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The catalytic domain of the calcium-dependent endoribonuclease EndoU from Homo sapiens was expressed in E. coli with 13 C and 15 N labeling. A nearly complete assignment of backbone 1 H, 15 N, and 13 C resonances was obtained, as well as a secondary structure prediction based on the assigned chemical shifts. The predicted secondary structures were almost identical to the published crystal structure of calcium-activated EndoU. This is the first NMR study of an eukaryotic member of the EndoU-like superfamily of ribonucleases., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Single domain antibody (sdAb) is only composed of a variable domain of the heavy-chain-only antibody, which is devoid of light chain and naturally occurring in camelids and cartilaginous fishes. Variable New Antigen Receptor (VNAR), a type of single domain antibody present in cartilaginous fishes such as sharks, is the smallest functional antigen-binding fragment found in nature. The unique features, including flexible paratope, high solubility and outstanding stability make VNAR a promising prospect in antibody drug development and structural biology research. However, VNAR's research has lagged behind camelid-derived sdAb, especially in the field of structural research. Here we report the 1 H, 15 N, 13 C resonance assignments of a VNAR derived from the immune library of Chiloscyllium plagiosum, termed B2-3, which recognizes the hyaluronan synthase. Analysis of the backbone chemical shifts demonstrates that the secondary structure of VNAR is predominately composed of β-sheets corresponding to around 40% of the B2-3 backbone. The Cβ chemical shift values of cysteine residues, combined with mass spectrometry data, clearly shows that B2-3 contains two pairs of disulfide bonds, which is import for protein stability. The assignments will be essential for determining the high resolution solution structure of B2-3 by NMR spectroscopy., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

A glycoside hydrolase family 45 (GH45) enzyme from the white-rot basidiomycete fungus Phanerochaete chrysosporium (PcCel45A) was expressed in Pichia pastoris with 13 C and 15 N labelling. A nearly complete assignment of 1 H, 13 C and 15 N backbone resonances was obtained, as well as the secondary structure prediction based on the assigned chemical shifts using the TALOS-N software. The predicted secondary structure was almost identical to previously published crystal structures of the same enzyme, except for differences in the termini of the sequence. This is the first NMR study using an isotopically labelled GH45 enzyme., (© 2024. The Author(s).)

The nucleocapsid (N) protein of SARS-CoV-2 is a multifunctional protein involved in nucleocapsid assembly and various regulatory functions. It is the most abundant protein during viral infection. Its functionality is closely related to its structure, which comprises two globular domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), flanked by intrinsically disordered regions. The linker between the NTD and CTD includes a Serine-Arginine rich (SR) region, which is crucial for the regulation of the N protein's function. Here, we report the near-complete assignment of the construct containing the NTD followed by the SR region (NTD-SR). Additionally, we describe the dynamic nature of the SR region and compare it with all other available chemical shift assignments reported for the SR region., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Klebsiella pneumoniae (Kp) poses an escalating threat to public health, particularly given its association with nosocomial infections and its emergence as a leading cause of neonatal sepsis, particularly in low- and middle-income countries (LMICs). Host cell adherence and biofilm formation of Kp is mediated by type 1 and type 3 fimbriae whose major fimbrial subunits are encoded by the fimA and mrkA genes, respectively. In this study, we focus on MrkA subunit, which is a 20 KDa protein whose 3D molecular structure remains elusive. We applied solution NMR to characterize a recombinant version of MrkA in which the donor strand segment situated at the protein's N-terminus is relocated to the C-terminus, preceded by a hexaglycine linker. This construct yields a self-complemented variant of MrkA. Remarkably, the self-complemented MrkA monomer loses its capacity to interact with other monomers and to extend into fimbriae structures. Here, we report the nearly complete assignment of the 13 C, 15 N labelled self-complemented MrkA monomer. Furthermore, an examination of its internal mobility unveiled that relaxation parameters are predominantly uniform across the polypeptide sequence, except for the glycine-rich region within loop 176-181. These data pave the way to a comprehensive structural elucidation of the MrkA monomer and to structurally map the molecular interaction regions between MrkA and antigen-induced antibodies., (© 2024. The Author(s).)

