In proteomics, postproline cleaving enzymes (PPCEs), such as Aspergillus niger prolyl endopeptidase (AnPEP) and neprosin, complement proteolytic tools because proline is a stop site for many proteases. But while aiming at using AnPEP in online proteolysis, we found that this enzyme also displayed specificity to reduced cysteine. By LC-MS/MS, we systematically analyzed AnPEP sources and conditions that could affect this cleavage preference. Postcysteine cleavage was blocked by cysteine modifications, including disulfide bond formation, oxidation, and alkylation. The last modification explains why this activity has remained undetected so far. In the same experimental paradigm, neprosin mimicked this cleavage specificity. Based on these findings, PPCEs cleavage preferences should be redefined from post-Pro/Ala to post-Pro/Ala/Cys. Moreover, this evidence demands reconsidering PPCEs applications, whether cleaving Cys-rich proteins or assessing Cys status in proteins, and calls for revisiting the proposed enzymatic mechanism of these proteases.

• Publikační typ
• časopisecké články MeSH

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

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

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

Isoprenoids are synthesized by the prenyltransferase superfamily, which is subdivided according to the product stereoisomerism and length. In short- and medium-chain isoprenoids, product length correlates with active site volume. However, enzymes synthesizing long-chain products and rubber synthases fail to conform to this paradigm, because of an unexpectedly small active site. Here, we focused on the human cis-prenyltransferase complex (hcis-PT), residing at the endoplasmic reticulum membrane and playing a crucial role in protein glycosylation. Crystallographic investigation of hcis-PT along the reaction cycle revealed an outlet for the elongating product. Hydrogen-deuterium exchange mass spectrometry analysis showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Last, using a fluorescence substrate analog, we show that product elongation and membrane association are closely correlated. Together, our results support direct membrane insertion of the elongating isoprenoid during catalysis, uncoupling active site volume from product length.

• Publikační typ
• časopisecké články 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

The limited information available on the structure of complexes involving transcription factors and cognate DNA response elements represents a major obstacle in the quest to understand their mechanism of action at the molecular level. We implemented a concerted structural proteomics approach, which combined hydrogen-deuterium exchange (HDX), quantitative protein-protein and protein-nucleic acid cross-linking (XL), and homology analysis, to model the structure of the complex between the full-length DNA binding domain (DBD) of Forkhead box protein O4 (FOXO4) and its DNA binding element (DBE). The results confirmed that FOXO4-DBD assumes the characteristic forkhead topology shared by these types of transcription factors, but its binding mode differs significantly from those of other members of the family. The results showed that the binding interaction stabilized regions that were rather flexible and disordered in the unbound form. Surprisingly, the conformational effects were not limited only to the interface between bound components, but extended also to distal regions that may be essential to recruiting additional factors to the transcription machinery. In addition to providing valuable new insights into the binding mechanism, this project provided an excellent evaluation of the merits of structural proteomics approaches in the investigation of systems that are not directly amenable to traditional high-resolution techniques.

• Klíčová slova
• DNA, FOXO4, cross-linking, molecular modeling, protein, protein-nucleic acid cross-linking, trans-dichlorodiamineplatinum(II), hydrogen-deuterium exchange, transcription factor, transplatin,
• MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• DNA chemie   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• molekulární struktura MeSH
• responzivní elementy MeSH
• transkripční faktory chemie   metabolismus   MeSH
• vodík-deuteriová výměna MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• DNA MeSH
• transkripční faktory MeSH

Filamentous hemagglutinin (FHA) mediates adherence and plays an important role in lower respiratory tract infections by pathogenic Bordetellae. The mature FHA proteins of B. pertussis (Bp-FHA) and the B. bronchiseptica (Bb-FHA) are generated by processing of the respective FhaB precursors by the autotransporter subtilisin-type protease SphB1. We have used bottom-up proteomics with differential 16O/18O labeling and show that despite high-sequence conservation of the corresponding FhaB segments, the mature Bp-FHA (~ 230 kDa) and Bb-FHA (~ 243 kDa) proteins are processed at different sites of FhaB, after the Ala-2348 and Lys-2479 residues, respectively. Moreover, protease surface accessibility probing by on-column (on-line) digestion of the Bp-FHA and Bb-FHA proteins yielded different peptide patterns, revealing structural differences in the N-terminal and C-terminal domains of the Bp-FHA and Bb-FHA proteins. These data indicate specific structural variations between the highly homologous FHA proteins.

• Klíčová slova
• Bordetella bronchiseptica, Bordetella pertussis, bacterial pathogenesis, mass spectrometry (MS), protein processing, serine protease,
• Publikační typ
• časopisecké články MeSH

Phosducin (Pdc) is a conserved phosphoprotein that, when unphosphorylated, binds with high affinity to the complex of βγ-subunits of G protein transducin (Gtβγ). The ability of Pdc to bind to Gtβγ is inhibited through its phosphorylation at S54 and S73 within the N-terminal domain (Pdc-ND) followed by association with the scaffolding protein 14-3-3. However, the molecular basis for the 14-3-3-dependent inhibition of Pdc binding to Gtβγ is unclear. By using small-angle x-ray scattering, high-resolution NMR spectroscopy, and limited proteolysis coupled with mass spectrometry, we show that phosphorylated Pdc and 14-3-3 form a complex in which the Pdc-ND region 45-80, which forms a part of Pdc's Gtβγ binding surface and contains both phosphorylation sites, is restrained within the central channel of the 14-3-3 dimer, with both 14-3-3 binding motifs simultaneously participating in protein association. The N-terminal part of Pdc-ND is likely located outside the central channel of the 14-3-3 dimer, but Pdc residues 20-30, which are also involved in Gtβγ binding, are positioned close to the surface of the 14-3-3 dimer. The C-terminal domain of Pdc is located outside the central channel and its structure is unaffected by the complex formation. These results indicate that the 14-3-3 protein-mediated inhibition of Pdc binding to Gtβγ is based on steric occlusion of Pdc's Gtβγ binding surface.

• MeSH
• difrakce rentgenového záření MeSH
• fosfoproteiny antagonisté a inhibitory   chemie   MeSH
• fosforylace MeSH
• krysa rodu Rattus MeSH
• maloúhlový rozptyl MeSH
• oční proteiny antagonisté a inhibitory   chemie   MeSH
• proteinové domény MeSH
• proteiny 14-3-3 chemie   metabolismus   MeSH
• proteiny vázající GTP - regulátory antagonisté a inhibitory   chemie   MeSH
• proteolýza MeSH
• protonová magnetická rezonanční spektroskopie MeSH
• sekundární struktura proteinů MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfoproteiny MeSH
• oční proteiny MeSH
• phosducin MeSH Prohlížeč
• proteiny 14-3-3 MeSH
• proteiny vázající GTP - regulátory MeSH