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Article ; Online: Native Top-Down Mass Spectrometry for Characterizing Sarcomeric Proteins Directly from Cardiac Tissue Lysate.

Chapman, Emily A / Li, Brad H / Krichel, Boris / Chan, Hsin-Ju / Buck, Kevin M / Roberts, David S / Ge, Ying

Journal of the American Society for Mass Spectrometry

2024 Volume 35, Issue 4, Page(s) 738–745

Abstract: Native top-down mass spectrometry (nTDMS) has emerged as a powerful structural biology tool that can localize post-translational modifications (PTMs), explore ligand-binding interactions, and elucidate the three-dimensional structure of proteins and ... ...

Abstract	Native top-down mass spectrometry (nTDMS) has emerged as a powerful structural biology tool that can localize post-translational modifications (PTMs), explore ligand-binding interactions, and elucidate the three-dimensional structure of proteins and protein complexes in the gas-phase. Fourier-transform ion cyclotron resonance (FTICR) MS offers distinct capabilities for nTDMS, owing to its ultrahigh resolving power, mass accuracy, and robust fragmentation techniques. Previous nTDMS studies using FTICR have mainly been applied to overexpressed recombinant proteins and protein complexes. Here, we report the first nTDMS study that directly analyzes human heart tissue lysate by direct infusion FTICR MS without prior chromatographic separation strategies. We have achieved comprehensive nTDMS characterization of cardiac contractile proteins that play critical roles in heart contraction and relaxation. Specifically, our results reveal structural insights into ventricular myosin light chain 2 (MLC-2v), ventricular myosin light chain 1 (MLC-1v), and alpha-tropomyosin (α-Tpm) in the sarcomere, the basic contractile unit of cardiac muscle. Furthermore, we verified the calcium (Ca
MeSH term(s)	Humans ; Sarcomeres/chemistry ; Proteins/chemistry ; Mass Spectrometry/methods ; Heart ; Myocardium/chemistry
Chemical Substances	Proteins
Language	English
Publishing date	2024-02-29
Publishing country	United States
Document type	Journal Article
ZDB-ID	1073671-2
ISSN	1879-1123 ; 1044-0305
ISSN (online)	1879-1123
ISSN	1044-0305
DOI	10.1021/jasms.3c00430
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Article ; Online: The kinetics of SARS-CoV-2 nsp7-11 polyprotein processing and impact on complexation with nsp16

Schamoni-Kast, Kira / Krichel, Boris / Damjanović, Tomislav / Kierspel, Thomas / Toker, Sibel / Uetrecht, Charlotte

bioRxiv

Abstract: In severe-acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, polyproteins (pp1a/pp1ab) are processed into non-structural proteins (nsps), which largely form the replication/transcription complex (RTC). The polyprotein processing and complex ...

Abstract	In severe-acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, polyproteins (pp1a/pp1ab) are processed into non-structural proteins (nsps), which largely form the replication/transcription complex (RTC). The polyprotein processing and complex formation is critical and offers potential therapeutic targets. However, the interplay of polyprotein processing and RTC-assembly are poorly understood. Here, we studied two key aspects: The influence of the pp1a terminal nsp11 on the order of polyprotein processing by viral main protease Mpro and the influence of polyprotein processing on core enzyme complex formation. We established a method based on native MS to determine rate constants k considering the structural environment. This enabled us to quantify the multi-reaction kinetics of coronavirus polyprotein processing for the first time. Our results serve as a blueprint for other multi-cleavage reactions. Further, it offers a detailed and quantifiable perspective to the dynamic reactions of SARS-CoV-2 polyprotein processing, which is required for development of novel antivirals.
Keywords	covid19
Language	English
Publishing date	2024-01-08
Publisher	Cold Spring Harbor Laboratory
Document type	Article ; Online
DOI	10.1101/2024.01.06.574466
Database	COVID19

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Article ; Online: The kinetics of SARS-CoV-2 nsp7-11 polyprotein processing and impact on complexation with nsp16