Monoclonal antibodies (mAbs) are biotherapeutics that have achieved outstanding success in treating many life-threatening and chronic diseases. The recognition of an antigen is mediated by the fragment antigen binding (Fab) regions composed by four different disulfide bridge-linked immunoglobulin domains. NMR is a powerful method to assess the integrity, the structure and interaction of Fabs, but site specific analysis has been so far hampered by the size of the Fabs and the lack of approaches to produce isotopically labeled samples. We proposed here an efficient in vitro method to produce [ 15 N, 13 C, 2 H]-labeled Fabs enabling high resolution NMR investigations of these powerful therapeutics. As an open system, the cell-free expression mode enables fine-tuned control of the redox potential in presence of disulfide bond isomerase to enhance the formation of native disulfide bonds. Moreover, inhibition of transaminases in the S30 cell-free extract offers the opportunity to produce perdeuterated Fab samples directly in 1 H 2 O medium, without the need for a time-consuming and inefficient refolding process. This specific protocol was applied to produce an optimally labeled sample of a therapeutic Fab, enabling the sequential assignment of 1 H N , 15 N, 13 C', 13 C α , 13 C β resonances of a full-length Fab. 90% of the backbone resonances of a Fab domain directed against the human LAMP1 glycoprotein were assigned successfully, opening new opportunities to study, at atomic resolution, Fabs' higher order structures, dynamics and interactions, using solution-state NMR., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Hepatocyte nuclear factor 1β (HNF1β) is a transcription factor that plays a key role in the development and function of the liver, pancreas, and kidney. HNF1β plays a key role in early vertebrate development and the morphogenesis of these organs. In humans, heterozygous mutations in the HNF1B gene can result in organ dysplasia, making it the most common cause of developmental renal diseases, including renal cysts, renal malformations, and familial hypoplastic glomerular cystic kidney disease. Pathogenic variants in the HNF1B gene are known to cause various diseases, including maturity-onset diabetes of the young and developmental renal diseases. This study presents the backbone resonance assignments of HNF1β POU S and POU HD domains, which are highly conserved domains required for the recognition of double-stranded DNA. Our data will be useful for NMR studies to verify the altered structures and functions of mutant HNF1B proteins that can induce developmental renal diseases, including renal cysts, renal malformations, and familial hypoplastic glomerular cystic kidney disease. This study will provide the structural basis for future studies to elucidate the molecular mechanisms underlying how mutations in HNF1β cause diseases., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The intraflagellar transport (IFT) machinery plays a crucial role in the bidirectional trafficking of components necessary for ciliary signaling, such as the Hedgehog, Wnt/PCR, and cAMP/PKA systems. Defects in some components of the IFT machinery cause dysfunction, leading to a wide range of human diseases and developmental disorders termed ciliopathies, such as nephronophthisis. The IFT machinery comprises three sub-complexes: BBsome, IFT-A, and IFT-B. The IFT protein 54 (IFT54) is an important component of the IFT-B sub-complex. In anterograde movement, IFT54 binds to active kinesin-II, walking along the cilia microtubule axoneme and carrying the dynein-2 complex in an inactive state, which works for retrograde movement. Several mutations in IFT54 are known to cause Senior-Loken syndrome, a ciliopathy. IFT54 possesses a divergent Calponin Homology (CH) domain termed as NN-CH domain at its N-terminus. However, several aspects of the function of the NN-CH domain of IFT54 are still obscure. Here, we report the 1 H, 15 N, and 13 C resonance assignments of the NN-CH domain of human IFT54 and its solution structure. The NN-CH domain of human IFT54 adopts essentially the α1-α2-α3-α4-α5 topology as that of mouse IFT54, whose structure was determined by X-ray crystallographic study. The structural information and assignments obtained in this study shed light on the molecular function of the NN-CH domain in IFT54., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Ricin is a potent plant toxin that targets the eukaryotic ribosome by depurinating an adenine from the sarcin-ricin loop (SRL), a highly conserved stem-loop of the rRNA. As a category-B agent for bioterrorism it is a prime target for therapeutic intervention with antibodies and enzyme blocking inhibitors since no effective therapy exists for ricin. Ricin toxin A subunit (RTA) depurinates the SRL by binding to the P-stalk proteins at a remote site. Stimulation of the N-glycosidase activity of RTA by the P-stalk proteins has been studied extensively by biochemical methods and by X-ray crystallography. The current understanding of RTA's depurination mechanism relies exclusively on X-ray structures of the enzyme in the free state and complexed with transition state analogues. To date we have sparse evidence of conformational dynamics and allosteric regulation of RTA activity that can be exploited in the rational design of inhibitors. Thus, our primary goal here is to apply solution NMR techniques to probe the residue specific structural and dynamic coupling active in RTA as a prerequisite to understand the functional implications of an allosteric network. In this report we present de novo sequence specific amide and sidechain methyl chemical shift assignments of the 267 residue RTA in the free state and in complex with an 11-residue peptide (P11) representing the identical C-terminal sequence of the ribosomal P-stalk proteins. These assignments will facilitate future studies detailing the propagation of binding induced conformational changes in RTA complexed with inhibitors, antibodies, and biologically relevant targets., (© 2024. The Author(s).)

In preparation for a detailed exploration of the structural and functional aspects of the Ser2Ala mutant of human carbonic anhydrase II, we present here almost complete sequence-specific resonance assignments for 1 H, 15 N, and 13 C. The mutation of serine to alanine at position 2, located in the N-terminal region of the enzyme, significantly alters the hydrophilic nature of the site, rendering it hydrophobic. Consequently, there is an underlying assumption that this mutation would repel water from the site. However, intriguingly, comparative analysis of the mutant structure with the wild type reveals minimal discernible differences. These assignments serve as the basis for in-depth studies on histidine dynamics, protonation states, and its intricate role in protein-water interactions and catalysis., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Human La-related protein 1 (HsLARP1) is involved in post-transcriptional regulation of certain 5' terminal oligopyrimidine (5'TOP) mRNAs as well as other mRNAs and binds to both the 5'TOP motif and the 3'-poly(A) tail of certain mRNAs. HsLARP1 is heavily involved in cell proliferation, cell cycle defects, and cancer, where HsLARP1 is significantly upregulated in malignant cells and tissues. Like all LARPs, HsLARP1 contains a folded RNA binding domain, the La motif (LaM). Our current understanding of post-transcriptional regulation that emanates from the intricate molecular framework of HsLARP1 is currently limited to small snapshots, obfuscating our understanding of the full picture on HsLARP1 functionality in post-transcriptional events. Here, we present the nearly complete resonance assignment of the LaM of HsLARP1, providing a significant platform for future NMR spectroscopic studies., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Human K-Ras protein, which is a member of the GTPase Ras family, hydrolyzes GTP to GDP and concomitantly converts from its active to its inactive state. It is a key oncoprotein, because several mutations, particularly those at residue position 12, occur with a high frequency in a wide range of human cancers. The K-Ras protein is therefore an important target for developing therapeutic anti-cancer agents. In this work we report the almost complete sequence-specific resonance assignments of wild-type and the oncogenic G12C and G12D mutants in the GTP-complexed active forms, including the functionally important Switch I and Switch II regions. These assignments serve as the basis for a comprehensive functional dynamics study of wild-type K-Ras and its G12 mutants., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Human small EDRK-rich factor protein SERF2 is a cellular driver of protein amyloid formation, a process that has been linked to neurodegenerative diseases including Alzheimer's and Parkinson's disease. SERF2 is a 59 amino acid protein, highly charged, and well conserved whose structure and physiological function is unclear. SERF family proteins including human SERF2 have shown a tendency to form fuzzy complexes with misfolded proteins such as α-Synuclein which has been linked to Parkinson's disease. SERF family proteins have been recently identified to bind nucleic acids, but the binding mechanism(s) remain enigmatic. Here, using multidimensional solution NMR, we report the 1 H, 15 N, and 13 C chemical shift assignments (~ 86% of backbone resonance assignments) for human SERF2. TALOS-N predicted secondary structure of SERF2 showed three very short helices (3-4 residues long) in the N-terminal region of the protein and a long helix in the C-terminal region spanning residues 37-46 which is consistent with the helical content indicated by circular dichroism spectroscopy. Paramagnetic relaxation enhancement NMR analysis revealed that a short C-terminal region E53-K55 is in the proximity of the N-terminus. Having the backbone assignment of SERF2 allowed us to probe its interaction with α-Synuclein and to identify the residues in SERF2 binding interfaces that likely promote α-Synuclein aggregation., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The backbone 1 H, 13 C and 15 N resonance assignment of Ubiquitin Specific Protease 7 catalytic domain (residues 208-554) was performed in its complex with a small molecule ligand and in its apo form as a reference. The amide 1 H- 15 N signal intensities were boosted by an amide hydrogen exchange protocol, where expressed 2 H, 13 C, 15 N-labeled protein was unfolded and re-folded to ensure exchange of amide deuterons to protons. The resonance assignments were used to determine chemical shift perturbations on ligand binding, which are consistent with the binding site observed by crystallography., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

