lifecycle and is responsible for breaking down and releasing vital functional viral proteins crucial for virus development and transmission. As a catalytically active dimer, its dimerization interface has become an attractive target for antiviral drug development. Recent research has extensively investigated the enzymatic activity of Mpro, focusing on its role in regulating the coronavirus replication complex and its significance in virus maturation and infectivity. Computational investigations have identified four druggable pockets, suggesting potential allosteric sites beyond the substrate-binding region. Empirical validation through site-directed alanine mutagenesis has targeted residues in both the active and allosteric regions and corroborated these predictions. Structural studies of drug target proteins can inform therapeutic approaches, with metadynamics simulations shedding light on the role of H163 in regulating Mpro function and providing insights into its dynamic equilibrium to the wild-type enzyme. Despite the efficacy of vaccines and drugs in mitigating SARS-CoV-2 spread, its ongoing viral evolution, selective pressures, and continued transmission pose challenges, potentially leading to resistant mutations. Phylogenetic analyses have indicated the existence of several resistant variations predating drug introduction to the human population, emphasizing the likelihood of drug spread. Hydrogen/deuterium-exchange mass spectrometry reveals the structural influence of the mutation. At the same time, clinical trials on 3CLPro inhibitors underscore the clinical significance of reduced enzymatic activity and offer avenues for future therapeutic exploration. Understanding the implications of 3CLPro mutations holds promise for shaping forthcoming therapeutic strategies against COVID-19. This review delves into factors influencing mutation rates and identifies areas warranting further investigation, providing a comprehensive overview of Mpro mutations, categorization, and terminology. Moreover, we examine their associations with clinical outcomes, illness severity, unresolved issues, and future research prospects, including their impact on vaccine efficacy and potential therapeutic targeting. Full article
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19 pages, 5159 KiB  
Article
Peptide Activator Stabilizes DJ-1 Structure and Enhances Its Activity
by Jing-Yuan Shih and Yuan-Hao Howard Hsu
Int. J. Mol. Sci. 2024, 25(20), 11075; https://doi.org/10.3390/ijms252011075 - 15 Oct 2024
Viewed by 671
Abstract
DJ-1 is a vital enzyme involved in the maintenance of mitochondrial health, and its mutation has been associated with an increased risk of Parkinson’s disease (PD). Effective regulation of DJ-1 activity is essential for the well-being of mitochondria, and DJ-1 is thus a [...] Read more.
DJ-1 is a vital enzyme involved in the maintenance of mitochondrial health, and its mutation has been associated with an increased risk of Parkinson’s disease (PD). Effective regulation of DJ-1 activity is essential for the well-being of mitochondria, and DJ-1 is thus a potential target for PD drug development. In this study, two peptides (15EEMETIIPVDVMRRA29 and 47SRDVVICPDA56) were utilized with the aim of enhancing the activity of DJ-1. The mechanisms underlying the activity enhancement by these two peptides were investigated using hydrogen/deuterium exchange mass spectrometry (HDXMS). The HDXMS results revealed distinct mechanisms. Peptide 1 obstructs the access of solvent to the dimer interface and stabilizes the α/β hydrolase structure, facilitating substrate binding to a stabilized active site. Conversely, peptide 2 induces a destabilization of the α/β hydrolase core, enhancing substrate accessibility and subsequently increasing DJ-1 activity. The binding of these two peptides optimizes the activity site within the dimeric structure. These findings offer valuable insights into the mechanisms underlying the activity enhancement of DJ-1 by the two peptides, potentially aiding the development of new drugs that can enhance the activity of DJ-1 and, consequently, advance PD treatment. Full article
(This article belongs to the Special Issue Biomolecular Structure, Function and Interactions)
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15 pages, 2512 KiB  
Article
Protein Microarrays for High Throughput Hydrogen/Deuterium Exchange Monitored by FTIR Imaging
by Joëlle De Meutter and Erik Goormaghtigh
Int. J. Mol. Sci. 2024, 25(18), 9989; https://doi.org/10.3390/ijms25189989 - 16 Sep 2024
Viewed by 779
Abstract
Proteins form the fastest-growing therapeutic class. Due to their intrinsic instability, loss of native structure is common. Structure alteration must be carefully evaluated as structural changes may jeopardize the efficiency and safety of the protein-based drugs. Hydrogen deuterium exchange (HDX) has long been [...] Read more.
