Hydrogen/deuterium exchange (HDX) followed by mass spectrometry detection (MS) provides a fast, reliable, and detailed solution for the assessment of a protein structure. It has been widely recognized as an indispensable tool and already approved by several regulatory agencies as a structural technique for the validation of protein biopharmaceuticals, including antibody-based drugs. Antibodies are of a key importance in life and medical sciences but considered to be challenging analytical targets because of their compact structure stabilized by disulfide bonds and due to the presence of glycosylation. Despite these difficulties, there are already numerous excellent studies describing MS-based antibody structure characterization. In this chapter, we describe a universal HDX-MS workflow. Deeper attention is paid to sample handling, optimization procedures, and feasibility stages, as these elements of the HDX experiment are crucial for obtaining reliable detailed and spatially well-resolved information.

• Klíčová slova
• Antibody, Biosimilars, Hydrogen/deuterium exchange, Mass spectrometry, Protein structure and dynamics, Proteolysis,
• MeSH
• deuterium MeSH
• hmotnostní spektrometrie MeSH
• protilátky * MeSH
• vodík/deuteriová výměna a hmotnostní spektrometrie * MeSH
• vodík MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• deuterium MeSH
• protilátky * MeSH
• vodík MeSH

Intrinsic protein dynamics contribute to their biological functions. Rational engineering of protein dynamics is extremely challenging with only a handful of successful examples. Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) represents a powerful technique for quantitative analysis of protein dynamics. Here we provide a detailed description of the preparation of protein samples, collection of high-quality data, and their in-depth analysis using various computational tools. We illustrate the application of HDX-MS for the study of protein dynamics in the rational engineering of flexible loops in the reconstructed ancestor of haloalkane dehalogenase and Renilla luciferase. These experiments provided unique and valuable data rigorously describing the modification of protein dynamics upon grafting of the loop-helix element. Tips and tricks are provided to stimulate the wider use of HDX-MS to study and engineer protein dynamics.

• Klíčová slova
• Ancestral luciferase, Hydrogen/deuterium exchange, LoopGrafter, Mass spectrometry, Protein dynamics, Protein engineering,
• MeSH
• deuterium chemie   MeSH
• hmotnostní spektrometrie metody   MeSH
• konformace proteinů MeSH
• vodík-deuteriová výměna * metody   MeSH
• vodík/deuteriová výměna a hmotnostní spektrometrie * MeSH
• vodík chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• deuterium MeSH
• vodík MeSH

Hydrogen/deuterium exchange (HDX) is a well-established analytical technique that enables monitoring of protein dynamics and interactions by probing the isotope exchange of backbone amides. It has virtually no limitations in terms of protein size, flexibility, or reaction conditions and can thus be performed in solution at different pH values and temperatures under controlled redox conditions. Thanks to its coupling with mass spectrometry (MS), it is also straightforward to perform and has relatively high throughput, making it an excellent complement to the high-resolution methods of structural biology. Given the recent expansion of artificial intelligence-aided protein structure modeling, there is considerable demand for techniques allowing fast and unambiguous validation of in silico predictions; HDX-MS is well-placed to meet this demand. Here we present a protocol for HDX-MS and illustrate its use in characterizing the dynamics and structural changes of a dimeric heme-containing oxygen sensor protein as it responds to changes in its coordination and redox state. This allowed us to propose a mechanism by which the signal (oxygen binding to the heme iron in the sensing domain) is transduced to the protein's functional domain.