A large proportion of human proteins contain post-translational modifications that cannot be synthesized by prokaryotes. Thus, mammalian expression systems are often employed to characterize structure/function relationships using NMR spectroscopy. Here we define the selective isotope labeling of secreted, post-translationally modified proteins using human embryonic kidney (HEK)293 cells. We determined that alpha-[ 15 N]- atoms from 10 amino acids experience minimal metabolic scrambling (C, F, H, K, M, N, R, T, W, Y). Two more interconvert to each other (G, S). Six others experience significant scrambling (A, D, E, I, L, V). We also demonstrate that tuning culture conditions suppressed V and I scrambling. These results define expectations for 15 N-labeling in HEK293 cells., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

NCYM is a cis-antisense gene of MYCN oncogene and encodes an oncogenic protein that stabilizes MYCN via inhibition of GSK3b. High NCYM expression levels are associated with poor clinical outcomes in human neuroblastomas, and NCYM overexpression promotes distant metastasis in animal models of neuroblastoma. Using vacuum-ultraviolet circular dichroism and small-angle X-ray scattering, we previously showed that NCYM has high flexibility with partially folded structures; however, further structural characterization is required for the design of anti-cancer agents targeting NCYM. Here we report the 1 H, 15 N and 13 C nuclear magnetic resonance assignments of NCYM. Secondary structure prediction using Secondary Chemical Shifts and TALOS-N analysis demonstrates that the structure of NCYM is essentially disordered, even though residues in the central region of the peptide clearly present a propensity to adopt a dynamic helical structure. This preliminary study provides foundations for further analysis of interaction between NCYM and potential partners., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

In this study, we collected 56 species of fishery organisms (including fish, crustaceans, cephalopods, gastropods, and bivalves) from four seasonal survey cruises at the Dachen fishery grounds. We measured the concentrations of seven heavy metals (Cd, Zn, Cu, Pb, Cr, As, and Hg) in these fisheries organisms. We determined their trophic levels using carbon and nitrogen stable isotope techniques. We analyzed the characteristics of heavy metal transfer in the food chain. The results showed significant differences in heavy metal concentrations among different species. Among all biological groups, bivalves and gastropods exhibited higher levels of heavy metal enrichment than other biological groups, while fish had the lowest levels of heavy metal enrichment. Heavy metals exhibited different patterns of nutritional transfer in the food chain. While Hg showed a biomagnification phenomenon in the food chain, it was not significant. Cd, Zn, Cu, Pb, Cr, and As exhibited a trend of biodilution with increasing nutritional levels, except for As, which showed no significant correlation with δ 15 N., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