Proteins form the fastest-growing therapeutic class. Due to their intrinsic instability, loss of native structure is common. Structure alteration must be carefully evaluated as structural changes may jeopardize the efficiency and safety of the protein-based drugs. Hydrogen deuterium exchange (HDX) has long been used to evaluate protein structure and dynamics. The rate of exchange constitutes a sensitive marker of the conformational state of the protein and of its stability. It is often monitored by mass spectrometry. Fourier transform infrared (FTIR) spectroscopy is another method with very promising capabilities. Combining protein microarrays with FTIR imaging resulted in high throughput HDX FTIR measurements. BaF2 slides bearing the protein microarrays were covered by another slide separated by a spacer, allowing us to flush the cell continuously with a flow of N2 gas saturated with 2H2O. Exchange occurred simultaneously for all proteins and single images covering ca. 96 spots of proteins that could be recorded on-line at selected time points. Each protein spot contained ca. 5 ng protein, and the entire array covered 2.5 × 2.5 mm2. Furthermore, HDX could be monitored in real time, and the experiment was therefore not subject to back-exchange problems. Analysis of HDX curves by inverse Laplace transform and by fitting exponential curves indicated that quantitative comparison of the samples is feasible. The paper also demonstrates how the whole process of analysis can be automatized to yield fast analyses. Full article
(This article belongs to the Special Issue Protein Structure Research 2024)
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20 pages, 3270 KiB  
Article
Chemical Synthesis and Structure–Activity Relationship Studies of the Coagulation Factor Xa Inhibitor Tick Anticoagulant Peptide from the Hematophagous Parasite Ornithodoros moubata
by Vincenzo De Filippis, Laura Acquasaliente, Andrea Pierangelini and Oriano Marin
Biomimetics 2024, 9(8), 485; https://doi.org/10.3390/biomimetics9080485 - 12 Aug 2024
Viewed by 1297
Abstract
Tick Anticoagulant Peptide (TAP), a 60-amino acid protein from the soft tick Ornithodoros moubata, inhibits activated coagulation factor X (fXa) with almost absolute specificity. Despite TAP and Bovine Pancreatic Trypsin Inhibitor (BPTI) (i.e., the prototype of the Kunitz-type protease inhibitors) sharing a [...] Read more.
Tick Anticoagulant Peptide (TAP), a 60-amino acid protein from the soft tick Ornithodoros moubata, inhibits activated coagulation factor X (fXa) with almost absolute specificity. Despite TAP and Bovine Pancreatic Trypsin Inhibitor (BPTI) (i.e., the prototype of the Kunitz-type protease inhibitors) sharing a similar 3D fold and disulphide bond topology, they have remarkably different amino acid sequence (only ~24% sequence identity), thermal stability, folding pathways, protease specificity, and even mechanism of protease inhibition. Here, fully active and correctly folded TAP was produced in reasonably high yields (~20%) by solid-phase peptide chemical synthesis and thoroughly characterised with respect to its chemical identity, disulphide pairing, folding kinetics, conformational dynamics, and fXa inhibition. The versatility of the chemical synthesis was exploited to perform structure–activity relationship studies on TAP by incorporating non-coded amino acids at positions 1 and 3 of the inhibitor. Using Hydrogen–Deuterium Exchange Mass Spectrometry, we found that TAP has a remarkably higher conformational flexibility compared to BPTI, and propose that these different dynamics could impact the different folding pathway and inhibition mechanisms of TAP and BPTI. Hence, the TAP/BPTI pair represents a nice example of divergent evolution, while the relative facility of TAP synthesis could represent a good starting point to design novel synthetic analogues with improved pharmacological profiles. Full article
(This article belongs to the Special Issue Biomimetic Approaches in Healthcare—Innovations Inspired by Nature)
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17 pages, 2672 KiB  
Article
Formulation Development of a COVID-19 Recombinant Spike Protein-Based Vaccine
by Emily Xiao, Clémentine Mirabel, Didier Clénet, Shaolong Zhu, Andrew James, Luciano Ettorre, Trevor Williams, Jason Szeto, Nausheen Rahman and Salvador Fernando Ausar
Vaccines 2024, 12(8), 830; https://doi.org/10.3390/vaccines12080830 - 23 Jul 2024
Viewed by 1310
Abstract
The purpose of this study was to develop a formulation for a recombinant prefusion spike protein vaccine against SARS-CoV-2. It was found that the spike protein was susceptible to aggregation due to mechanical stress. Therefore, formulation studies were initiated focused on screening pharmaceutical [...] Read more.