Schamoni-Kast, Kira / Krichel, Boris / Damjanovic, Tomislav / Kierspel, Thomas / Toker, Sibel / Uetrecht, Charlotte

bioRxiv

Abstract	In severe-acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, polyproteins (pp1a/pp1ab) are processed into non-structural proteins (nsps), which largely form the replication/transcription complex (RTC). The polyprotein processing and complex formation is critical and offers potential therapeutic targets. However, the interplay of polyprotein processing and RTC-assembly are poorly understood. Here, we studied two key aspects: The influence of the pp1a terminal nsp11 on the order of polyprotein processing by viral main protease Mpro and the influence of polyprotein processing on core enzyme complex formation. We established a method based on native MS to determine rate constants k considering the structural environment. This enabled us to quantify the multi-reaction kinetics of coronavirus polyprotein processing for the first time. Our results serve as a blueprint for other multi-cleavage reactions. Further, it offers a detailed and quantifiable perspective to the dynamic reactions of SARS-CoV-2 polyprotein processing, which is required for development of novel antivirals.
Keywords	covid19
Language	English
Publishing date	2024-01-08
Publisher	Cold Spring Harbor Laboratory
Document type	Article ; Online
DOI	10.1101/2024.01.06.574466
Database	COVID19

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Book ; Online ; Thesis: In vitro processing and complex formation of coronavirus polyprotein NSP7-10 region

Krichel, Boris [Verfasser] / Uetrecht, Charlotte [Akademischer Betreuer]

2020

Author's details	Boris Krichel ; Betreuer: Charlotte Uetrecht
Keywords	Biowissenschaften, Biologie ; Life Science, Biology
Subject code	sg570
Language	English
Publisher	Staats- und Universitätsbibliothek Hamburg
Publishing place	Hamburg
Document type	Book ; Online ; Thesis
Database	Digital theses on the web

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Article ; Online: Processing of the SARS-CoV pp1a/ab nsp7-10 region.

Krichel, Boris / Falke, Sven / Hilgenfeld, Rolf / Redecke, Lars / Uetrecht, Charlotte

The Biochemical journal

2020 Volume 477, Issue 5, Page(s) 1009–1019

Abstract: Severe acute respiratory syndrome coronavirus is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/transcription complex, essential steps include processing of the conserved ... ...

Abstract	Severe acute respiratory syndrome coronavirus is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/transcription complex, essential steps include processing of the conserved polyprotein nsp7-10 region by the main protease Mpro and subsequent complex formation of the released nsp's. Here, we analyzed processing of the coronavirus nsp7-10 region using native mass spectrometry showing consumption of substrate, rise and fall of intermediate products and complexation. Importantly, there is a clear order of cleavage efficiencies, which is influenced by the polyprotein tertiary structure. Furthermore, the predominant product is an nsp7+8(2 : 2) hetero-tetramer with nsp8 scaffold. In conclusion, native MS, opposed to other methods, can expose the processing dynamics of viral polyproteins and the landscape of protein interactions in one set of experiments. Thereby, new insights into protein interactions, essential for generation of viral progeny, were provided, with relevance for development of antivirals.
MeSH term(s)	Coronavirus 3C Proteases ; Coronavirus Infections/genetics ; Coronavirus RNA-Dependent RNA Polymerase ; Cysteine Endopeptidases/chemistry ; Cysteine Endopeptidases/genetics ; Fluorescence Resonance Energy Transfer ; Protein Structure, Secondary ; RNA-Binding Proteins/chemistry ; RNA-Binding Proteins/genetics ; Sequence Alignment/methods ; Viral Nonstructural Proteins/chemistry ; Viral Nonstructural Proteins/genetics ; Viral Regulatory and Accessory Proteins/chemistry ; Viral Regulatory and Accessory Proteins/genetics ; Virus Replication/physiology
Chemical Substances	NS8 protein, SARS-CoV-2 ; NSP10 protein, SARS-CoV-2 ; NSP9 protein, SARS-CoV-2 ; RNA-Binding Proteins ; Viral Nonstructural Proteins ; Viral Regulatory and Accessory Proteins ; Coronavirus RNA-Dependent RNA Polymerase (EC 2.7.7.48) ; NSP7 protein, SARS-CoV-2 (EC 2.7.7.48) ; Cysteine Endopeptidases (EC 3.4.22.-) ; Coronavirus 3C Proteases (EC 3.4.22.28)
Keywords	covid19
Language	English
Publishing date	2020-02-29
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2969-5
ISSN	1470-8728 ; 0006-2936 ; 0306-3275 ; 0264-6021
ISSN (online)	1470-8728
ISSN	0006-2936 ; 0306-3275 ; 0264-6021
DOI	10.1042/BCJ20200029
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Article ; Online: The crystal structure of mycobacterial epoxide hydrolase A.