It is essential to study the sources of suspended particulate organic matter (SPOM) for a comprehensive understanding of the carbon and nitrogen cycles in reservoir located in desert grassland areas. Atmospheric deposition is an important pathway for human and natural nutrients to enter water ecosystems. Previous studies have focused on hotspot areas, but neglected the impact of atmospheric deposition on reservoirs in desert grassland regions with little rainfall, long freezing periods, and a dusty climate. In this study, we measured the contents of organic carbon, total nitrogen, the isotopic composition (δ 13 C and δ 15 N), and the ratio of total organic carbon to total nitrogen (C/N ratios) for SPOM in the reservoir and its watershed across both dry and wet seasons using carbon and nitrogen isotope techniques. We also analyzed the sources of SPOM in the reservoir using the end-member mixing model. The results showed that the variation range in SPOM for δ 13 C was - 28.3 to - 21.8‰; for δ 15 N, it was 2.3 to 8.7‰; and the C/N ratios were 8.12 to 19.12. The variation range of δ 13 C for atmospheric particulate matter was - 20.0 to - 25.0‰; for δ 15 N, it was 2.3 to 11.9‰; and the C/N ratios ranged from 7.22 to 17.81. The main sources of carbon in the SPOM were atmospheric deposition and terrestrial C3 plants. The origins were significantly different between the wet and dry periods. Atmospheric nitrogen deposition and soil erosion were the primary sources of particulate nitrogen in the reservoir., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Recent studies have demonstrated that there exists significant variability in amino acid (AA) δ 15 N values of terrestrial plants, discriminating among plant types (i.e., legume seeds, grasses, tree leaves) as well as tissues of the same plant. For the first time, we investigate the potential of the spacing between the δ 15 N values of different AAs to differentiate between plant types and thus elucidate their relative importance in herbivore diet. Using principal component analysis, we show that it is possible to distinguish among five plant categories-cereal grains, rachis, legume seeds, herbaceous plants, and woody plants-whose consumption has different implications for understanding herbivore ecology and management practices. Furthermore, we were able to correctly classify the herbaceous plant diet of modern cattle using AA δ 15 N values of their tooth dentine adjusted for trophic enrichment. The AA δ 15 N patterns of wild and domestic herbivores from archaeological sites seem to be consistent with diets comprised predominantly of herbaceous plants, but there is variation in AA δ 15 N values among individuals that may reflect differing inputs of other plant types. The variation in AA δ 15 N values does not necessarily reflect the variation in herbivore bulk collagen δ 13 C and δ 15 N values, indicating that AA δ 15 N values have the potential to provide additional insights into plant dietary sources compared to bulk tissue isotope values alone. Future work should focus on defining trophic enrichment factors for a wider range of terrestrial herbivores and expanding libraries of primary producer AA δ 15 N values., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Understanding how intraspecific variation in the use of prey resources impacts energy metabolism has strong implications for predicting long-term fitness and is critical for predicting population-to-community level responses to environmental change. Here, we examine the energetic consequences of variable prey resource use in a widely distributed marine carnivore, juvenile sand tiger sharks (Carcharias taurus). We used carbon and nitrogen isotope analysis to identify three primary prey resource pools-demersal omnivores, pelagic forage, and benthic detritivores and estimated the proportional assimilation of each resource using Bayesian mixing models. We then quantified how the utilization of these resource pools impacted the concentrations of six plasma lipids and how this varied by ontogeny. Sharks exhibited variable reliance on two of three predominant prey resource pools: demersal omnivores and pelagic forage. Resource use variation was a strong predictor of energetic condition, whereby individuals more reliant upon pelagic forage exhibited higher blood plasma concentrations of very low-density lipoproteins, cholesterol, and triglycerides. These findings underscore how intraspecific variation in resource use may impact the energy metabolism of animals, and more broadly, that natural and anthropogenically driven fluctuations in prey resources could have longer term energetic consequences., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A coupled methodology of nitrogen isotopes, hydrogeochemical characterization, multivariate statistical analysis, and SIAR Bayesian modeling has been employed to identify the sources of NO 3 - and N transformation processes in three alluvial aquifers (Schinos, Thiva, and Central Evia) located in central Greece where geogenic Cr(VI) co-occurs with agricultural activity and rural development. Hexavalent chromium concentrations exceed 50 μg/L in many sampling stations of the studied groundwater bodies, while nitrate contamination is evident in all three study areas with concentrations well over 50 mg/L. The mean δ 15 N-NO 3 - and δ 18 Ο-NO 3 - values are 6.67 ± 1.77‰ and 2.68 ± 1.77‰ in C. Evia, 8.72 ± 4.74‰ and 3.96 ± 4.57‰ in Schinos and 4.44 ± 1.71‰ and 2.91 ± 1.02‰ in Thiva, respectively. Domestic sewage and N-bearing fertilizers are contributing in various degrees to the observed nitrification which is the dominant transformation process of N in the studied aquifers. Multivariate statistics indicated that the main processes identified in the study areas are salinization, silicate dissolution, and groundwater contamination due to fertilizer use. It is suggested that ultramafic rock-related alluvial aquifers must be closely monitored in terms of nutrient inputs as an effective measure for controlling Cr(VI) release in groundwater., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Nitrate (NO 3 - ) pollution is a common phenomenon in karst underground rivers, which are important water sources in karst landscapes. For drinking water safety and environmental protection, it is crucial to accurately identify NO 3 - sources and their migration and transformation processes in the Lihu Underground River. In this study, water samples of the Lihu Underground River in Guangxi were collected in May 2014, October 2014, January 2015, and July 2015, and water chemical and dual isotopic (δ 15 N-NO 3 - and δ 18 O-NO 3 - ) approaches were used to evaluate the NO 3 - characteristics and sources in the Lihu Underground River. The concentration of NO 3 - in the Lihu Underground River ranged from 1.16 to 19.78 mg·L -1 , with an average of 9.30 mg·L -1 , which is more than 37% of the WHO standard (10 mg·L -1 ). The concentrations of NO 3 - in the wet season (May 2014 and July 2015) were slightly lower than those in the dry season (from October 2014 to January 2015) at most sampling sites due to dilution effects. The migration and transformation processes of NO 3 - were analyzed by comparing the measured and calculated concentrations of NO 3 - in the Lihu Underground River. In the dry season (from October 2014 to January 2015), the variation in NO 3 - concentration upstream and midstream of the Lihu Underground River was affected by exogenous input and nitrification. From midstream to the outlet of Xiaolongdong, it is affected by self-purification factors, including physical processes, chemical processes, and biological processes. In the wet season (May 2014 and July 2015), the dilution and mixing effects were the main factors controlling the variation in NO 3 - concentration in the Lihu Underground River. The contribution rates of potential NO 3 - sources (incl. atmospheric precipitation (AP), NO 3 - fertilizer (NF), NH 4 + in fertilizer and rainfall (NFA), soil organic nitrogen (SON), and manure and sewage (M&S)) were quantitatively evaluated by using the IsoSource model. The results showed that in May 2014, the main sources of NO 3 - were M&S and NF, with contribution rates of 46% and 41%, respectively. In October 2014, NO 3 - sources were M&S with a contribution rate of 47%, followed by NFA with a contribution rate of 31%. In January 2015, NO 3 - sources in groundwater were M&S, with a contribution rate of 53%, followed by NFA (34%). In July 2015, the main NO 3 - sources were M&S and NF, whose contribution rates were 54% and 39%, respectively., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Nitrogen isotope (δ 15 N) analysis of bulk tissues and individual amino acids (AA) can be used to assess how consumers maintain nitrogen balance with broad implications for predicting individual fitness. For elasmobranchs, a ureotelic taxa thought to be constantly nitrogen limited, the isotopic effects associated with nitrogen-demanding events such as prolonged gestation remain unknown. Given the linkages between nitrogen isotope variation and consumer nitrogen balance, we used AA δ 15 N analysis of muscle and liver tissue collected from female bonnethead sharks (Sphyrna tiburo, n = 16) and their embryos (n = 14) to explore how nitrogen balance may vary across gestation. Gestational stage was a strong predictor of bulk tissue and AA δ 15 N values in pregnant shark tissues, decreasing as individuals neared parturition. This trend was observed in trophic (e.g., Glx, Ala, Val), source (e.g., Lys), and physiological (e.g., Gly) AAs. Several potential mechanisms may explain these results including nitrogen conservation, scavenging, and bacterially mediated breakdown of urea to free ammonia that is used to synthesize AAs. We observed contrasting patterns of isotopic discrimination in embryo tissues, which generally became enriched in 15 N throughout development. This was attributed to greater excretion of nitrogenous waste in more developed embryos, and the role of physiologically sensitive AAs (i.e., Gly and Ser) to molecular processes such as nucleotide synthesis. These findings underscore how AA isotopes can quantify shifts in nitrogen balance, providing unequivocal evidence for the role of physiological condition in driving δ 15 N variation in both bulk tissues and individual AAs., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Nitrate (NO 3 - ) loss and enrichment in water bodies caused by fertilization are a major environmental problem in agricultural areas. However, the quantitative contribution of different NO 3 - sources, especially chemical fertilizers (CF) and soil organic nitrogen (SON), to NO 3 - runoff loss remains unclear. In this study, a systematic investigation of NO 3 - runoff and its sources was conducted in a subtropical agricultural watershed located in Yujiang County, Jiangxi Province, China. A semi-monthly sampling was performed at the inlet and outlet from March 2018 to February 2019. Hydrochemical and dual NO 3 - isotope ( 15  N and 18 O) approaches were combined to estimate the NO 3 - runoff loss and quantify the contribution of different sources with a Bayesian isotope mixing model. Source apportionment by Stable Isotope Analysis in R (SIAR) suggested that NO 3 - in runoff was mainly derived from nitrification of ammonium (NH 4 + ) mineralized from SON (37-52%) and manure/sewage (M&S) (25-47%), while the contribution of CF was relatively small (14-25%). The contribution of various sources showed seasonal variations, with a greater contribution of CF in the wet growing season (March to August). Compared with the inlet which contributed 37-40% to runoff NO 3 - , SON contributed more at the outlet (49-52%). Denitrification in the runoff was small and appeared to be confined to the dry season (September to February), with an estimated NO 3 - loss of 2.73 kg N ha -1 . The net NO 3 - runoff loss of the watershed was 34.5 kg N ha -1  yr -1 , accounting for 15% of the annual fertilization rate (229 kg N ha -1  yr -1 ). Besides M&S (22%), fertilization and remineralization of SON (CF + SON) were the main sources for the NO 3 - runoff loss (78%), suggesting accelerated nitrification of NH 4 + from CF (24%) and SON mineralization (54%). Our study indicates that NO 3 - runoff loss in subtropical agricultural watersheds is dominated by nonpoint source pollution from fertilization. SON played a more important role than CF. Besides, the contribution of sewage should not be neglected. Our data suggest that a combination of more rational fertilizer N application (CF), better management of SON, and better treatment of domestic sewage could alleviate NO 3 - pollution in subtropical China., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The New Zealand huia (Heteralocha acutirostris) had the most extreme bill sexual dimorphism among modern birds. Given the quick extinction of the species, the cause of the dimorphism could only be hypothesised to reflect different trophic niches and reduce male/female competition. We tested that hypothesis by combining museum specimens, geometric morphometrics, and isotopic analyses. We used geometric morphometrics to describe bill shape; measured bulk (δ 15 N bulk ) and (δ 13 C bulk ) values from feather as proxies of the birds' foraging habitat and diet; and compared compound-specific stable isotopes analyses (CSIA) of nitrogen in amino acids (δ 15 N AA ) in male-female pairs to estimate their trophic position. Sexes had significantly different, but overlapping feather δ 15 N bulk and δ 13 C bulk values, but δ 15 N AA indicated identical trophic positions and δ 15 N bulk was not related to bill shape. Trophic position was less variable among females, consistent with a specialised foraging behaviour and, thus, supporting a partial male/female foraging segregation., (© 2021. The Author(s).)