The purpose of this study was to develop a formulation for a recombinant prefusion spike protein vaccine against SARS-CoV-2. It was found that the spike protein was susceptible to aggregation due to mechanical stress. Therefore, formulation studies were initiated focused on screening pharmaceutical excipients capable of preventing this. The screening of a panel of potential stabilizing conditions found that Tween 20 could inhibit mechanically induced aggregation. A concentration-dependent study indicated that a higher concentration of Tween 20 (0.2% v/v) was required to prevent conformational changes in the trimer. The conformational changes induced by mechanical stress were characterized by size exclusion chromatography (SEC) and hydrogen–deuterium exchange mass spectrometry (HDX-MS), indicating the formation of an extended trimeric conformation that was also unable to bind to antibodies directed to the S2 domain. Long-term stability modeling, using advanced kinetic analysis, indicated that the formulation containing 0.2% (v/v) Tween 20 at a neutral pH was predicted to be stable for at least two years at 2 °C to 8 °C. Additional stabilizer screening conducted by thermal shift assay indicated that sucrose and glycerol were able to significantly increase the spike protein melting temperature (Tm) and improve the overall thermostability of the spike protein in a short-term stability study. Thus, while 0.2% (v/v) Tween 20 was sufficient to prevent aggregation and to maintain spike protein stability under refrigeration, the addition of sucrose further improved vaccine thermostability. Altogether, our study provides a systematic approach to the formulation of protein-based COVID-19 vaccine and highlights the impact of mechanical stress on the conformation of the spike protein and the significance of surfactants and stabilizers in maintaining the structural and functional integrity of the spike protein. Full article
(This article belongs to the Section COVID-19 Vaccines and Vaccination)
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21 pages, 3608 KiB  
Article
Characterization of the Charge Heterogeneity of a Monoclonal Antibody That Binds to Both Cation Exchange and Anion Exchange Columns under the Same Binding Conditions
by Ming-Ching Hsieh, Jingming Zhang, Liangjie Tang, Cheng-Yen Huang, Yang Shen, Alice Matathia, Jun Qian and Babita Saxena Parekh
Antibodies 2024, 13(3), 52; https://doi.org/10.3390/antib13030052 - 30 Jun 2024
Viewed by 1899
Abstract
Therapeutic antibodies play an important role in the public healthcare system to treat patients with a variety of diseases. Protein characterization using an array of analytical tools provides in-depth information for drug quality, safety, efficacy, and the further understanding of the molecule. A [...] Read more.
Therapeutic antibodies play an important role in the public healthcare system to treat patients with a variety of diseases. Protein characterization using an array of analytical tools provides in-depth information for drug quality, safety, efficacy, and the further understanding of the molecule. A therapeutic antibody candidate MAB1 exhibits unique binding properties to both cation and anion exchange columns at neutral pH. This uniqueness disrupts standard purification processes and necessitates adjustments in manufacturing. This study identifies that the charge heterogeneity of MAB1 is primarily due to the N-terminal cyclization of glutamine to pyroglutamine and, to a lesser extent, succinimide intermediate, deamidation, and C-terminal lysine. Using three approaches, i.e., deferential chemical labeling, H/D exchange, and molecular modeling, the binding to anion exchange resins is attributed to negatively charged patches on the antibody’s surface, involving specific carboxylic acid residues. The methodologies shown here can be extended to study protein binding orientation in column chromatography. Full article
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20 pages, 2693 KiB  
Article
A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer
by Nora D. Mineva, Stefania Pianetti, Sonia G. Das, Srimathi Srinivasan, Nicolas M. Billiald and Gail E. Sonenshein
Pharmaceutics 2024, 16(4), 536; https://doi.org/10.3390/pharmaceutics16040536 - 13 Apr 2024
Cited by 3 | Viewed by 2038
Abstract
New targeted treatments are urgently needed to improve triple-negative breast cancer (TNBC) patient survival. Previously, we identified the cell surface protein A Disintegrin And Metalloprotease 8 (ADAM8) as a driver of TNBC tumor growth and spread via its metalloproteinase and disintegrin (MP and [...] Read more.