Schulz, Eike C / Henderson, Sara R / Illarionov, Boris / Crosskey, Thomas / Southall, Stacey M / Krichel, Boris / Uetrecht, Charlotte / Fischer, Markus / Wilmanns, Matthias

Scientific reports

2020 Volume 10, Issue 1, Page(s) 16539

Abstract: The human pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis resulting in over 1 million fatalities every year, despite decades of research into the development of new anti-TB compounds. Unlike most other organisms M. tuberculosis ...

Abstract	The human pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis resulting in over 1 million fatalities every year, despite decades of research into the development of new anti-TB compounds. Unlike most other organisms M. tuberculosis has six putative genes for epoxide hydrolases (EH) of the α/β-hydrolase family with little known about their individual substrates, suggesting functional significance for these genes to the organism. Due to their role in detoxification, M. tuberculosis EH's have been identified as potential drug targets. Here, we demonstrate epoxide hydrolase activity of M. thermoresistibile epoxide hydrolase A (Mth-EphA) and report its crystal structure in complex with the inhibitor 1,3-diphenylurea at 2.0 Å resolution. Mth-EphA displays high sequence similarity to its orthologue from M. tuberculosis and generally high structural similarity to α/β-hydrolase EHs. The structure of the inhibitor bound complex reveals the geometry of the catalytic residues and the conformation of the inhibitor. Comparison to other EHs from mycobacteria allows insight into the active site plasticity with respect to substrate specificity. We speculate that mycobacterial EHs may have a narrow substrate specificity providing a potential explanation for the genetic repertoire of epoxide hydrolase genes in M. tuberculosis.
MeSH term(s)	Carbanilides ; Crystallization ; Epoxide Hydrolases/chemistry ; Epoxide Hydrolases/genetics ; Epoxide Hydrolases/physiology ; Genes, Bacterial/genetics ; Inactivation, Metabolic/genetics ; Mycobacterium tuberculosis/enzymology ; Mycobacterium tuberculosis/genetics ; Substrate Specificity
Chemical Substances	Carbanilides ; Epoxide Hydrolases (EC 3.3.2.-)
Language	English
Publishing date	2020-10-06
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-020-73452-y
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Article: Processing of the SARS-CoV pp1a/ab nsp7-10 region

Krichel, Boris / Falke, Sven / Hilgenfeld, Rolf / Redecke, Lars / Uetrecht, Charlotte

Biochem J

Abstract	Severe acute respiratory syndrome coronavirus is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/transcription complex, essential steps include processing of the conserved polyprotein nsp7-10 region by the main protease Mpro and subsequent complex formation of the released nsp's. Here, we analyzed processing of the coronavirus nsp7-10 region using native mass spectrometry showing consumption of substrate, rise and fall of intermediate products and complexation. Importantly, there is a clear order of cleavage efficiencies, which is influenced by the polyprotein tertiary structure. Furthermore, the predominant product is an nsp7+8(2 : 2) hetero-tetramer with nsp8 scaffold. In conclusion, native MS, opposed to other methods, can expose the processing dynamics of viral polyproteins and the landscape of protein interactions in one set of experiments. Thereby, new insights into protein interactions, essential for generation of viral progeny, were provided, with relevance for development of antivirals.
Keywords	covid19
Publisher	WHO
Document type	Article
Note	WHO #Covidence: #827308
Database	COVID19

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Article ; Online: Structural mass spectrometry goes viral.

Dülfer, Jasmin / Kadek, Alan / Kopicki, Janine-Denise / Krichel, Boris / Uetrecht, Charlotte

Advances in virus research

2019 Volume 105, Page(s) 189–238

Abstract: Over the last 20 years, mass spectrometry (MS), with its ability to analyze small sample amounts with high speed and sensitivity, has more and more entered the field of structural virology, aiming to investigate the structure and dynamics of viral ... ...