• Klíčová slova
• Globin-coupled histidine kinase, Heme-containing oxygen sensors, Hydrogen/deuterium exchange, Ligand binding, Mass spectrometry, Protein conformational dynamics, Signal transduction,
• MeSH
• deuterium MeSH
• hem chemie   MeSH
• hemoproteiny * MeSH
• hmotnostní spektrometrie metody   MeSH
• kyslík metabolismus   MeSH
• umělá inteligence MeSH
• vodík-deuteriová výměna metody   MeSH
• vodík chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• deuterium MeSH
• hem MeSH
• hemoproteiny * MeSH
• kyslík MeSH
• vodík MeSH

Protein hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) can be used to study interactions of proteins with various ligands, to describe the effects of mutations, or to reveal structural responses of proteins to different experimental conditions. It is often described as a method with virtually no limitations in terms of protein size or sample composition. While this is generally true, there are, however, ligands or buffer components that can significantly complicate the analysis. One such compound, that can make HDX-MS troublesome, is DNA. In this chapter, we will focus on the analysis of protein-DNA interactions, describe the detailed protocol, and point out ways to overcome the complications arising from the presence of DNA.

• Klíčová slova
• DNA, Hydrogen/deuterium exchange, Protein–DNA binding, Structural mass spectrometry, Transcription factor,
• MeSH
• analýza dat MeSH
• chromatografie kapalinová MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• DNA chemie   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• lidé MeSH
• transkripční faktory MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vodík/deuteriová výměna a hmotnostní spektrometrie * metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• DNA MeSH
• transkripční faktory MeSH

Lytic polysaccharide monooxygenases (LPMOs) are industrially important oxidoreductases employed in lignocellulose saccharification. Using advanced time-resolved mass spectrometric techniques, we elucidated the structural determinants for substrate-mediated stabilization of the fungal LPMO9C from Neurosporacrassa during catalysis. LPMOs require a reduction in the active-site copper for catalytic activity. We show that copper reduction in NcLPMO9C leads to structural rearrangements and compaction around the active site. However, longer exposure to the reducing agent ascorbic acid also initiated an uncoupling reaction of the bound oxygen species, leading to oxidative damage, partial unfolding, and even fragmentation of NcLPMO9C. Interestingly, no changes in the hydrogen/deuterium exchange rate were detected upon incubation of oxidized or reduced LPMO with crystalline cellulose, indicating that the LPMO-substrate interactions are mainly side-chain mediated and neither affect intraprotein hydrogen bonding nor induce significant shielding of the protein surface. On the other hand, we observed a protective effect of the substrate, which slowed down the autooxidative damage induced by the uncoupling reaction. These observations further complement the picture of structural changes during LPMO catalysis.

• Klíčová slova
• hydrogen/deuterium exchange mass spectrometry, lignocellulose degradation, lytic polysaccharide monooxygenase, oxidative amino acid modification, peptide bond cleavage, reactive oxygen species,
• MeSH
• celulosa chemie   MeSH
• fungální proteiny chemie   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• hmotnostní spektrometrie MeSH
• katalytická doména MeSH
• katalýza MeSH
• koncentrace vodíkových iontů MeSH
• konformace proteinů MeSH
• kyslík chemie   MeSH
• lignin chemie   MeSH
• měď chemie   MeSH
• Neurospora crassa enzymologie   MeSH
• oxidační stres MeSH
• oxidoreduktasy chemie   MeSH
• oxygenasy se smíšenou funkcí chemie   MeSH
• polysacharidy chemie   MeSH
• reaktivní formy kyslíku chemie   MeSH
• substrátová specifita MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• celulosa MeSH
• fungální proteiny MeSH
• kyslík MeSH
• lignin MeSH
• lignocellulose MeSH Prohlížeč
• měď MeSH
• oxidoreduktasy MeSH
• oxygenasy se smíšenou funkcí MeSH
• polysacharidy MeSH
• reaktivní formy kyslíku MeSH