The SARS-CoV-2 virus is the cause of the respiratory disease COVID-19. As of today, therapeutic interventions in severe COVID-19 cases are still not available as no effective therapeutics have been developed so far. Despite the ongoing development of a number of effective vaccines, therapeutics to fight the disease once it has been contracted will still be required. Promising targets for the development of antiviral agents against SARS-CoV-2 can be found in the viral RNA genome. The 5'- and 3'-genomic ends of the 30 kb SCoV-2 genome are highly conserved among Betacoronaviruses and contain structured RNA elements involved in the translation and replication of the viral genome. The 40 nucleotides (nt) long highly conserved stem-loop 4 (5_SL4) is located within the 5'-untranslated region (5'-UTR) important for viral replication. 5_SL4 features an extended stem structure disrupted by several pyrimidine mismatches and is capped by a pentaloop. Here, we report extensive 1 H, 13 C, 15 N and 31 P resonance assignments of 5_SL4 as the basis for in-depth structural and ligand screening studies by solution NMR spectroscopy., (© 2021. The Author(s).)

The human UDP-glucuronosyltransferase (UGT) family of enzymes catalyze the covalent addition of glucuronic acid to a wide range of compounds, generally rendering them inactive. Although important for clearance of environmental toxins and metabolites, UGT activation can lead to inappropriate glucuronidation of therapeutics underlying drug resistance. Indeed, 50% of medications are glucuronidated. To better understand this mode of resistance, we studied the UGT2B7 enzyme associated with glucuronidation of cancer drugs such as Tamoxifen and Sorafenib. We report 1 H,  13 C and 15 N backbone (> 90%) and side-chain assignments (~ 78% completeness according to CYANA) for the C-terminal domain of UGT2B7 (UGT2B7-C). Given the biomedical importance of this family of enzymes, our assignments will provide a key tool for improving understanding of the biochemical basis for substrate selectivity and other aspects of enzyme activity. This in turn will inform on drug design to overcome UGT-related drug resistance., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Spider silk is renowned for its excellent mechanical properties. Among six types of silk and one silk glue produced by different abdominal glands for various purposes, tubuliform (eggcase) silk is unique due to its high serine and low glycine content. Eggcase silk is spun from at least two spidroins, tubuliform spidroin 1 (TuSp1) and TuSp2. TuSp1 and TuSp2 were identified as the major and the minor components in tubuliform glands, respectively. TuSp2 consists of multiple repetitive (RP) domains with short terminal tails and shares very limited homology to all known spidroins. Here we report backbone and side chain resonance assignments of TuSp2-RP as a basis for structural and functional studies on eggcase silk formation., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