Abstract	Over the last 20 years, mass spectrometry (MS), with its ability to analyze small sample amounts with high speed and sensitivity, has more and more entered the field of structural virology, aiming to investigate the structure and dynamics of viral proteins as close to their native environment as possible. The use of non-perturbing labels in hydrogen-deuterium exchange MS allows for the analysis of interactions between viral proteins and host cell factors as well as their dynamic responses to the environment. Cross-linking MS, on the other hand, can analyze interactions in viral protein complexes and identify virus-host interactions in cells. Native MS allows transferring viral proteins, complexes and capsids into the gas phase and has broken boundaries to overcome size limitations, so that now even the analysis of intact virions is possible. Different MS approaches not only inform about size, stability, interactions and dynamics of virus assemblies, but also bridge the gap to other biophysical techniques, providing valuable constraints for integrative structural modeling of viral complex assemblies that are often inaccessible by single technique approaches. In this review, recent advances are highlighted, clearly showing that structural MS approaches in virology are moving towards systems biology and ever more experiments are performed on cellular level.
MeSH term(s)	Capsid/chemistry ; Capsid/metabolism ; Macromolecular Substances/chemistry ; Macromolecular Substances/metabolism ; Mass Spectrometry/methods ; Protein Interaction Mapping/methods ; Viral Proteins/chemistry ; Viral Proteins/metabolism
Chemical Substances	Macromolecular Substances ; Viral Proteins
Language	English
Publishing date	2019-08-27
Publishing country	United States
Document type	Journal Article
ZDB-ID	195-8
ISSN	1557-8399 ; 0065-3527
ISSN (online)	1557-8399
ISSN	0065-3527
DOI	10.1016/bs.aivir.2019.07.003
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Article ; Online: Processing of the SARS-CoV pp1a/ab nsp7-10 region

Krichel, Boris / Falke, Sven / Hilgenfeld, Rolf / Redecke, Lars / Uetrecht, Charlotte

bioRxiv

Abstract: Severe acute respiratory syndrome coronavirus (SARS-CoV) is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/transcription complex (RTC), essential steps include processing ... ...

Abstract	Severe acute respiratory syndrome coronavirus (SARS-CoV) is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/transcription complex (RTC), essential steps include processing of the conserved polyprotein nsp7-10 region by the main protease Mpro and subsequent complex formation of the released nsp’s. Here, we analyzed processing of the coronavirus nsp7-10 region using native mass spectrometry showing consumption of substrate, rise and fall of intermediate products and complexation. Importantly, there is a clear order of cleavage efficiencies, which is influenced by the polyprotein tertiary structure. Furthermore, the predominant product is an nsp7+8(2:2) hetero-tetramer with nsp8 scaffold. In conclusion, native MS, opposed to other methods, can expose the processing dynamics of viral polyproteins and the landscape of protein interactions in one set of experiments. Thereby, new insights into protein interactions, essential for generation of viral progeny, were provided, with relevance for development of antivirals.
Keywords	covid19
Publisher	BioRxiv; MedRxiv
Document type	Article ; Online
DOI	10.1101/860049
Database	COVID19

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Article: MASH Native: A Unified Solution for Native Top-Down Proteomics Data Processing.

bioRxiv : the preprint server for biology

2023

Abstract: Native top-down proteomics (nTDP) integrates native mass spectrometry (nMS) with top-down proteomics (TDP) to provide comprehensive analysis of protein complexes together with proteoform identification and characterization. Despite significant advances ... ...

Abstract	Native top-down proteomics (nTDP) integrates native mass spectrometry (nMS) with top-down proteomics (TDP) to provide comprehensive analysis of protein complexes together with proteoform identification and characterization. Despite significant advances in nMS and TDP software developments, a unified and user-friendly software package for analysis of nTDP data remains lacking. Herein, we have developed MASH Native to provide a unified solution for nTDP to process complex datasets with database searching capabilities in a user-friendly interface. MASH Native supports various data formats and incorporates multiple options for deconvolution, database searching, and spectral summing to provide a one-stop shop for characterizing both native protein complexes and proteoforms. The MASH Native app, video tutorials, written tutorials and additional documentation are freely available for download at https://labs.wisc.edu/gelab/MASH_Explorer/MASHNativeSoftware.php . All data files shown in user tutorials are included with the MASH Native software in the download .zip file.
Language	English
Publishing date	2023-01-03
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.01.02.522513
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