Phosphorylation in the Ser/Arg-rich region of the nucleocapsid of SARS-CoV-2 regulates phase separation by inhibiting self-association of a distant helix.

The nucleocapsid protein (N) of SARS-CoV-2 is essential for virus replication, genome packaging, evading host immunity, and virus maturation. N is a multidomain protein composed of an independently folded monomeric N-terminal domain that is the primary site for RNA binding and a dimeric C-terminal domain that is essential for efficient phase separation and condensate formation with RNA. The domains are separated by a disordered Ser/Arg-rich region preceding a self-associating Leu-rich helix. Phosphorylation in the Ser/Arg region in infected cells decreases the viscosity of N:RNA condensates promoting viral replication and host immune evasion. The molecular level effect of phosphorylation, however, is missing from our current understanding. Using NMR spectroscopy and analytical ultracentrifugation, we show that phosphorylation destabilizes the self-associating Leu-rich helix 30 amino-acids distant from the phosphorylation site. NMR and gel shift assays demonstrate that RNA binding by the linker is dampened by phosphorylation, whereas RNA binding to the full-length protein is not significantly affected presumably due to retained strong interactions with the primary RNA-binding domain. Introducing a switchable self-associating domain to replace the Leu-rich helix confirms the importance of linker self-association to droplet formation and suggests that phosphorylation not only increases solubility of the positively charged elongated Ser/Arg region as observed in other RNA-binding proteins but can also inhibit self-association of the Leu-rich helix. These data highlight the effect of phosphorylation both at local sites and at a distant self-associating hydrophobic helix in regulating liquid-liquid phase separation of the entire protein.

DNA-based assay for calorimetric determination of protein concentrations in pure or mixed solutions.

It was recently reported that values of the transition heat capacities, as measured by differential scanning calorimetry, for two globular proteins and a short DNA hairpin in NaCl buffer are essentially equivalent, at equal concentrations (mg/mL). To validate the broad applicability of this phenomenon, additional evidence for this equivalence is presented that reveals it does not depend on DNA sequence, buffer salt, or transition temperature (Tm). Based on the equivalence of transition heat capacities, a calorimetric method was devised to determine protein concentrations in pure and complex solutions. The scheme uses direct comparisons between the thermodynamic stability of a short DNA hairpin standard of known concentration, and thermodynamic stability of protein solutions of unknown concentrations. Sequences of two DNA hairpins were designed to confer a near 20°C difference in their Tm values. In all cases, evaluated protein concentrations determined from the DNA standard curves agreed with the UV-Vis concentration for monomeric proteins. For multimeric proteins evaluated concentrations were greater than determined by UV-Vis suggesting the calorimetric approach can also be an indicator of molecular stoichiometry.

RNA structure and multiple weak interactions balance the interplay between RNA binding and phase separation of SARS-CoV-2 nucleocapsid.

The nucleocapsid (N) protein of SARS-CoV-2 binds viral RNA, condensing it inside the virion, and phase separating with RNA to form liquid-liquid condensates. There is little consensus on what differentiates sequence-independent N-RNA interactions in the virion or in liquid droplets from those with specific genomic RNA (gRNA) motifs necessary for viral function inside infected cells. To identify the RNA structures and the N domains responsible for specific interactions and phase separation, we use the first 1,000 nt of viral RNA and short RNA segments designed as models for single-stranded and paired RNA. Binding affinities estimated from fluorescence anisotropy of these RNAs to the two-folded domains of N (the NTD and CTD) and comparison to full-length N demonstrate that the NTD binds preferentially to single-stranded RNA, and while it is the primary RNA-binding site, it is not essential to phase separation. Nuclear magnetic resonance spectroscopy identifies two RNA-binding sites on the NTD: a previously characterized site and an additional although weaker RNA-binding face that becomes prominent when binding to the primary site is weak, such as with dsRNA or a binding-impaired mutant. Phase separation assays of nucleocapsid domains with double-stranded and single-stranded RNA structures support a model where multiple weak interactions, such as with the CTD or the NTD's secondary face promote phase separation, while strong, specific interactions do not. These studies indicate that both strong and multivalent weak N-RNA interactions underlie the multifunctional abilities of N.