New targeted treatments are urgently needed to improve triple-negative breast cancer (TNBC) patient survival. Previously, we identified the cell surface protein A Disintegrin And Metalloprotease 8 (ADAM8) as a driver of TNBC tumor growth and spread via its metalloproteinase and disintegrin (MP and DI) domains. In proof-of-concept studies, we demonstrated that a monoclonal antibody (mAb) that simultaneously inhibits both domains represents a promising therapeutic approach. Here, we screened a hybridoma library using a multistep selection strategy, including flow cytometry for Ab binding to native conformation protein and in vitro cell-based functional assays to isolate a novel panel of highly specific human ADAM8 dual MP and DI inhibitory mAbs, called ADPs. The screening of four top candidates for in vivo anti-cancer activity in an orthotopic MDA-MB-231 TNBC model of ADAM8-driven primary growth identified two lead mAbs, ADP2 and ADP13. Flow cytometry, hydrogen/deuterium exchange–mass spectrometry (HDX-MS) and alanine (ALA) scanning mutagenesis revealed that dual MP and DI inhibition was mediated via binding to the DI. Further testing in mice showed ADP2 and ADP13 reduce aggressive TNBC characteristics, including locoregional regrowth and metastasis, and improve survival, demonstrating strong therapeutic potential. The continued development of these mAbs into an ADAM8-targeted therapy could revolutionize TNBC treatment. Full article
(This article belongs to the Special Issue Advances in Anticancer Agent, 2nd Edition)
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15 pages, 3672 KiB  
Article
Higher-Order Structure of Adeno-Associated Virus Serotype 8 by Hydrogen/Deuterium Exchange Mass Spectrometry
by Tomohiko Ikeda, Yuki Yamaguchi, Hiroaki Oyama, Aoba Matsushita, Yasuo Tsunaka, Mitsuko Fukuhara, Tetsuo Torisu and Susumu Uchiyama
Viruses 2024, 16(4), 585; https://doi.org/10.3390/v16040585 - 10 Apr 2024
Cited by 2 | Viewed by 2304
Abstract
The higher-order structure (HOS) is a critical quality attribute of recombinant adeno-associated viruses (rAAVs). Evaluating the HOS of the entire rAAV capsid is challenging because of the flexibility and/or less folded nature of the VP1 unique (VP1u) and VP1/VP2 common regions, which are [...] Read more.
The higher-order structure (HOS) is a critical quality attribute of recombinant adeno-associated viruses (rAAVs). Evaluating the HOS of the entire rAAV capsid is challenging because of the flexibility and/or less folded nature of the VP1 unique (VP1u) and VP1/VP2 common regions, which are structural features essential for these regions to exert their functions following viral infection. In this study, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used for the structural analysis of full and empty rAAV8 capsids. We obtained 486 peptides representing 85% sequence coverage. Surprisingly, the VP1u region showed rapid deuterium uptake even though this region contains the phospholipase A2 domain composed primarily of α-helices. The comparison of deuterium uptake between full and empty capsids showed significant protection from hydrogen/deuterium exchange in the full capsid at the channel structure of the 5-fold symmetry axis. This corresponds to cryo-electron microscopy studies in which the extended densities were observed only in the full capsid. In addition, deuterium uptake was reduced in the VP1u region of the full capsid, suggesting the folding and/or interaction of this region with the encapsidated genome. This study demonstrated HDX-MS as a powerful method for probing the structure of the entire rAAV capsid. Full article
(This article belongs to the Special Issue Adeno-Associated Virus (AAV) for Gene Therapy)
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14 pages, 2983 KiB  
Article
Epitope Mapping of Japanese Encephalitis Virus Neutralizing Antibodies by Native Mass Spectrometry and Hydrogen/Deuterium Exchange
by Jagat Adhikari, James Heffernan, Melissa Edeling, Estefania Fernandez, Prashant N. Jethva, Michael S. Diamond, Daved H. Fremont and Michael L. Gross
Biomolecules 2024, 14(3), 374; https://doi.org/10.3390/biom14030374 - 20 Mar 2024
Viewed by 2459
Abstract
Japanese encephalitis virus (JEV) remains a global public health concern due to its epidemiological distribution and the existence of multiple strains. Neutralizing antibodies against this infection have shown efficacy in in vivo studies. Thus, elucidation of the epitopes of neutralizing antibodies can aid [...] Read more.