During the maturation of the HIV-1 particle, the Gag polyprotein is cleaved by the viral protease into several proteins: matrix (MA), capsid (CA), spacer peptide 1 (SP1), nucleocapsid (NC), spacer peptide 2 (SP2) and p6. After cleavage, these proteins rearrange to form infectious viral particles. The final cleavage by the protease occurs between CA and SP1 and is the limiting step for the maturation of the particle. The CA-SP1 junction is the target of HIV-1 maturation inhibitors. CA is responsible for the formation of the viral capsid which protects the viral RNA inside. The SP1 domain is essential for viral assembly and infectivity, it is flexible and in helix-coil equilibrium. The presence of NC allows the SP1 domain to be less dynamic. The perturbation of the natural coil-helix equilibrium to helix interferes with protease cleavage and leads to non-completion of viral maturation. In this work, two mutations, W316A and M317A, that abolish the oligomerization of CA were introduced into the protein. The HIV-1 CA CTD W316A, M317A -SP1-NC which contains the C-terminal monomeric mutant of CA, SP1 and NC was produced to study the mechanism of action of HIV-1 maturation inhibitors. Here we report the backbone assignment of the protein CA CTD W316A, M317A -SP1-NC. These results will be useful to study the interaction between HIV-1 Gag and HIV-1 maturation inhibitors., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Type II secretion systems (T2SS) allow Gram-negative bacteria to transport toxins and enzymes from the periplasm to the external milieu, and are thus important for the pathogenicity of bacteria. To drive secretion, T2SS assemble filaments called pseudopili closely related to bacterial type IV pili. These filaments are non-covalent polymers of proteins that are assembled by an inner membrane complex called the assembly platform connected to a cytoplasmic ATPase motor. In the Klebsiella oxytoca T2SS, the PulL protein from the assembly platform is essential for pseudopilus assembly and protein secretion. However, its role in these processes is not well understood. To decipher the molecular basis of PulL function, we used solution NMR to study its structure and interactions with other components of the machinery. Here as a first step, we report the 1 H, 15  N and 13 C backbone and side-chain chemical shift assignments of the C-terminal periplasmic domain of PulL and its secondary structure based on NMR data., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7-33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1 H, 13 C and 15 N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair., (© 2021. The Author(s).)

SET (TAF-1β/I2PP2A) is a ubiquitously expressed, multifunctional protein that plays a role in regulating diverse cellular processes, including cell cycle progression, migration, apoptosis, transcription, and DNA repair. SET expression is ubiquitous across all cell types. However, it is overexpressed or post-translationally modified in several solid tumors and blood cancers, where expression levels are correlated with worsening clinical outcomes. SET exerts its oncogenic effects primarily through the formation of antagonistic protein complexes with the tumor suppressor, protein phosphatase 2A (PP2A), and the well-known metastasis suppressor, nm23-H1. PP2A inhibition is often observed as a secondary driver of tumorigenesis and metastasis in human cancers. Preclinical studies have shown that the pharmacological reactivation of PP2A combined with potent inhibitors of the primary driver oncogene produces synergistic cell death and decreased drug resistance. Therefore, the development of novel inhibitors of the SET-PP2A interaction presents an attractive approach to reactivation of PP2A, and thereby, tumor suppression. NMR provides a unique platform to investigate protein targets in their natively folded state to identify protein and small-molecule ligands and report on the protein internal dynamics. The backbone 1 H N , 13 C, and 15 N NMR resonance assignments were completed for the 204 amino acid nucleosome assembly protein-1 (NAP-1) domain of the human SET oncoprotein (residues 23-225). These assignments provide a vital first step toward the development of novel PP2A reactivators via SET-selective inhibition., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The current COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has become a worldwide health crisis, necessitating coordinated scientific research and urgent identification of new drug targets for treatment of COVID-19 lung disease. The covid19-nmr consortium seeks to support drug development by providing publicly accessible NMR data on the viral RNA elements and proteins. The SARS-CoV-2 genome comprises a single RNA of about 30 kb in length, in which 14 open reading frames (ORFs) have been annotated, and encodes approximately 30 proteins. The first two-thirds of the SARS-CoV-2 genome is made up of two large overlapping open-reading-frames (ORF1a and ORF1b) encoding a replicase polyprotein, which is subsequently cleaved to yield 16 so-called non-structural proteins. The non-structural protein 1 (Nsp1), which is considered to be a major virulence factor, suppresses host immune functions by associating with host ribosomal complexes at the very end of its C-terminus. Furthermore, Nsp1 facilitates initiation of viral RNA translation via an interaction of its N-terminal domain with the 5' untranslated region (UTR) of the viral RNA. Here, we report the near-complete backbone chemical-shift assignments of full-length SARS-CoV-2 Nsp1 (19.8 kDa), which reveal the domain organization, secondary structure and backbone dynamics of Nsp1, and which will be of value to further NMR-based investigations of both the biochemical and physiological functions of Nsp1., (© 2021. The Author(s).)

The sequence-specific backbone assignment of the mitogen-activated protein kinase (MAPK) binding domain of the dual-specificity phosphatase 1 (DUSP1) has been accomplished using a uniformly [ 13 C, 15 N]-labeled protein. These assignments will facilitate further studies of DUSP1 in the presence of inhibitors/ligands to target MAPK associated diseases and provide further insights into the function of dual-specificity phosphatase 1 in MAPK regulation., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Human prion disease, also known as transmissible spongiform encephalopathy (TSEs), is caused by the conformational conversion of the normal cellular prion protein (PrP C ) into the scrapie form (PrP Sc ). Pathogenic point mutations of prion proteins typically facilitate conformational conversion and lead to inherited prion diseases. A previous study has demonstrated that the pathogenic G131V mutation of human prion protein (HuPrP) brings in Gerstmann-Sträussler-Scheinker syndrome. However, the three-dimensional structure and dynamic features of the HuPrP(G131V) mutant remain unclear. It is expected that the determination of these structural bases will be beneficial to the pathogenic mechanistic understanding of G131V-related prion diseases. Here, we performed 1 H, 15 N, 13 C backbone and side-chain resonance assignments of the G131V mutant of HuPrP(91-231) by using heteronuclear multi-dimensional NMR spectroscopy, and predicted the secondary structural elements and order parameters of the protein based on the assigned backbone chemical shifts. Our work lays the necessary foundation for further structural determination, dynamics characterization, and intermolecular interaction assay for the G131V mutant., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