Hydrothermal Alkaline Treatment (HALT) of Foam Fractionation Concentrate Derived from PFAS-Contaminated Groundwater.

While foam fractionation (FF) process has emerged as a promising technology for removal of per- and polyfluoroalkyl substances (PFASs) from contaminated groundwater, management of the resulting foam concentrates with elevated concentrations of PFASs (e.g., >1 g/L) remains a challenge. Here, we applied hydrothermal alkaline treatment (HALT) to two foam concentrates derived from FF field demonstration projects that treated aqueous film-forming foam (AFFF)-impacted groundwater. Results showed >90% degradation and defluorination within 90 min of treatment (350 °C, 1 M NaOH) of all 62 PFASs (including cations, anions, and zwitterions) identified in foam concentrates. Observed rate constants for degradation of individual perfluoroalkyl sulfonates (PFSAs, CnF2n+1-SO3-), the most recalcitrant class of PFASs, in both foam concentrates were similar to values measured previously in other aqueous matrices, indicating that elevated initial PFAS concentrations (e.g., PFHxSinit = 0.55 g/L), dissolved organic carbon (DOC; up to 4.5 g/L), and salt levels (e.g., up to 325 mg/L chloride) do not significantly affect PFAS reaction kinetics. DOC was partially mineralized by treatment, but a fraction (∼15%) was recalcitrant. Spectroscopic characterization revealed molecular features of the HALT-recalcitrant DOC fraction, and nontarget high-resolution mass spectrometry tentatively identified 129 nonfluorinated HALT-recalcitrant molecules. Analysis of process energy requirements shows that treating PFAS-contaminated foam concentrates with HALT would add minimally (<5%) to the overall energy requirements of an integrated FF-HALT treatment train.

Calorimetric analysis using DNA thermal stability to determine protein concentration.

It was recently reported for two globular proteins and a short DNA hairpin in NaCl buffer that values of the transition heat capacities, Cp,DNA and Cp,PRO, for equal concentrations (mg/mL) of DNA and proteins, are essentially equivalent (differ by less than 1%). Additional evidence for this equivalence is presented that reveals this phenomenon does not depend on DNA sequence, buffer salt, or Tm. Sequences of two DNA hairpins were designed to confer a near 20°C difference in their Tm's. For the molecules, in NaCl and CsCl buffer the evaluated Cp,PRO and Cp,DNA were equivalent. Based on the equivalence of transition heat capacities, a calorimetric method was devised to determine protein concentrations in pure and complex solutions. The scheme uses direct comparisons between the thermodynamic stability of a short DNA hairpin standard of known concentration, and thermodynamic stability of protein solutions of unknown concentrations. In all cases, evaluated protein concentrations determined from the DNA standard curve agreed with the UV-Vis concentration for monomeric proteins. For samples of multimeric proteins, streptavidin (tetramer), Herpes Simplex Virus glycoprotein D (trimer/dimer), and a 16 base pair DNA duplex (dimer), evaluated concentrations were greater than determined by UV-Vis by factors of 3.94, 2.65, and 2.15, respectively.

The Dysferlin C2A Domain Binds PI(4,5)P2 and Penetrates Membranes.

Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca2+ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.

Outcomes of Revision Arthroscopic Posterior Labral Repair and Capsulorrhaphy: A Systematic Review.