Japanese encephalitis virus (JEV) remains a global public health concern due to its epidemiological distribution and the existence of multiple strains. Neutralizing antibodies against this infection have shown efficacy in in vivo studies. Thus, elucidation of the epitopes of neutralizing antibodies can aid in the design and development of effective vaccines against different strains of JEV. Here, we describe a combination of native mass spectrometry (native-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to complete screening of eight mouse monoclonal antibodies (MAbs) against JEV E-DIII to identify epitope regions. Native-MS was used as a first pass to identify the antibodies that formed a complex with the target antigen, and it revealed that seven of the eight monoclonal antibodies underwent binding. Native mass spectra of a MAb (JEV-27) known to be non-binding showed broad native-MS peaks and poor signal, suggesting the protein is a mixture or that there are impurities in the sample. We followed native-MS with HDX-MS to locate the binding sites for several of the complex-forming antibodies. This combination of two mass spectrometry-based approaches should be generally applicable and particularly suitable for screening of antigen–antibody and other protein–protein interactions when other traditional approaches give unclear results or are difficult, unavailable, or need to be validated. Full article
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16 pages, 2209 KiB  
Article
Hydrophilic Interaction Liquid Chromatography–Hydrogen/Deuterium Exchange–Mass Spectrometry (HILIC-HDX-MS) for Untargeted Metabolomics
by Tomas Cajka, Jiri Hricko, Stanislava Rakusanova, Kristyna Brejchova, Michaela Novakova, Lucie Rudl Kulhava, Veronika Hola, Michaela Paucova, Oliver Fiehn and Ondrej Kuda
Int. J. Mol. Sci. 2024, 25(5), 2899; https://doi.org/10.3390/ijms25052899 - 1 Mar 2024
Cited by 3 | Viewed by 2175
Abstract
Liquid chromatography with mass spectrometry (LC-MS)-based metabolomics detects thousands of molecular features (retention time–m/z pairs) in biological samples per analysis, yet the metabolite annotation rate remains low, with 90% of signals classified as unknowns. To enhance the metabolite annotation rates, [...] Read more.
Liquid chromatography with mass spectrometry (LC-MS)-based metabolomics detects thousands of molecular features (retention time–m/z pairs) in biological samples per analysis, yet the metabolite annotation rate remains low, with 90% of signals classified as unknowns. To enhance the metabolite annotation rates, researchers employ tandem mass spectral libraries and challenging in silico fragmentation software. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) may offer an additional layer of structural information in untargeted metabolomics, especially for identifying specific unidentified metabolites that are revealed to be statistically significant. Here, we investigate the potential of hydrophilic interaction liquid chromatography (HILIC)-HDX-MS in untargeted metabolomics. Specifically, we evaluate the effectiveness of two approaches using hypothetical targets: the post-column addition of deuterium oxide (D2O) and the on-column HILIC-HDX-MS method. To illustrate the practical application of HILIC-HDX-MS, we apply this methodology using the in silico fragmentation software MS-FINDER to an unknown compound detected in various biological samples, including plasma, serum, tissues, and feces during HILIC-MS profiling, subsequently identified as N1-acetylspermidine. Full article
(This article belongs to the Section Biochemistry)
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17 pages, 4558 KiB  
Article
Recognition of Aedes aegypti Mosquito Saliva Protein LTRIN by the Human Receptor LTβR for Controlling the Immune Response
by Su Ning Loh, Ian Russell Anthony, Edem Gavor, Xin Shan Lim, R. Manjunatha Kini, Yu Keung Mok and J. Sivaraman
Biology 2024, 13(1), 42; https://doi.org/10.3390/biology13010042 - 12 Jan 2024
Viewed by 2287
Abstract
Salivary proteins from mosquitoes have received significant attention lately due to their potential to develop therapeutic treatments or vaccines for mosquito-borne diseases. Here, we report the characterization of LTRIN (lymphotoxin beta receptor inhibitor), a salivary protein known to enhance the pathogenicity of ZIKV [...] Read more.
