Structural transitions modulate the chaperone activities of Grp94.

Hsp90s are ATP-dependent chaperones that collaborate with co-chaperones and Hsp70s to remodel client proteins. Grp94 is the ER Hsp90 homolog essential for folding multiple secretory and membrane proteins. Grp94 interacts with the ER Hsp70, BiP, although the collaboration of the ER chaperones in protein remodeling is not well understood. Grp94 undergoes large-scale conformational changes that are coupled to chaperone activity. Within Grp94, a region called the pre-N domain suppresses ATP hydrolysis and conformational transitions to the active chaperone conformation. In this work, we combined in vivo and in vitro functional assays and structural studies to characterize the chaperone mechanism of Grp94. We show that Grp94 directly collaborates with the BiP chaperone system to fold clients. Grp94's pre-N domain is not necessary for Grp94-client interactions. The folding of some Grp94 clients does not require direct interactions between Grp94 and BiP in vivo, suggesting that the canonical collaboration may not be a general chaperone mechanism for Grp94. The BiP co-chaperone DnaJB11 promotes the interaction between Grp94 and BiP, relieving the pre-N domain suppression of Grp94's ATP hydrolysis activity. In structural studies, we find that ATP binding by Grp94 alters the ATP lid conformation, while BiP binding stabilizes a partially closed Grp94 intermediate. Together, BiP and ATP push Grp94 into the active closed conformation for client folding. We also find that nucleotide binding reduces Grp94's affinity for clients, which is important for productive client folding. Alteration of client affinity by nucleotide binding may be a conserved chaperone mechanism for a subset of ER chaperones.

A hydrophilic microenvironment in the substrate-translocating groove of the YidC membrane insertase is essential for enzyme function.

The YidC family of proteins are membrane insertases that catalyze the translocation of the periplasmic domain of membrane proteins via a hydrophilic groove located within the inner leaflet of the membrane. All homologs have a strictly conserved, positively charged residue in the center of this groove. In Bacillus subtilis, the positively charged residue has been proposed to be essential for interacting with negatively charged residues of the substrate, supporting a hypothesis that YidC catalyzes insertion via an early-step electrostatic attraction mechanism. Here, we provide data suggesting that the positively charged residue is important not for its charge but for increasing the hydrophilicity of the groove. We found that the positively charged residue is dispensable for Escherichia coli YidC function when an adjacent residue at position 517 was hydrophilic or aromatic, but was essential when the adjacent residue was apolar. Additionally, solvent accessibility studies support the idea that the conserved positively charged residue functions to keep the top and middle of the groove sufficiently hydrated. Moreover, we demonstrate that both the E. coli and Streptococcus mutans YidC homologs are functional when the strictly conserved arginine is replaced with a negatively charged residue, provided proper stabilization from neighboring residues. These combined results show that the positively charged residue functions to maintain a hydrophilic microenvironment in the groove necessary for the insertase activity, rather than to form electrostatic interactions with the substrates.

The role of native cysteine residues in the oligomerization of KCNQ1 channels.

KCNQ1, the major component of the slow-delayed rectifier potassium channel, is responsible for repolarization of cardiac action potential. Mutations in this channel can lead to a variety of diseases, most notably long QT syndrome. It is currently unknown how many of these mutations change channel function and structure on a molecular level. Since tetramerization is key to proper function and structure of the channel, it is likely that mutations modify the stability of KCNQ1 oligomers. Presently, the C-terminal domain of KCNQ1 has been noted as the driving force for oligomer formation. However, truncated versions of this protein lacking the C-terminal domain still tetramerize. Therefore, we explored the role of native cysteine residues in a truncated construct of human KCNQ1, amino acids 100-370, by blocking potential interactions of cysteines with a nitroxide based spin label. Mobility of the spin labels was investigated with continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. The oligomerization state was examined by gel electrophoresis. The data provide information on tetramerization of human KCNQ1 without the C-terminal domain. Specifically, how blocking the side chains of native cysteines residues reduces oligomerization. A better understanding of tetramer formation could provide improved understanding of the molecular etiology of long QT syndrome and other diseases related to KCNQ1.