Nepenthes regulates enzyme activities by sensing stimuli from the insect prey. Protein is the best inductor mimicking the presence of an insect prey. Carnivorous plants of the genus Nepenthes have evolved passive pitcher traps for prey capture. In this study, we investigated the ability of chemical signals from a prey (chitin, protein, and ammonium) to induce transcription and synthesis of digestive enzymes in Nepenthes × Mixta. We used real-time PCR and specific antibodies generated against the aspartic proteases nepenthesins, and type III and type IV chitinases to investigate the induction of digestive enzyme synthesis in response to different chemical stimuli from the prey. Transcription of nepenthesins was strongly induced by ammonium, protein and live prey; chitin induced transcription only very slightly. This is in accordance with the amount of released enzyme and proteolytic activity in the digestive fluid. Although transcription of type III chitinase was induced by all investigated stimuli, a significant accumulation of the enzyme in the digestive fluid was found mainly after protein and live prey addition. Protein and live prey were also the best inducers for accumulation of type IV chitinase in the digestive fluid. Although ammonium strongly induced transcription of all investigated genes probably through membrane depolarization, strong acidification of the digestive fluid affected stability and abundance of both chitinases in the digestive fluid. The study showed that the proteins are universal inductors of enzyme activities in carnivorous pitcher plants best mimicking the presence of insect prey. This is not surprising, because proteins are a much valuable source of nitrogen, superior to chitin. Extensive vesicular activity was observed in prey-activated glands.

• Klíčová slova
• Carnivorous plant, Chitin, Chitinase, Enzyme, Nepenthesin, Pitcher plant, Protease,
• MeSH
• Caryophyllales enzymologie   fyziologie   ultrastruktura   MeSH
• chitin metabolismus   MeSH
• chlorid amonný farmakologie   MeSH
• enzymy genetika   metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• masožravci MeSH
• membránové potenciály MeSH
• regulace genové exprese u rostlin * MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• sérový albumin hovězí metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chitin MeSH
• chlorid amonný MeSH
• enzymy MeSH
• rostlinné proteiny MeSH
• sérový albumin hovězí MeSH

Celiac disease is triggered by partially digested gluten proteins. Enzyme therapies that complete protein digestion in vivo could support a gluten-free diet, but the barrier to completeness is high. Current options require enzyme amounts on the same order as the protein meal itself. In this study, we evaluated proteolytic components of the carnivorous pitcher plant (Nepenthes spp.) for use in this context. Remarkably low doses enhance gliadin solubilization rates, and degrade gliadin slurries within the pH and temporal constraints of human gastric digestion. Potencies in excess of 1200:1 (substrate-to-enzyme) are achieved. Digestion generates small peptides through nepenthesin and neprosin, the latter a novel enzyme defining a previously-unknown class of prolyl endoprotease. The digests also exhibit reduced TG2 conversion rates in the immunogenic regions of gliadin, providing a twin mechanism for evading T-cell recognition. When sensitized and dosed with enzyme-treated gliadin, NOD/DQ8 mice did not show intestinal inflammation, when compared to mice challenged with only pepsin-treated gliadin. The low enzyme load needed for effective digestion suggests that gluten detoxification can be achieved in a meal setting, using metered dosing based on meal size. We demonstrate this by showing efficient antigen processing at total substrate-to-enzyme ratios exceeding 12,000:1.

• MeSH
• bezlepková dieta * MeSH
• celiakie enzymologie   imunologie   terapie   MeSH
• Drosophila metabolismus   MeSH
• enzymoterapie * MeSH
• gliadin metabolismus   MeSH
• gluteny metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• myši inbrední NOD MeSH
• myši MeSH
• protein-glutamin:amin-gama-glutamyltransferasa 2 MeSH
• proteiny vázající GTP metabolismus   MeSH
• proteolýza MeSH
• transglutaminasy metabolismus   MeSH
• zánět imunologie   metabolismus   prevence a kontrola   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• gliadin MeSH
• gluteny MeSH
• protein-glutamin:amin-gama-glutamyltransferasa 2 MeSH
• proteiny vázající GTP MeSH
• transglutaminasy MeSH