Background: Failure rates up to 14% have been reported after arthroscopic posterior capsulolabral repair. It is unknown if revision arthroscopic posterior capsulolabral stabilization has inferior restoration of stability and return to sport when compared with primary repair. Optimal management of failed posterior capsulolabral stabilization is unknown. Purpose: To report outcomes of revision posterior capsulolabral repair and factors that contribute to failure and to determine optimal management of failed posterior stabilization procedures. Study Design: Systematic review; Level of evidence, 4. Methods: A computerized search of the PubMed, EMBASE, and Web of Science databases and manual screening of selected article reference lists were performed in January 2022. Randomized controlled trial, cohort, case-control, and case series studies reporting clinical outcomes of revision arthroscopic posterior capsulolabral repair were eligible. Patient characteristics, indications for revision, intraoperative findings, surgical techniques, and patient-reported outcomes were recorded. Owing to heterogeneity of reported outcomes, data were summarized and presented without pooled statistics. Results: Only 3 of the 990 identified studies met inclusion criteria. The included studies encompassed 26 revision arthroscopic posterior capsulolabral repairs, with follow-up ranging from 2.3 to 5.3 years. The failed index procedure was arthroscopic capsulolabral repair with suture anchors (n = 22) or posterior thermal capsulorrhaphy (n = 4). The primary indications for revision were recurrent instability and pain. Six patients experienced recurrent instability after revision. Patient satisfaction ranged from 15% to 25%. Conclusion: This systematic review of 3 studies demonstrated that the incidence of persistent pain and recurrent instability after revision arthroscopic posterior shoulder stabilization is common, and despite slight improvement in patient-reported outcomes, many patients are dissatisfied with their clinical outcomes. Revision arthroscopic posterior shoulder stabilization appears to have a significant failure rate, and there is need for additional prospective studies to help determine the best intervention for these patients.

Autonomous Synthesis of Functional, Permanently Phosphorylated Proteins for Defining the Interactome of Monomeric 14-3-3ζ.

14-3-3 proteins are dimeric hubs that bind hundreds of phosphorylated "clients" to regulate their function. Installing stable, functional mimics of phosphorylated amino acids into proteins offers a powerful strategy to study 14-3-3 function in cellular-like environments, but a previous genetic code expansion (GCE) system to translationally install nonhydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with CH2, site-specifically into proteins has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into Escherichia coli a six-step biosynthetic pathway that produces nhpSer from phosphoenolpyruvate. Using this autonomous "PermaPhos" expression system, we produce three biologically relevant proteins with nhpSer and confirm that nhpSer mimics the effects of phosphoserine for activating GSK3ß phosphorylation of the SARS-CoV-2 nucleocapsid protein, promoting 14-3-3/client complexation, and monomerizing 14-3-3 dimers. Then, to understand the biological function of these phosphorylated 14-3-3ζ monomers (containing nhpSer at Ser58), we isolate its interactome from HEK293T lysates and compare it with that of wild-type 14-3-3ζ. These data identify two new subsets of 14-3-3 client proteins: (i) those that selectively bind dimeric 14-3-3ζ and (ii) those that selectively bind monomeric 14-3-3ζ. We discover that monomeric-but not dimeric-14-3-3ζ interacts with cereblon, an E3 ubiquitin-ligase adaptor protein of pharmacological interest.

Linker Length Drives Heterogeneity of Multivalent Complexes of Hub Protein LC8 and Transcription Factor ASCIZ.