Formation of styrene maleic acid lipid nanoparticles (SMALPs) using SMA thin film on a substrate.

Despite the important role of membrane proteins in biological function and physiology, studying them remains challenging because of limited biomimetic systems for the protein to remain in its native membrane environment. Cryo electron microscopy (Cryo-EM) is emerging as a powerful tool for analyzing the structure of membrane proteins. However, Cryo-EM and other membrane protein analyses are better studied in a native lipid bilayer. Although traditional, mimetic systems have disadvantages that limit their use in the study of membrane proteins. As an alternative, styrene-maleic acid copolymers are used to form nanoparticles with POPC:POPG lipids. Traditional characterization of these styrene maleic acid lipid nanoparticles (SMALPs) includes dynamic light scattering (DLS), electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and transmission electron microscopy (TEM). In this study a new method was developed that utilizes SMALPs using a styrene-maleic acid copolymer (SMA) thin film on a TEM grid, acting as a substrate. By directly adding POPC:POPG lipid vesicles to the SMA coated grid SMALPs can be formed, visualized, and characterized by TEM without the need to make them in solution prior to imaging. We envision these functionalized grids could aid in single particle specimen preparation, increasing the efficiency of structural biology and biophysical techniques such as Cryo-EM.

Simple Derivatization of RAFT-Synthesized Styrene-Maleic Anhydride Copolymers for Lipid Disk Formulations.

Styrene-maleic acid copolymers have received significant attention because of their ability to interact with lipid bilayers and form styrene-maleic acid copolymer lipid nanoparticles (SMALPs). However, these SMALPs are limited in their chemical diversity, with only phenyl and carboxylic acid functional groups, resulting in limitations because of sensitivity to low pH and high concentrations of divalent metals. To address this limitation, various nucleophiles were reacted with the anhydride unit of well-defined styrene-maleic anhydride copolymers in order to assess the potential for a new lipid disk nanoparticle-forming species. These styrene-maleic anhydride copolymer derivatives (SMADs) can form styrene-maleic acid derivative lipid nanoparticles (SMADLPs) when they interact with lipid molecules. Polymers were synthesized, purified, characterized by Fourier-transform infrared spectroscopy, gel permeation chromatography, and nuclear magnetic resonance and then used to make disk-like SMADLPs, whose sizes were measured by dynamic light scattering (DLS). The SMADs form lipid nanoparticles, observable by DLS and transmission electron microscopy, and were used to reconstitute a spin-labeled transmembrane protein, KCNE1. The polymer method reported here is facile and scalable and results in functional and robust polymers capable of forming lipid nanodisks that are stable against a wide pH range and 100 mM magnesium.

Probing the Dynamics and Structural Topology of the Reconstituted Human KCNQ1 Voltage Sensor Domain (Q1-VSD) in Lipid Bilayers Using Electron Paramagnetic Resonance Spectroscopy.

KCNQ1 (Kv7.1 or KvLQT1) is a potassium ion channel protein found in the heart, ear, and other tissues. In complex with the KCNE1 accessory protein, it plays a role during the repolarization phase of the cardiac action potential. Mutations in the channel have been associated with several diseases, including congenital deafness and long QT syndrome. Nuclear magnetic resonance (NMR) structural studies in detergent micelles and a cryo-electron microscopy structure of KCNQ1 from Xenopus laevis have shown that the voltage sensor domain (Q1-VSD) of the channel has four transmembrane helices, S1-S4, being overall structurally similar with other VSDs. In this study, we describe a reliable method for the reconstitution of Q1-VSD into (POPC/POPG) lipid bilayer vesicles. Site-directed spin labeling electron paramagnetic resonance spectroscopy was used to probe the structural dynamics and topology of several residues of Q1-VSD in POPC/POPG lipid bilayer vesicles. Several mutants were probed to determine their location and corresponding immersion depth (in angstroms) with respect to the membrane. The dynamics of the bilayer vesicles upon incorporation of Q1-VSD were studied using 31P solid-state NMR spectroscopy by varying the protein:lipid molar ratios confirming the interaction of the protein with the bilayer vesicles. Circular dichroism spectroscopic data showed that the α-helical content of Q1-VSD is higher for the protein reconstituted in vesicles than in previous studies using DPC detergent micelles. This study provides insight into the structural topology and dynamics of Q1-VSD reconstituted in a lipid bilayer environment, forming the basis for more advanced structural and functional studies.