Nepenthesins are aspartic proteases secreted by carnivorous pitcher plants of the genus Nepenthes. They significantly differ in sequence from other plant aspartic proteases. This difference, which provides more cysteine residues in the structure of nepenthesins, may contribute to their unique stability profile. Recombinantly produced nepenthesin 1 (rNep1) from N. gracilis in complex with pepstatin A was crystallized under two different crystallization conditions using a newly formulated low-pH crystallization screen. The diffraction data were processed to 2.9 and 2.8 Å resolution, respectively. The crystals belonged to space group P212121, with unit-cell parameters a = 86.63, b = 95.90, c = 105.40 Å, α = β = γ = 90° and a = 86.28, b = 97.22, c = 103.78 Å, α = β = γ = 90°, respectively. Matthews coefficient and solvent-content calculations suggest the presence of two molecules of rNep1 in the asymmetric unit. Here, the details of the crystallization experiment and analysis of the X-ray data are reported.

• Klíčová slova
• Nepenthes gracilis, aspartic proteases, low-pH crystallization screen, nepenthesins,
• MeSH
• aspartátové endopeptidasy chemie   MeSH
• koncentrace vodíkových iontů MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• Magnoliopsida enzymologie   MeSH
• pepstatiny chemie   MeSH
• rostlinné proteiny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aspartátové endopeptidasy MeSH
• pepstatin MeSH Prohlížeč
• pepstatiny MeSH
• rostlinné proteiny MeSH

The virulence of Plasmodium falciparum is linked to the ability of infected erythrocytes (IE) to adhere to the vascular endothelium, mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). In this article, we report the functional characterization of an mAb that recognizes a panel of PfEMP1s and inhibits ICAM-1 binding. The 24E9 mouse mAb was raised against PFD1235w DBLβ3_D4, a domain from the group A PfEMP1s associated with severe malaria. 24E9 recognizes native PfEMP1 expressed on the IE surface and shows cross-reactivity with and cross-inhibition of the ICAM-1 binding capacity of domain cassette 4 PfEMP1s. 24E9 Fab fragments bind DBLβ3_D4 with nanomolar affinity and inhibit ICAM-1 binding of domain cassette 4-expressing IE. The antigenic regions targeted by 24E9 Fab were identified by hydrogen/deuterium exchange mass spectrometry and revealed three discrete peptides that are solvent protected in the complex. When mapped onto a homology model of DBLβ3_D4, these cluster to a defined, surface-exposed region on the convex surface of DBLβ3_D4. Mutagenesis confirmed that the site most strongly protected is necessary for 24E9 binding, which is consistent with a low-resolution structure of the DBLβ3_D4::24E9 Fab complex derived from small-angle x-ray scattering. The convex surface of DBLβ3_D4 has previously been shown to contain the ICAM-1 binding site of DBLβ domains, suggesting that the mAb acts by occluding the ICAM-1 binding surface. Conserved epitopes, such as those targeted by 24E9, are promising candidates for the inclusion in a vaccine interfering with ICAM-1-specific adhesion of group A PfEMP1 expressed by P. falciparum IE during severe malaria.

• MeSH
• antigeny protozoální imunologie   MeSH
• buněčná adheze MeSH
• cévní endotel metabolismus   parazitologie   MeSH
• epitopy imunologie   MeSH
• erytrocytární membrána imunologie   MeSH
• erytrocyty parazitologie   MeSH
• hybridomy MeSH
• kultivované buňky MeSH
• mezibuněčná adhezivní molekula-1 imunologie   MeSH
• molekulární sekvence - údaje MeSH
• monoklonální protilátky imunologie   MeSH
• myši MeSH
• Plasmodium falciparum imunologie   MeSH
• protilátky protozoální imunologie   MeSH
• protozoální proteiny imunologie   MeSH
• terciární struktura proteinů MeSH
• tropická malárie imunologie   parazitologie   MeSH
• vazebná místa protilátek imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny protozoální MeSH
• epitopy MeSH
• erythrocyte membrane protein 1, Plasmodium falciparum MeSH Prohlížeč
• mezibuněčná adhezivní molekula-1 MeSH
• monoklonální protilátky MeSH
• protilátky protozoální MeSH
• protozoální proteiny MeSH