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.

Preparation of a Well-Defined and Stable ß-Barrel Pore-Forming Aß42 Oligomer.

The formation of amyloid-ß peptide (Aß) oligomers at the cellular membrane is considered a crucial process that underlies neurotoxicity in Alzheimer's disease (AD). To obtain structural information on this type of oligomers, we were inspired by membrane protein approaches used to stabilize, characterize, and analyze the function of such proteins. Using these approaches, we developed conditions under which Aß42, the Aß variant most strongly linked to the aetiology of AD, assembles into an oligomer that inserts into lipid bilayers as a well-defined pore and adopts a specific structure with characteristics of a ß-barrel arrangement. We named this oligomer ß-barrel Pore-Forming Aß42 Oligomer (ßPFOAß42). Here, we describe detailed protocols for its preparation and characterization. We expect ßPFOAß42 to be useful in establishing the involvement of membrane-associated Aß oligomers in AD.

Aß42 assembles into specific ß-barrel pore-forming oligomers in membrane-mimicking environments.

The formation of amyloid-ß peptide (Aß) oligomers at the cellular membrane is considered to be a crucial process underlying neurotoxicity in Alzheimer's disease (AD). Therefore, it is critical to characterize the oligomers that form within a membrane environment. To contribute to this characterization, we have applied strategies widely used to examine the structure of membrane proteins to study the two major Aß variants, Aß40 and Aß42. Accordingly, various types of detergent micelles were extensively screened to identify one that preserved the properties of Aß in lipid environments-namely the formation of oligomers that function as pores. Remarkably, under the optimized detergent micelle conditions, Aß40 and Aß42 showed different behavior. Aß40 aggregated into amyloid fibrils, whereas Aß42 assembled into oligomers that inserted into lipid bilayers as well-defined pores and adopted a specific structure with characteristics of a ß-barrel arrangement that we named ß-barrel pore-forming Aß42 oligomers (ßPFOsAß42). Because Aß42, relative to Aß40, has a more prominent role in AD, the higher propensity of Aß42 to form ßPFOs constitutes an indication of their relevance in AD. Moreover, because ßPFOsAß42 adopt a specific structure, this property offers an unprecedented opportunity for testing a hypothesis regarding the involvement of ßPFOs and, more generally, membrane-associated Aß oligomers in AD.