The Turner syndrome research registry: Creating equipoise between investigators and participants.

To address knowledge gaps about Turner syndrome (TS) associated disease mechanisms, the Turner Syndrome Society of the United States created the Turner Syndrome Research Registry (TSRR), a patient-powered registry for girls and women with TS. More than 600 participants, parents or guardians completed a 33-item foundational survey that included questions about demographics, medical conditions, psychological conditions, sexuality, hormonal therapy, patient and provider knowledge about TS, and patient satisfaction. The TSRR platform is engineered to allow individuals living with rare conditions and investigators to work side-by-side. The purpose of this article is to introduce the concept, architecture, and currently available content of the TSRR, in anticipation of inviting proposals to utilize registry resources.

Solid phase synthesis and spectroscopic characterization of the active and inactive forms of bacteriophage S21 pinholin protein.

The mechanism for the lysis pathway of double-stranded DNA bacteriophages involves a small hole-forming class of membrane proteins, the holins. This study focuses on a poorly characterized class of holins, the pinholin, of which the S21 protein of phage ϕ21 is the prototype. Here we report the first in vitro synthesis of the wildtype form of the S21 pinholin, S2168, and negative-dominant mutant form, S21IRS, both prepared using solid phase peptide synthesis and studied using biophysical techniques. Both forms of the pinholin were labeled with a nitroxide spin label and successfully incorporated into both bicelles and multilamellar vesicles which are membrane mimetic systems. Circular dichroism revealed the two forms were both >80% alpha helical, in agreement with the predictions based on the literature. The molar ellipticity ratio [θ]222/[θ]208 for both forms of the pinholin was 1.4, suggesting a coiled-coil tertiary structure in the bilayer consistent with the proposed oligomerization step in models for the mechanism of hole formation. 31P solid-state NMR spectroscopic data on pinholin indicate a strong interaction of both forms of the pinholin with the membrane headgroups. The 31P NMR data has an axially symmetric line shape which is consistent with lamellar phase proteoliposomes lipid mimetics.

Utilization of 13C-labeled amino acids to probe the α-helical local secondary structure of a membrane peptide using electron spin echo envelope modulation (ESEEM) spectroscopy.

Electron spin echo envelope modulation (ESEEM) spectroscopy in combination with site-directed spin labeling (SDSL) has been established as a valuable biophysical technique to provide site-specific local secondary structure of membrane proteins. This pulsed electron paramagnetic resonance (EPR) method can successfully distinguish between α-helices, ß-sheets, and 310-helices by strategically using 2H-labeled amino acids and SDSL. In this study, we have explored the use of 13C-labeled residues as the NMR active nuclei for this approach for the first time. 13C-labeled d5-valine (Val) or 13C-labeled d6-leucine (Leu) were substituted at a specific Val or Leu residue (i), and a nitroxide spin label was positioned 2 or 3 residues away (denoted i-2 and i-3) on the acetylcholine receptor M2δ (AChR M2δ) in a lipid bilayer. The 13C ESEEM peaks in the FT frequency domain data were observed for the i-3 samples, and no 13C peaks were observed in the i-2 samples. The resulting spectra were indicative of the α-helical local secondary structure of AChR M2δ in bicelles. This study provides more versatility and alternative options when using this ESEEM approach to study the more challenging recombinant membrane protein secondary structures.

A Budding-Defective M2 Mutant Exhibits Reduced Membrane Interaction, Insensitivity to Cholesterol, and Perturbed Interdomain Coupling.

