Disordered Ferlin C2A-C2B Linkers Bind Membranes and Encode Small Linear Motifs.

Ferlins are vesicle trafficking proteins composed of folded C2 domains conjugated by linkers which are largely disordered. Although a role for the C2 domains as calcium sensors has been established it remains unclear whether the linkers function beyond acting as passive spacers. We examined the C2A-C2B linker sequences of vertebrate ferlins and found both putative short linear motifs (SLiMs) as well as membrane binding sequences for members of the protein family.

Practical Guidance on Selecting Analytical Methods for PFAS in Semiconductor Manufacturing Wastewater.

The focus of this review is to provide an overview of the nomenclature, structure, and properties of perfluoroalkyl and polyfluoroalkyl substances (PFAS) that dictate the selection of analytical methods for analyzing PFAS in treated semiconductor wastewater. The review is organized by introducing the fundamental concepts of how structure dictates the physical-chemical properties of PFAS and how these properties determine the suitability and applicability of standardized analytical methods for individual PFAS as well as methods for total fluorine. Structures for PFAS measured in semiconductor wastewater or known to be in use by industry are given with tables intended as guidance for method selection.

Nitration-driven structural changes in Hsp90 linked to gain of pathological functions.

Protein tyrosine (Y) nitration is an oxidative modification that occurs in pathological conditions such as neurodegenerative diseases and solid tumors. Depending on the location of the tyrosine residue, nitration can modify protein structure and function and affect cellular processes. We previously showed that site-specific nitration of the molecular chaperone heat shock protein 90 (Hsp90) leads to distinct pathological gain-of-function that cannot be compensated or overcome by native Hsp90.

LC8 enhances 53BP1 foci through heterogeneous bridging of 53BP1 oligomers.

53BP1 is a key player in DNA repair and together with BRCA1 regulate selection of DNA double-strand break repair mechanisms. Localization of DNA repair factors to sites of DNA damage by 53BP1 is controlled by its oligomerization domain (OD) and binding to LC8, a hub protein that functions to dimerize >100 clients. Here, we show that 53BP1 OD is a trimer, an unusual finding for LC8 clients which are all dimers or tetramers.

Changes in microbial community structure during adaptation of kombucha symbiotic culture of bacteria and yeast to fermentation of sweet and acid whey.

Whey is a liquid byproduct from the dairy industry that is not fully utilized and can be problematic to dispose of. Based on its composition, there is potential to upcycle whey into fermented beverages for human consumption. Most focus to date has been upon alcoholic fermentation to generate alcohol for distillation, or use of kefir grains to make acidic beverages.

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.

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.

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.

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, and , 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 T. Sequences of two DNA hairpins were designed to confer a near 20°C difference in their T's.

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.

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.

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 CH, site-specifically into proteins has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into a six-step biosynthetic pathway that produces nhpSer from phosphoenolpyruvate.

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.

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 p150 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.

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 ( = 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 F 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).

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.

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 N- (and C)-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.

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.

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.

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.

PermaPhos : 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 a biosynthetic pathway that produces nhpSer from the central metabolite phosphoenolpyruvate.

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 (P) a coiled-coil multimerization domain (P), similar to the well-characterized paramyxovirus phosphoproteins from measles and Sendai viruses.

Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins.

A significant number of proteins possess sizable intrinsically disordered regions (IDRs). Due to the dynamic nature of IDRs, NMR spectroscopy is often the tool of choice for characterizing these segments. However, the application of NMR to IDRs is often hindered by their instability, spectral overlap and resonance assignment difficulties.