Salivary proteins from mosquitoes have received significant attention lately due to their potential to develop therapeutic treatments or vaccines for mosquito-borne diseases. Here, we report the characterization of LTRIN (lymphotoxin beta receptor inhibitor), a salivary protein known to enhance the pathogenicity of ZIKV by interrupting the LTβR-initiated NF-κB signaling pathway and, therefore, diminish the immune responses. We demonstrated that the truncated C-terminal LTRIN (ΔLTRIN) is a dimeric protein with a stable alpha helix-dominant secondary structure, which possibly aids in withstanding the temperature fluctuations during blood-feeding events. ΔLTRIN possesses two Ca2+ binding EF-hand domains, with the second EF-hand motif playing a more significant role in interacting with LTβR. Additionally, we mapped the primary binding regions of ΔLTRIN on LTβR using hydrogen–deuterium exchange mass spectrometry (HDX-MS) and identified that 91QEKAHIAEHMDVPIDTSKMSEQELQFHY118 from the N-terminal of ΔLTRIN is the major interacting region. Together, our studies provide insight into the recognition of LTRIN by LTβR. This finding may aid in a future therapeutic and transmission-blocking vaccine development against ZIKV. Full article
(This article belongs to the Section Biochemistry and Molecular Biology)
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19 pages, 3926 KiB  
Review
Significance of Histidine Hydrogen–Deuterium Exchange Mass Spectrometry in Protein Structural Biology
by Masaru Miyagi and Takashi Nakazawa
Biology 2024, 13(1), 37; https://doi.org/10.3390/biology13010037 - 9 Jan 2024
Viewed by 3226
Abstract
Histidine residues play crucial roles in shaping the function and structure of proteins due to their unique ability to act as both acids and bases. In other words, they can serve as proton donors and acceptors at physiological pH. This exceptional property is [...] Read more.
Histidine residues play crucial roles in shaping the function and structure of proteins due to their unique ability to act as both acids and bases. In other words, they can serve as proton donors and acceptors at physiological pH. This exceptional property is attributed to the side-chain imidazole ring of histidine residues. Consequently, determining the acid-base dissociation constant (Ka) of histidine imidazole rings in proteins often yields valuable insights into protein functions. Significant efforts have been dedicated to measuring the pKa values of histidine residues in various proteins, with nuclear magnetic resonance (NMR) spectroscopy being the most commonly used technique. However, NMR-based methods encounter challenges in assigning signals to individual imidazole rings and require a substantial amount of proteins. To address these issues associated with NMR-based approaches, a mass-spectrometry-based method known as histidine hydrogen–deuterium exchange mass spectrometry (His-HDX-MS) has been developed. This technique not only determines the pKa values of histidine imidazole groups but also quantifies their solvent accessibility. His-HDX-MS has proven effective across diverse proteins, showcasing its utility. This review aims to clarify the fundamental principles of His-HDX-MS, detail the experimental workflow, explain data analysis procedures and provide guidance for interpreting the obtained results. Full article
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12 pages, 8915 KiB  
Article
Enhancement of SARS-CoV-2 Infection via Crosslinking of Adjacent Spike Proteins by N-Terminal Domain-Targeting Antibodies
by Tina Lusiany, Tohru Terada, Jun-ichi Kishikawa, Mika Hirose, David Virya Chen, Fuminori Sugihara, Hendra Saputra Ismanto, Floris J. van Eerden, Songling Li, Takayuki Kato, Hisashi Arase, Matsuura Yoshiharu, Masato Okada and Daron M. Standley
Viruses 2023, 15(12), 2421; https://doi.org/10.3390/v15122421 - 13 Dec 2023
Cited by 1 | Viewed by 2023
Abstract
The entry of SARS-CoV-2 into host cells is mediated by the interaction between the spike receptor-binding domain (RBD) and host angiotensin-converting enzyme 2 (ACE2). Certain human antibodies, which target the spike N-terminal domain (NTD) at a distant epitope from the host cell binding [...] Read more.