Influenza A M2 is a membrane-associated protein with a C-terminal amphipathic helix that plays a cholesterol-dependent role in viral budding. An M2 mutant with alanine substitutions in the C-terminal amphipathic helix is deficient in viral scission. With the goal of providing atomic-level understanding of how the wild-type protein functions, we used a multipronged site-directed spin labeling electron paramagnetic resonance spectroscopy (SDSL-EPR) approach to characterize the conformational properties of the alanine mutant. We spin-labeled sites in the transmembrane (TM) domain and the C-terminal amphipathic helix (AH) of wild-type (WT) and mutant M2, and collected information on line shapes, relaxation rates, membrane topology, and distances within the homotetramer in membranes with and without cholesterol. Our results identify marked differences in the conformation and dynamics between the WT and the alanine mutant. Compared to WT, the dominant population of the mutant AH is more dynamic, shallower in the membrane, and has altered quaternary arrangement of the C-terminal domain. While the AH becomes more dynamic, the dominant population of the TM domain of the mutant is immobilized. The presence of cholesterol changes the conformation and dynamics of the WT protein, while the alanine mutant is insensitive to cholesterol. These findings provide new insight into how M2 may facilitate budding. We propose the AH-membrane interaction modulates the arrangement of the TM helices, effectively stabilizing a conformational state that enables M2 to facilitate viral budding. Antagonizing the properties of the AH that enable interdomain coupling within M2 may therefore present a novel strategy for anti-influenza drug design.

Tuning the size of styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) using RAFT polymerization for biophysical studies.

Characterization of membrane proteins is challenging due to the difficulty in mimicking the native lipid bilayer with properly folded and functional membrane proteins. Recently, styrene-maleic acid (StMA) copolymers have been shown to facilitate the formation of disc-like lipid bilayer mimetics that maintain the structural and dynamic integrity of membrane proteins. Here we report the controlled synthesis and characterization of StMA containing block copolymers. StMA polymers with different compositions and molecular weights were synthesized and characterized by size exclusion chromatography (SEC). These polymers act as macromolecular surfactants for 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol (POPG) lipids, forming disc like structures of the lipids with the polymer wrapping around the hydrophobic lipid edge. A combination of dynamic light scattering (DLS), solid-state nuclear magnetic resonance (SSNMR) spectroscopy, and transmission electron microscopy (TEM) was used to characterize the size of the nanoparticles created using these StMA polymers. At a weight ratio of 1.25:1 StMA to lipid, the nanoparticle size created is 28+1nm for a 2:1 ratio, 10+1nm for a 3:1 StMA ratio and 32+1nm for a 4:1 StMA ratio independent of the molecular weight of the polymer. Due to the polymer acting as a surfactant that forms disc like nanoparticles, we term these StMA based block copolymers "RAFT SMALPs". RAFT SMALPs show promise as a new membrane mimetic with different nanoscale sizes, which can be used for a wide variety of biophysical studies of membrane proteins.

Probing the interaction of the potassium channel modulating KCNE1 in lipid bilayers via solid-state NMR spectroscopy.

KCNE1 is known to modulate the voltage-gated potassium channel α subunit KCNQ1 to generate slowly activating potassium currents. This potassium channel is essential for the cardiac action potential that mediates a heartbeat as well as the potassium ion homeostasis in the inner ear. Therefore, it is important to know the structure and dynamics of KCNE1 to better understand its modulatory role. Previously, the Sanders group solved the three-dimensional structure of KCNE1 in LMPG micelles, which yielded a better understanding of this KCNQ1/KCNE1 channel activity. However, research in the Lorigan group showed different structural properties of KCNE1 when incorporated into POPC/POPG lipid bilayers as opposed to LMPG micelles. It is hence necessary to study the structure of KCNE1 in a more native-like environment such as multi-lamellar vesicles. In this study, the dynamics of lipid bilayers upon incorporation of the membrane protein KCNE1 were investigated using 31 P solid-state nuclear magnetic resonance (NMR) spectroscopy. Specifically, the protein/lipid interaction was studied at varying molar ratios of protein to lipid content. The static 31 P NMR and T1 relaxation time were investigated. The 31 P NMR powder spectra indicated significant perturbations of KCNE1 on the phospholipid headgroups of multi-lamellar vesicles as shown from the changes in the 31 P spectral line shape and the chemical shift anisotropy line width. 31 P T1 relaxation times were shown to be reversely proportional to the molar ratios of KCNE1 incorporated. The 31 P NMR data clearly indicate that KCNE1 interacts with the membrane. Copyright © 2017 John Wiley & Sons, Ltd.

