Accessing isotopically labeled proteins containing genetically encoded phosphoserine for NMR with optimized expression conditions

Phosphoserine (pSer) sites are primarily located within disordered protein regions, making it difficult to experimentally ascertain their effects on protein structure and function. Therefore, the production of 15N- (and 13C)-labeled proteins with site-specifically encoded pSer for NMR studies is essential to uncover molecular mechanisms of protein regulation by phosphorylation. While genetic code expansion technologies for the translational installation of pSer in Escherichia coli are well established and offer a powerful strategy to produce site-specifically phosphorylated proteins, methodologies to adapt them to minimal or isotope-enriched media have not been described. This shortcoming exists because pSer genetic code expansion expression hosts require the genomic DELTAserB mutation, which increases pSer bioavailability but also imposes serine auxotrophy, preventing growth in minimal media used for isotopic labeling of recombinant proteins. Here, by testing different media supplements, we restored normal BL21(DE3) DELTAserB growth in labeling media but subsequently observed an increase of phosphatase activity and mis-incorporation not typically seen in standard rich media. After rounds of optimization and adaption of a high-density culture protocol, we were able to obtain >=10 mg/L homogenously labeled, phosphorylated superfolder GFP. To demonstrate the utility of this method, we also produced the intrinsically disordered serine/arginine-rich region of the SARS-CoV-2 Nucleocapsid protein labeled with 15N and pSer at the key site S188 and observed the resulting peak shift due to phosphorylation by 2D and 3D heteronuclear single quantum correlation analyses. We propose this cost-effective methodology will pave the way for more routine access to pSer-enriched proteins for 2D and 3D NMR analyses. GCE4All Biomedical Technology Development and Dissemination Center was supported by National Institute of General Medical Science, OSU NMR Facility funded in part by the National Institutes of Health, the Medical Research Foundation at OHSU and the Collins Medical Trust, National Science Foundation EAGER, and by the M. J. Murdock Charitable Trust.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Modifications in SARS-CoV-2 N Linker Region Regulate RNA Interactions and Phase Separation

The N protein of the SARS-CoV-2 virion is critical for viral genome packaging via RNA binding and regulation of viral transcription at the replication-transcription complex (RTC). The N protein can be divided into five main domains, and the central region is the linker, which is predicted to be primarily disordered and has not been heavily studied. The linker is Serine-Arginine Rich, which is phosphorylated at multiple sites by host kinases during infection, thereby promoting the N protein's role in viral transcription. Phosphorylation is a critical process for the regulation of many cellular processes and can provide recognition sites for binding complexes. In a study that examined the recognition of the SARS-CoV-2 N protein by the human 14-3-3 protein, the linker was found to contain critical phosphosites for 14-3-3 binding. The goals of this project are to determine the structure, dynamics, and RNA interactions of the Serine-Arginine Rich linker region. To accomplish this, we performed Nuclear Magnetic Resonance spectroscopy (NMR) experiments to analyze the structure of the linker region of the N protein and its ability to bind viral RNA. NMR confirms predictions that the linker is not entirely unstructured and it is able to bind RNA. The linker region of the N protein with phosphoserine incorporated at S188 was also examined via an NMR titration experiment with 1-1000 RNA. Compared to wild type, the incorporation of phosphorylation decreases binding. Other biophysical techniques such as Analytical Ultracentrifugation (AUC) and Multi-Angle Light Scattering (MALS) are used to identify the association state of the linker and the size of the resulting protein-RNA complex. We are currently working to biophysically characterize the structure, dynamics, and viral RNA binding ability of a mutation found in the Delta and Omicron variants: the R203M linker, which have been shown to enhance viral infectivity. This work was supported by the NSF EAGER grant NSF/ MCB 2034446 and URSA Engage. Support to facilities includes the Oregon State University NMR Facility funded in part by NIH, HEI Grant 1S10OD018518, and by the M. J. Murdock Charitable Trust grant # 2014162.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Accessing isotopically labeled proteins containing genetically encoded phosphoserine for NMR with optimized expression conditions.

Phosphoserine (pSer) sites are primarily located within disordered protein regions, making it difficult to experimentally ascertain their effects on protein structure and function. Therefore, the production of 15N- (and 13C)-labeled proteins with site-specifically encoded pSer for NMR studies is essential to uncover molecular mechanisms of protein regulation by phosphorylation. While genetic code expansion technologies for the translational installation of pSer in Escherichia coli are well established and offer a powerful strategy to produce site-specifically phosphorylated proteins, methodologies to adapt them to minimal or isotope-enriched media have not been described. This shortcoming exists because pSer genetic code expansion expression hosts require the genomic ΔserB mutation, which increases pSer bioavailability but also imposes serine auxotrophy, preventing growth in minimal media used for isotopic labeling of recombinant proteins. Here, by testing different media supplements, we restored normal BL21(DE3) ΔserB growth in labeling media but subsequently observed an increase of phosphatase activity and mis-incorporation not typically seen in standard rich media. After rounds of optimization and adaption of a high-density culture protocol, we were able to obtain ≥10 mg/L homogenously labeled, phosphorylated superfolder GFP. To demonstrate the utility of this method, we also produced the intrinsically disordered serine/arginine-rich region of the SARS-CoV-2 Nucleocapsid protein labeled with 15N and pSer at the key site S188 and observed the resulting peak shift due to phosphorylation by 2D and 3D heteronuclear single quantum correlation analyses. We propose this cost-effective methodology will pave the way for more routine access to pSer-enriched proteins for 2D and 3D NMR analyses.