KIN is a DNA/RNA-binding protein conserved evolutionarily from yeast to humans and expressed ubiquitously in mammals. It is an essential nuclear protein involved in numerous cellular processes, such as DNA replication, class-switch recombination, cell cycle regulation, and response to UV or ionizing radiation-induced DNA damage. The C-terminal region of the human KIN (hKIN) protein is composed of an SH3-like tandem domain, which is crucial for the anti-proliferation effect of the full-length protein. Herein, we present the 1 H, 15 N, and 13 C resonances assignment of the backbone and side chains for the SH3-like tandem domain of the hKIN protein, as well as the secondary structure prediction based on the assigned chemical shifts using TALOS-N software. This work prepares the ground for future studies of RNA-binding and backbone dynamics., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Thioredoxins are ubiquitous and conserved small proteins. The redox-active site is composed of highly conserved Cys32 and Cys35. In higher eukaryotes, thioredoxin evolved to a gain of function in nitrosative control, with 3 extra cysteines, Cys62, Cys69, and Cys73. Human thioredoxin 1 (hTrx) is directly involved in cellular signal transduction through S-nitrosation. The understanding of the mechanism of S-nitrosation is essential. Here we produced a mutant of hTrx containing only Cys62 (C62only). We report the almost full 1H, 15N, and 13C chemical shift assignment of the reduced and S-nitrosated C62only. This study will help to measure the reactivity Cys62 toward S-nitrosants and the stability of S-nitrosated Cys62., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

DNA double-strand breaks (DSBs) represent the most cytotoxic DNA lesions, as-if mis- or unrepaired-they can cause cell death or lead to genome instability, which in turn can cause cancer. DSBs are repaired by two major pathways termed homologous recombination and non-homologous end-joining (NHEJ). NHEJ is responsible for repairing the vast majority of DSBs arising in human cells. Defects in NHEJ factors are also associated with microcephaly, primordial dwarfism and immune deficiencies. One of the key proteins important for mediating NHEJ is XRCC4. XRCC4 is a dimer, with the dimer interface mediated by an extended coiled-coil. The N-terminal head domain forms a mixed alpha-beta globular structure. Numerous factors interact with the C-terminus of the coiled-coil domain, which is also associated with significant self-association between XRCC4 dimers. A range of construct lengths of human XRCC4 were expressed and purified, and the 1-164 variant had the best NMR properties, as judged by consistent linewidths, and chemical shift dispersion. In this work we report the 1 H, 15  N and 13 C backbone resonance assignments of human XRCC4 in the solution form of the 1-164 construct. Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 156 of 161 assignable residues of XRCC4 were assigned to resonances in the TROSY spectrum, with an additional 11 resonances assigned to His-Tag residues. Prediction of solution secondary structure from a chemical shift analysis using the TALOS + webserver is in good agreement with the published X-ray crystal structures of this protein., (© 2021. The Author(s).)

Tuberculosis (TB), a lethal disease caused by Mycobacterium tuberculosis (Mtb) infection, develops multidrug-resistance and needs new drugs for effective treatment. As a ribosome maturation factor protein, RimM plays an essential role in the bacterial ribosome assembly and is a potential target for antibiotics against TB. RimM is involved in the incorporation of ribosomal protein S19 into the 30 S ribosomal subunit, where the C-terminal domain of RimM is speculated to bind S19. However, the structure and dynamics features of MtbRimM remain unclear to date. Herein, we report the NMR assignments for the 1 H, 13 C, 15 N backbone and side-chain resonances of the C-terminal domain of MtbRimM. We also provide the prediction of its secondary structure and order parameters. Our work lays the basis for solution structure, dynamics and functional studies on MtbRimM in future, and provides clues for the anti-tuberculosis drug development., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

In the present study, the performance of a set of density functionals: BP86, PBE, OLYP, BEEF, PBEpow, TPSS, SCAN, PBEGXPBE, M06L, MN15L, B3LYP, PBE0, mPW1PW, B97, BHandHLYP, mPW1PW, B98, TPSS0, PBE1KCIS, SCAN0, M06, M06-2X, MN15, CAM-B3LYP, ωB97x, B2PLYP, and the B3LYP/N07D and PBE/N07D schemes in the calculation of the 14 N anisotropic hyperfine coupling (HFC) constants of a set of 23 nitroxide radicals is evaluated. The results are compared with those obtained with the DLPNO-CCSD method and experimental HFC values. Harmonic contribution to the 14 N HFC vibrational correction was calculated at the revPBE0/def2-TZVPP level and included in the evaluation. With the vibrational correction, the DLPNO-CCSD method yielded HFC values in good agreement with the experiment (mean absolute deviation (MAD) = 0.3 G for the dipole-dipole contribution and MAD = 0.8 G for the contact coupling contribution). The best DFT results are obtained using the M06 functional with MAD = 0.2 G for the dipole-dipole contribution and MAD = 0.7 G for the contact coupling contribution. In general, vibrational correction significantly improved most DFT functionals' performance but did not change its overall ranking.

The non-structural protein nsp3 from SARS-CoV-2 plays an essential role in the viral replication transcription complex. Nsp3a constitutes the N-terminal domain of nsp3, comprising a ubiquitin-like folded domain and a disordered acidic chain. This region of nsp3a has been linked to interactions with the viral nucleoprotein and the structure of double membrane vesicles. Here, we report the backbone resonance assignment of both domains of nsp3a. The study is carried out in the context of the international covid19-nmr consortium, which aims to characterize SARS-CoV-2 proteins and RNAs, providing for example NMR chemical shift assignments of the different viral components. Our assignment will provide the basis for the identification of inhibitors and further functional and interaction studies of this essential protein.

SARS-CoV-2 RNA, nsP3c (non-structural Protein3c) spans the sequence of the so-called SARS Unique Domains (SUDs), first observed in SARS-CoV. Although the function of this viral protein is not fully elucidated, it is believed that it is crucial for the formation of the replication/transcription viral complex (RTC) and of the interaction of various viral "components" with the host cell; thus, it is essential for the entire viral life cycle. The first two SUDs, the so-called SUD-N (the N-terminal domain) and SUD-M (domain following SUD-N) domains, exhibit topological and conformational features that resemble the nsP3b macro (or "X") domain. Indeed, they are all folded in a three-layer α/β/α sandwich structure, as revealed through crystallographic structural investigation of SARS-CoV SUDs, and they have been attributed to different substrate selectivity as they selectively bind to oligonucleotides. On the other hand, the C-terminal SUD (SUD-C) exhibit much lower sequence similarities compared to the SUD-N & SUD-M, as reported in previous crystallographic and NMR studies of SARS-CoV. In the absence of the 3D structures of SARS-CoV-2, we report herein the almost complete NMR backbone and side-chain resonance assignment ( 1 H, 13 C, 15 N) of SARS-CoV-2 SUD-M and SUD-C proteins, and the NMR chemical shift-based prediction of their secondary structure elements. These NMR data will set the base for further understanding at the atomic-level conformational dynamics of these proteins and will allow the effective screening of a large number of small molecules as binders with potential biological impact on their function.