LC8, a ubiquitous and highly conserved hub protein, binds over 100 proteins involved in numerous cellular functions, including cell death, signaling, tumor suppression, and viral infection. LC8 binds intrinsically disordered proteins (IDPs), and although several of these contain multiple LC8 binding motifs, the effects of multivalency on complex formation are unclear. Drosophila ASCIZ has seven motifs that vary in sequence and inter-motif linker lengths, especially within subdomain QT2-4 containing the second, third, and fourth LC8 motifs. Using isothermal-titration calorimetry, analytical-ultracentrifugation, and native mass-spectrometry of QT2-4 variants, with methodically deactivated motifs, we show that inter-motif spacing and specific motif sequences combine to control binding affinity and compositional heterogeneity of multivalent duplexes. A short linker separating strong and weak motifs results in stable duplexes but forms off-register structures at high LC8 concentrations. Contrastingly, long linkers engender lower cooperativity and heterogeneous complexation at low LC8 concentrations. Accordingly, two-mers, rather than the expected three-mers, dominate negative-stain electron-microscopy images of QT2-4. Comparing variants containing weak-strong and strong-strong motif combinations demonstrates sequence also regulates IDP/LC8 assembly. The observed trends persist for trivalent ASCIZ subdomains: QT2-4, with long and short linkers, forms heterogeneous complexes, whereas QT4-6, with similar mid-length linkers, forms homogeneous complexes. Implications of linker length variations for function are discussed.

Multivalent binding of the hub protein LC8 at a newly discovered site in 53BP1.

Tumor suppressor p53 binding protein 1 (53BP1) is a scaffolding protein involved in poly-ADP ribose polymerase inhibitor hypersensitivity in BRCA1-negative cancers. 53BP1 plays a critical role in the DNA damage response and relies on its oligomerization to create foci that promote repair of DNA double-strand breaks. Previous work shows that mutation of either the oligomerization domain or the dynein light chain 8 (LC8)-binding sites of 53BP1 results in reduced accumulation of 53BP1 at double-strand breaks. Mutation of both abolishes focus formation almost completely. Here, we show that, contrary to current literature, 53BP1 contains three LC8-binding sites, all of which are conserved in mammals. Isothermal titration calorimetry measuring binding affinity of 53BP1 variants with LC8 shows that the third LC8-binding site has a high affinity and can bind LC8 in the absence of other sites. NMR titrations confirm that the third site binds LC8 even in variants that lack the other LC8-binding sites. The third site is the closest to the oligomerization domain of 53BP1, and its discovery would challenge our current understanding of the role of LC8 in 53BP1 function.

Multivalency, autoinhibition, and protein disorder in the regulation of interactions of dynein intermediate chain with dynactin and the nuclear distribution protein.

As the only major retrograde transporter along microtubules, cytoplasmic dynein plays crucial roles in the intracellular transport of organelles and other cargoes. Central to the function of this motor protein complex is dynein intermediate chain (IC), which binds the three dimeric dynein light chains at multivalent sites, and dynactin p150Glued and nuclear distribution protein (NudE) at overlapping sites of its intrinsically disordered N-terminal domain. The disorder in IC has hindered cryo-electron microscopy and X-ray crystallography studies of its structure and interactions. Here we use a suite of biophysical methods to reveal how multivalent binding of the three light chains regulates IC interactions with p150Glued and NudE. Using IC from Chaetomium thermophilum, a tractable species to interrogate IC interactions, we identify a significant reduction in binding affinity of IC to p150Glued and a loss of binding to NudE for constructs containing the entire N-terminal domain as well as for full-length constructs when compared to the tight binding observed with short IC constructs. We attribute this difference to autoinhibition caused by long-range intramolecular interactions between the N-terminal single α-helix of IC, the common site for p150Glued, and NudE binding, and residues closer to the end of the N-terminal domain. Reconstitution of IC subcomplexes demonstrates that autoinhibition is differentially regulated by light chains binding, underscoring their importance both in assembly and organization of IC, and in selection between multiple binding partners at the same site.