The entry of SARS-CoV-2 into host cells is mediated by the interaction between the spike receptor-binding domain (RBD) and host angiotensin-converting enzyme 2 (ACE2). Certain human antibodies, which target the spike N-terminal domain (NTD) at a distant epitope from the host cell binding surface, have been found to augment ACE2 binding and enhance SARS-CoV-2 infection. Notably, these antibodies exert their effect independently of the antibody fragment crystallizable (Fc) region, distinguishing their mode of action from previously described antibody-dependent infection-enhancing (ADE) mechanisms. Building upon previous hypotheses and experimental evidence, we propose that these NTD-targeting infection-enhancing antibodies (NIEAs) achieve their effect through the crosslinking of neighboring spike proteins. In this study, we present refined structural models of NIEA fragment antigen-binding region (Fab)–NTD complexes, supported by molecular dynamics simulations and hydrogen–deuterium exchange mass spectrometry (HDX-MS). Furthermore, we provide direct evidence confirming the crosslinking of spike NTDs by NIEAs. Collectively, our findings advance our understanding of the molecular mechanisms underlying NIEAs and their impact on SARS-CoV-2 infection. Full article
(This article belongs to the Special Issue Molecular Epidemiology of SARS-CoV-2: 2nd Edition)
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10 pages, 1775 KiB  
Article
Antibody Binding Captures High Energy State of an Antigen: The Case of Nsp1 SARS-CoV-2 as Revealed by Hydrogen–Deuterium Exchange Mass Spectrometry
by Ravi Kant, Nawneet Mishra and Michael L. Gross
Int. J. Mol. Sci. 2023, 24(24), 17342; https://doi.org/10.3390/ijms242417342 - 11 Dec 2023
Cited by 1 | Viewed by 1178
Abstract
We describe an investigation using structural mass spectrometry (MS) of the impact of two antibodies, 15497 and 15498, binding the highly flexible SARS-CoV-2 Nsp1 protein. We determined the epitopes and paratopes involved in the antibody–protein interactions by using hydrogen–deuterium exchange MS (HDX-MS). Notably, [...] Read more.
We describe an investigation using structural mass spectrometry (MS) of the impact of two antibodies, 15497 and 15498, binding the highly flexible SARS-CoV-2 Nsp1 protein. We determined the epitopes and paratopes involved in the antibody–protein interactions by using hydrogen–deuterium exchange MS (HDX-MS). Notably, the Fab (Fragment antigen binding) for antibody 15498 captured a high energy form of the antigen exhibiting significant conformational changes that added flexibility over most of the Nsp1 protein. The Fab for antibody 15497, however, showed usual antigen binding behavior, revealing local changes presumably including the binding site. These findings illustrate an unusual antibody effect on an antigen and are consistent with the dynamic nature of the Nsp1 protein. Our studies suggest that this interaction capitalizes on the high flexibility of Nsp1 to undergo conformational change and be trapped in a higher energy state by binding with a specific antibody. Full article
(This article belongs to the Special Issue Virus Engineering and Applications: 2nd Edition)
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15 pages, 2839 KiB  
Review
Advances in Mass Spectrometry on Membrane Proteins
by Hsin-Chieh Yang, Weikai Li, Jie Sun and Michael L. Gross
Membranes 2023, 13(5), 457; https://doi.org/10.3390/membranes13050457 - 24 Apr 2023
Cited by 1 | Viewed by 3762
Abstract
Understanding the higher-order structure of membrane proteins (MPs), which are vital for numerous biological processes, is crucial for comprehending their function. Although several biophysical approaches have been used to study the structure of MPs, limitations exist owing to the proteins’ dynamic nature and [...] Read more.
Understanding the higher-order structure of membrane proteins (MPs), which are vital for numerous biological processes, is crucial for comprehending their function. Although several biophysical approaches have been used to study the structure of MPs, limitations exist owing to the proteins’ dynamic nature and heterogeneity. Mass spectrometry (MS) is emerging as a powerful tool for investigating membrane protein structure and dynamics. Studying MPs using MS, however, must meet several challenges including the lack of stability and solubility of MPs, the complexity of the protein–membrane system, and the difficulty of digestion and detection. To meet these challenges, recent advances in MS have engendered opportunities in resolving the dynamics and structures of MP. This article reviews achievements over the past few years that enable the study of MPs by MS. We first introduce recent advances in hydrogen deuterium exchange and native mass spectrometry for MPs and then focus on those footprinting methods that report on protein structure. Full article
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