Among the proteins encoded by the SARS-CoV-2 RNA, nsP3 (non-structural Protein3) is the largest multi-domain protein. Its role is multifaceted and important for the viral life cycle. Nonetheless, regarding the specific role of each domain there are many aspects of their function that have to be investigated. SARS Unique Domains (SUDs), constitute the nsP3c region of the nsP3, and were observed for the first time in SARS-CoV. Two of them, namely SUD-N (the first SUD) and the SUD-M (sequential to SUD-N), exhibit structural homology with nsP3b ("X" or macro domain); indeed all of them are folded in a three-layer α/β/α sandwich. On the contrary, they do not exhibit functional similarities, like ADP-ribose binding properties and ADP-ribose hydrolase activity. There are reports that suggest that these two SUDs may exhibit a binding selectivity towards G-oligonucleotides, a feature which may contribute to the characterization of their role in the formation of the replication/transcription viral complex (RTC) and of the interaction of various viral "components" with the host cell. While the structures of these domains of SARS-CoV-2 have not been determined yet, SUDs interaction with oligonucleotides and/or RNA molecules may provide a platform for drug discovery. Here, we report the almost complete NMR backbone and side-chain resonance assignment ( 1 H, 13 C, 15 N) of SARS-CoV-2 SUD-N protein, and the NMR chemical shift-based prediction of the secondary structure elements. These data may be exploited for its 3D structure determination and the screening of chemical compounds libraries, which may alter SUD-N function.

The current outbreak of the highly infectious COVID-19 respiratory disease is caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). To fight the pandemic, the search for promising viral drug targets has become a cross-border common goal of the international biomedical research community. Within the international Covid19-NMR consortium, scientists support drug development against SARS-CoV-2 by providing publicly available NMR data on viral proteins and RNAs. The coronavirus nucleocapsid protein (N protein) is an RNA-binding protein involved in viral transcription and replication. Its primary function is the packaging of the viral RNA genome. The highly conserved architecture of the coronavirus N protein consists of an N-terminal RNA-binding domain (NTD), followed by an intrinsically disordered Serine/Arginine (SR)-rich linker and a C-terminal dimerization domain (CTD). Besides its involvement in oligomerization, the CTD of the N protein (N-CTD) is also able to bind to nucleic acids by itself, independent of the NTD. Here, we report the near-complete NMR backbone chemical shift assignments of the SARS-CoV-2 N-CTD to provide the basis for downstream applications, in particular site-resolved drug binding studies.

The Betacoronavirus SARS-CoV-2 non-structural protein Nsp9 is a 113-residue protein that is essential for viral replication, and consequently, a potential target for the development of therapeutics against COVID19 infections. To capture insights into the dynamics of the protein's backbone in solution and accelerate the identification and mapping of ligand-binding surfaces through chemical shift perturbation studies, the backbone 1 H, 13 C, and 15 N NMR chemical shifts for Nsp9 have been extensively assigned. These assignments were assisted by the preparation of an ~ 70% deuterated sample and residue-specific, 15 N-labelled samples (V, L, M, F, and K). A major feature of the assignments was the "missing" amide resonances for N96-L106 in the 1 H- 15 N HSQC spectrum, a region that comprises almost the complete C-terminal α-helix that forms a major part of the homodimer interface in the crystal structure of SARS-CoV-2 Nsp9, suggesting this region either undergoes intermediate motion in the ms to μs timescale and/or is heterogenous. These "missing" amide resonances do not unambiguously appear in the 1 H- 15 N HSQC spectrum of SARS-CoV-2 Nsp9 collected at a concentration of 0.0007 mM. At this concentration, at the detection limit, native mass spectrometry indicates the protein is exclusively in the monomeric state, suggesting the intermediate motion in the C-terminal of Nsp9 may be due to intramolecular dynamics. Perhaps this intermediate ms to μs timescale dynamics is the physical basis for a previously suggested "fluidity" of the C-terminal helix that may be responsible for homophilic (Nsp9-Nsp9) and postulated heterophilic (Nsp9-Unknown) protein-protein interactions.

The C-terminally truncated Y145Stop variant of prion protein (PrP23-144) has been linked to a heritable prionopathy in humans and is also capable of triggering a transmissible prion disease in mice. PrP23-144 can be converted from soluble monomeric form to amyloid under physiological conditions, providing an in vitro model for investigating the molecular basis of amyloid strains and cross-seeding barriers. Here, we use magic-angle spinning solid-state NMR to establish the sequential backbone and sidechain 13 C and 15 N chemical shift assignments for amyloid fibrils formed by the A117V and M129V mutants of human PrP23-144, which in the context of full length PrP in vivo are among the specific residues associated with development of Gerstmann-Straüssler-Scheinker disease. The chemical shift data are utilized to identify amino acids comprising the rigid amyloid core regions and to predict the protein secondary structures for human PrP23-144 A117V and M129V fibrils.

Coronaviruses have become of great medical and scientific interest because of the Covid-19 pandemic. The hCoV-HKU1 is an endemic betacoronavirus that causes mild respiratory symptoms, although the infection can progress to severe lung disease and death. During viral replication, a discontinuous transcription of the genome takes place, producing the subgenomic messenger RNAs. The nucleocapsid protein (N) plays a pivotal role in the regulation of this process, acting as an RNA chaperone and participating in the nucleocapsid assembly. The isolated N-terminal domain of protein N (N-NTD) specifically binds to the transcriptional regulatory sequences and control the melting of the double-stranded RNA. Here, we report the resonance assignments of the N-NTD of HKU1-CoV.