Motor proteins are the freight trains of the cell, transporting large molecular cargo from one location to another using an array of 'roads' known as microtubules. These hollow tubes are oriented, with one extremity (the plus-end) growing faster than the other (the minus-end). While over 40 different motor proteins travel towards the plus-end of microtubules, just one is responsible for moving cargo in the opposite direction. This protein, called dynein, performs a wide range of functions which must be carefully regulated, often through changes in the shape and interactions of various dynein segments. The intermediate chain is one of the essential subunits that form dynein, and it acts as a binding site for a range of molecular actors. In particular, it connects the three other dynein subunits (known as the light chains) to the dynein heavy chain containing the motor domain. It also binds to two non-dynein proteins: NudE, which helps to organise microtubules, and the p150^Glued region of dynactin, a protein required for dynein activity. Despite their distinct roles, p150^Glued and NudE attach to the same region of the intermediate chain, a highly flexible 'unstructured' segment which is difficult to study. How the binding of p150^Glued and NudE is regulated has therefore remained unsolved. In response, Jara et al. decided to investigate how the three dynein light chains may help to control interactions between the intermediate chain and non-dynein proteins. They used more stable versions of dynein, NudE and dynactin (from a fungus that grows at high temperatures) to produce the various subcomplexes formed by the intermediate chain, the three dynein light chains, and parts of p150^Glued and NudE. A suite of biophysical techniques was applied to study these structures, as they are challenging to capture using traditional approaches. This revealed that the unstructured region of the intermediate chain can fold back on itself, bringing together its two extremities; such folding blocks the p150^Glued and NudE binding site. This obstruction is cleared when the light chains bind to the intermediate chain, demonstrating how these three subunits can regulate dynein activity. In humans, mutations in dynein are associated with a range of serious neurological and muscular diseases. The work by Jara et al. brings new insight into the way this protein works; more importantly, it describes how to combine several biophysical techniques to study non-structured proteins, offering a blueprint that is likely to be relevant for a wide range of scientists.

Paints: A Source of Volatile PFAS in Air─Potential Implications for Inhalation Exposure.

Paints are widely used in indoor settings yet there are no data for volatile per- and polyfluoroalkyl substances (PFAS) for paints or knowledge if paints are potentially important sources of human exposure to PFAS. Different commercial paints (n = 27) were collected from local hardware stores and analyzed for volatile PFAS by gas chromatography-mass spectrometry (GC-MS), nonvolatile PFAS by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-qTOF), and total fluorine by 19F nuclear magnetic resonance spectroscopy (NMR). Diluted paint required clean up to remove 6:2 fluorotelomer phosphate diester (diPAP), which thermally transforms into 6:2 FTOH at 280 °C (GC inlet temperature). Only 6:2 FTOH (0.9-83 µg/g) and 6:2 diPAP (0.073-58 µg/g) were found in five exterior and nine interior paints and only accounted for a maximum of 17% of total fluorine. Upon drying, 40% of the FTOH mass was lost, and the loss was verified by measurements of the cumulative FTOH mass measured in the air of a small, confined space over a 3 h period. Based on the liquid paint results, the ConsExpo model was used for potential exposure assessment and one commercial paint exceeded the chosen reference dose (5 µg/kg-day) for children and adults, indicating the potential for human exposure during painting.

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.

Evolution avoids a pathological stabilizing interaction in the immune protein S100A9.

Stability constrains evolution. While much is known about constraints on destabilizing mutations, less is known about the constraints on stabilizing mutations. We recently identified a mutation in the innate immune protein S100A9 that provides insight into such constraints. When introduced into human S100A9, M63F simultaneously increases the stability of the protein and disrupts its natural ability to activate Toll-like receptor 4. Using chemical denaturation, we found that M63F stabilizes a calcium-bound conformation of hS100A9. We then used NMR to solve the structure of the mutant protein, revealing that the mutation distorts the hydrophobic binding surface of hS100A9, explaining its deleterious effect on function. Hydrogen-deuterium exchange (HDX) experiments revealed stabilization of the region around M63F in the structure, notably Phe37. In the structure of the M63F mutant, the Phe37 and Phe63 sidechains are in contact, plausibly forming an edge-face π-stack. Mutating Phe37 to Leu abolished the stabilizing effect of M63F as probed by both chemical denaturation and HDX. It also restored the biological activity of S100A9 disrupted by M63F. These findings reveal that Phe63 creates a molecular staple with Phe37 that stabilizes a nonfunctional conformation of the protein, thus disrupting function. Using a bioinformatic analysis, we found that S100A9 proteins from different organisms rarely have Phe at both positions 37 and 63, suggesting that avoiding a pathological stabilizing interaction indeed constrains S100A9 evolution. This work highlights an important evolutionary constraint on stabilizing mutations, namely, that they must avoid inappropriately stabilizing nonfunctional protein conformations.

