6 research outputs found
Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications
The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form
Biological NMR of the Intrinsically Disordered Proteins Alpha-Synuclein and Prothymosin Alpha
Structural insights into α-synuclein monomer–fibril interactions
Protein aggregation into amyloid fibrils is associated with multiple neurodegenerative diseases, including Parkinson’s disease. Kinetic data and biophysical characterization have shown that the secondary nucleation pathway highly accelerates aggregation via the absorption of monomeric protein on the surface of amyloid fibrils. Here, we used NMR and electron paramagnetic resonance spectroscopy to investigate the interaction of monomeric α-synuclein (α-Syn) with its fibrillar form. We demonstrate that α-Syn monomers interact transiently via their positively charged N terminus with the negatively charged flexible C-terminal ends of the fibrils. These intermolecular interactions reduce intramolecular contacts in monomeric α-Syn, yielding further unfolding of the partially collapsed intrinsically disordered states of α-Syn along with a possible increase in the local concentration of soluble α-Syn and alignment of individual monomers on the fibril surface. Our data indicate that intramolecular unfolding critically contributes to the aggregation kinetics of α-Syn during secondary nucleation.ISSN:0027-8424ISSN:1091-649
Protein structure determination in human cells by in-cell NMR and a reporter system to optimize protein delivery or transexpression
Most experimental methods for structural biology proceed in vitro and therefore the contribution of the intracellular environment on protein structure and dynamics is absent. Studying proteins at atomic resolution in living mammalian cells has been elusive due to the lack of methodologies. In-cell nuclear magnetic resonance spectroscopy (in-cell NMR) is an emerging technique with the power to do so. Here, we improved current methods of in-cell NMR by the development of a reporter system that allows monitoring the delivery of exogenous proteins into mammalian cells, a process that we called here “transexpression”. The reporter system was used to develop an efficient protocol for in-cell NMR which enables spectral acquisition with higher quality for both disordered and folded proteins. With this method, the 3D atomic resolution structure of the model protein GB1 in human cells was determined with a backbone root-mean-square deviation (RMSD) of 1.1 Å.ISSN:2399-364
Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications
The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.ISSN:2296-889
Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications
The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.This work was supported by Goethe University (Corona funds),
the DFG-funded CRC: “Molecular Principles of RNA-Based
Regulation,” DFG infrastructure funds (project numbers:
277478796, 277479031, 392682309, 452632086, 70653611), the
state of Hesse (BMRZ), the Fondazione CR Firenze (CERM),
and the IWB-EFRE-program 20007375. This project has
received funding from the European Union’s Horizon 2020
research and innovation program under Grant Agreement No.
871037. AS is supported by DFG Grant SCHL 2062/2-1 and by the
JQYA at Goethe through project number 2019/AS01. Work in the
lab of KV was supported by a CoRE grant from the University of
New Hampshire. The FLI is a member of the Leibniz Association
(WGL) and financially supported by the Federal Government of
Germany and the State of Thuringia. Work in the lab of RM was
supported by NIH (2R01EY021514) and NSF (DMR-2002837).
BN-B was supported by theNSF GRFP.MCwas supported byNIH
(R25 GM055246 MBRS IMSD), and MS-P was supported by the
HHMI Gilliam Fellowship. Work in the labs of KJ and KT was
supported by Latvian Council of Science Grant No. VPP-COVID
2020/1-0014. Work in the UPAT’s lab was supported by the
INSPIRED (MIS 5002550) project, which is implemented under
the Action “Reinforcement of the Research and Innovation
Infrastructure,” funded by the Operational Program
“Competitiveness, Entrepreneurship and Innovation” (NSRF
2014–2020) and cofinanced by Greece and the EU (European
Regional Development Fund) and the FP7 REGPOT CT-2011-
285950–“SEE-DRUG” project (purchase of UPAT’s 700MHz
NMR equipment). Work in the CM-G lab was supported by
the Helmholtz society. Work in the lab of ABö was supported
by the CNRS, the French National Research Agency (ANR, NMRSCoV2-
ORF8), the Fondation de la Recherche Médicale (FRM,
NMR-SCoV2-ORF8), and the IR-RMN-THC Fr3050 CNRS.
Work in the lab of BM was supported by the Swiss National
Science Foundation (Grant number 200020_188711), the
Günthard Stiftung für Physikalische Chemie, and the ETH
Zurich. Work in the labs of ABö and BM was supported by a
common grant from SNF (grant 31CA30_196256). This work was
supported by the ETHZurich, the grant ETH40 18 1, and the grant
Krebsliga KFS 4903 08 2019. Work in the lab of the IBS Grenoble
was supported by the Agence Nationale de Recherche (France)
RA-COVID SARS2NUCLEOPROTEIN and European Research
Council Advanced Grant DynamicAssemblies. Work in the
CA lab was supported by Patto per il Sud della Regione
Siciliana–CheMISt grant (CUP G77B17000110001). Part of
this work used the platforms of the Grenoble Instruct-ERIC
center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the
Grenoble Partnership for Structural Biology (PSB), supported
by FRISBI (ANR-10-INBS-05-02) and GRAL, financed within
the University Grenoble Alpes graduate school (Ecoles
Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-
0003). Work at the UW-Madison was supported by grant
numbers NSF MCB2031269 and NIH/NIAID AI123498. MM
is a Ramón y Cajal Fellow of the Spanish AEI-Ministry of
Science and Innovation (RYC2019-026574-I), and a “La
Caixa” Foundation (ID 100010434) Junior Leader Fellow
(LCR/BQ/PR19/11700003). Funded by project COV20/00764
fromthe Carlos III Institute of Health and the SpanishMinistry
of Science and Innovation to MMand DVL. VDJ was supported
by the Boehringer Ingelheim Fonds. Part of this work used the
resources of the Italian Center of Instruct-ERIC at the CERM/
CIRMMP infrastructure, supported by the Italian Ministry for
University and Research (FOE funding). CF was supported by
the Stiftung Polytechnische Gesellschaft. Work in the lab of
JH was supported by NSF (RAPID 2030601) and NIH
(R01GM123249).Peer reviewe