Computation-Assisted Identification of Bioactive Compounds in Botanical Extracts: A Case Study of Anti-Inflammatory Natural Products from Hops.

The slow pace of discovery of bioactive natural products can be attributed to the difficulty in rapidly identifying them in complex mixtures such as plant extracts. To overcome these hurdles, we explored the utility of two machine learning techniques, i.e., Elastic Net and Random Forests, for identifying the individual anti-inflammatory principle(s) of an extract of the inflorescences of the hops (Humulus lupulus) containing hundreds of natural products. We fractionated a hop extract by column chromatography to obtain 40 impure fractions, determined their anti-inflammatory activity using a macrophage-based bioassay that measures inhibition of iNOS-mediated formation of nitric oxide, and characterized the chemical composition of the fractions by flow-injection HRAM mass spectrometry and LC-MS/MS. Among the top 10 predictors of bioactivity were prenylated flavonoids and humulones. The top Random Forests predictor of bioactivity, xanthohumol, was tested in pure form in the same bioassay to validate the predicted result (IC50 7 µM). Other predictors of bioactivity were identified by spectral similarity with known hop natural products using the Global Natural Products Social Networking (GNPS) algorithm. Our machine learning approach demonstrated that individual bioactive natural products can be identified without the need for extensive and repetitive bioassay-guided fractionation of a plant extract.

Postoperative bleeding and leaks in sleeve gastrectomy are independent of both staple height and staple line oversewing.

BACKGROUND: Over 100,000 sleeve gastrectomy procedures are performed annually in the USA. Despite technological advances, postoperative bleeding and gastric staple line leak are complications of this procedure. We analyzed patient-specific and perioperative factors to determine their association with these complications. METHODS: We performed a retrospective cohort analysis of patients who underwent sleeve gastrectomy between 2005 and 2019 at our institution. Patient demographics, comorbidities, and procedure details including type of energy device, staple type, staple height, staple line oversewing, and staple line clipping were compared using multiple logistic regression for combined postoperative complications (blood transfusion, bleeding, and staple line leak). Postoperative bleeding was defined by requiring blood transfusion and/or re-operation to control bleeding. Staple line leak was confirmed radiographically. RESULTS: There were 1213 patients who underwent sleeve gastrectomy. Fifty-two high-risk patients were excluded due to cirrhosis, end-stage renal disease, and anticoagulation use for left ventricular assist device. Of the remaining 1161 patients, twenty-five (2.2%) received postoperative blood transfusion, nine (0.8%) had postoperative bleeding, two (0.2%) had staple line leak, and twenty-eight patients (2.4%) had combined postoperative complications. The median age was significantly higher for patients with combined postoperative complications (43 vs 49; p = 0.02). There was no difference in postoperative blood transfusion, bleeding, staple line leak, or combined postoperative complication with different energy devices (p = 0.92), staple types (p = 0.21), staple heights (p = 0.50), or staple line suturing/clipping (p = 0.95). In addition, there was no difference in bleeding when comparing staple line sewing techniques (p = 0.44). Predictably, patients with combined postoperative complications had increased length of stay (3 days vs 1 day; p < 0.001). CONCLUSION: Sleeve gastrectomy procedure has tremendous variability in technique and devices used. We observed no difference in the combined postoperative complications of bleeding or staple line leak with respect to different energy devices, staple height, or oversewing of the gastric staple line. Patient selection is crucial, as patient age and coagulopathic comorbidities were found to lead to higher combined postoperative complications.

Native state fluctuations in a peroxiredoxin active site match motions needed for catalysis.

Peroxiredoxins are ubiquitous enzymes that detoxify peroxides and regulate redox signaling. During catalysis, a "peroxidatic" cysteine (CP) in the conserved active site reduces peroxide while being oxidized to a CP-sulfenate, prompting a local unfolding event that enables formation of a disulfide with a second, "resolving" cysteine. Here, we use nuclear magnetic resonance spectroscopy to probe the dynamics of the CP-thiolate and disulfide forms of Xanthomonas campestris peroxiredoxin Q. Chemical exchange saturation transfer behavior of the resting enzyme reveals 26 residues in and around the active site exchanging at a rate of 72 s-1 with a locally unfolded, high-energy (2.5% of the population) state. This unequivocally establishes that a catalytically relevant local unfolding equilibrium exists in the enzyme's CP-thiolate form. Also, faster motions imply an active site instability that could promote local unfolding and, based on other work, be exacerbated by CP-sulfenate formation so as to direct the enzyme along a functional catalytic trajectory.

PermaPhos Ser : autonomous synthesis of functional, permanently phosphorylated proteins.

Installing stable, functional mimics of phosphorylated amino acids into proteins offers a powerful strategy to study protein regulation. Previously, a genetic code expansion (GCE) system was developed to translationally install non-hydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with carbon, but it has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into Escherichia coli a biosynthetic pathway that produces nhpSer from the central metabolite phosphoenolpyruvate. Using this "PermaPhos Ser " system - an autonomous 21-amino acid E. coli expression system for incorporating nhpSer into target proteins - we show that nhpSer faithfully mimics the effects of phosphoserine in three stringent test cases: promoting 14-3-3/client complexation, disrupting 14-3-3 dimers, and activating GSK3ß phosphorylation of the SARS-CoV-2 nucleocapsid protein. This facile access to nhpSer containing proteins should allow nhpSer to replace Asp and Glu as the go-to pSer phosphomimetic for proteins produced in E. coli .

Human Parainfluenza Virus 3 Phosphoprotein Is a Tetramer and Shares Structural and Interaction Features with Ebola Phosphoprotein VP35.

The human parainfluenza virus 3 (HPIV3) poses a risk for pneumonia development in young children and immunocompromised patients. To investigate mechanisms of HPIV3 pathogenesis, we characterized the association state and host protein interactions of HPIV3 phosphoprotein (HPIV3 P), an indispensable viral polymerase cofactor. Sequence analysis and homology modeling predict that HPIV3 P possesses a long, disordered N-terminal tail (PTAIL) a coiled-coil multimerization domain (PMD), similar to the well-characterized paramyxovirus phosphoproteins from measles and Sendai viruses. Using a recombinantly expressed and purified construct of PMD and PTAIL, we show that HPIV3 P in solution is primarily an alpha-helical tetramer that is stable up to 60 °C. Pulldown and isothermal titration calorimetry experiments revealed that HPIV3 P binds the host hub protein LC8, and turbidity experiments demonstrated a new role for LC8 in increasing the solubility of HPIV3 P in the presence of crowding agents such as RNA. For comparison, we show that the multimerization domain of the Zaire Ebola virus phosphoprotein VP35 is also a tetramer and binds LC8 but with significantly higher affinity. Comparative analysis of the domain architecture of various virus phosphoproteins in the order Mononegavirales show multiple predicted and verified LC8 binding motifs, suggesting its prevalence and importance in regulating viral phosphoprotein structures. Our work provides evidence for LC8 binding to phosphoproteins with multiple association states, either tetrameric, as in the HPIV3 and Ebola phosphoproteins shown here, or dimeric as in rabies virus phosphoprotein. Taken together the data suggest that the association states of a virus-specific phosphoprotein and the complex formed by binding of the phosphoprotein to host LC8 are important regulators of viral function.