Characterizing the structure of lipodisq nanoparticles for membrane protein spectroscopic studies.

Membrane protein spectroscopic studies are challenging due to the difficulty introduced in preparing homogenous and functional hydrophobic proteins incorporated into a lipid bilayer system. Traditional membrane mimics such as micelles or liposomes have proved to be powerful in solubilizing membrane proteins for biophysical studies, however, several drawbacks have limited their applications. Recently, a nanosized complex termed lipodisq nanoparticles was utilized as an alternative membrane mimic to overcome these caveats by providing a homogeneous lipid bilayer environment. Despite all the benefits that lipodisq nanoparticles could provide to enhance the biophysical studies of membrane proteins, structural characterization in different lipid compositions that closely mimic the native membrane environment is still lacking. In this study, the formation of lipodisq nanoparticles using different weight ratios of POPC/POPG lipids to SMA polymers was characterized via solid-state nuclear magnetic resonance (SSNMR) spectroscopy and dynamic light scattering (DLS). A critical weight ratio of (1/1.25) for the complete solubilization of POPC/POPG vesicles has been observed and POPC/POPG vesicles turned clear instantaneously upon the addition of the SMA polymer. The size of lipodisq nanoparticles formed from POPC/POPG lipids at this weight ratio of (1/1.25) was found to be about 30 nm in radius. We also showed that upon the complete solubilization of POPC/POPG vesicles by SMA polymers, the average size of the lipodisq nanoparticles is weight ratio dependent, when more SMA polymers were introduced, smaller lipodisq nanoparticles were obtained. The results of this study will be helpful for a variety of biophysical experiments when specific size of lipid disc is required. Further, this study will provide a proper path for researchers working on membrane proteins to obtain pertinent structure and dynamic information in a physiologically relevant membrane mimetic environment.

Cholesterol-Dependent Conformational Exchange of the C-Terminal Domain of the Influenza A M2 Protein.

The C-terminal amphipathic helix of the influenza A M2 protein plays a critical cholesterol-dependent role in viral budding. To provide atomic-level detail on the impact cholesterol has on the conformation of M2 protein, we spin-labeled sites right before and within the C-terminal amphipathic helix of the M2 protein. We studied the spin-labeled M2 proteins in membranes both with and without cholesterol. We used a multipronged site-directed spin-label electron paramagnetic resonance (SDSL-EPR) approach and collected data on line shapes, relaxation rates, accessibility of sites to the membrane, and distances between symmetry-related sites within the tetrameric protein. We demonstrate that the C-terminal amphipathic helix of M2 populates at least two conformations in POPC/POPG 4:1 bilayers. Furthermore, we show that the conformational state that becomes more populated in the presence of cholesterol is less dynamic, less membrane buried, and more tightly packed than the other state. Cholesterol-dependent changes in M2 could be attributed to the changes cholesterol induces in bilayer properties and/or direct binding of cholesterol to the protein. We propose a model consistent with all of our experimental data that suggests that the predominant conformation we observe in the presence of cholesterol is relevant for the understanding of viral budding.

Probing Structural Dynamics and Topology of the KCNE1 Membrane Protein in Lipid Bilayers via Site-Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy.

KCNE1 is a single transmembrane protein that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in the genes encoding either protein can result in diseases such as congenital deafness, long QT syndrome, ventricular tachyarrhythmia, syncope, and sudden cardiac death. Despite the biological significance of KCNE1, the structure and dynamic properties of its physiologically relevant native membrane-bound state are not fully understood. In this study, the structural dynamics and topology of KCNE1 in bilayered lipid vesicles was investigated using site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. A 53-residue nitroxide EPR scan of the KCNE1 protein sequence including all 27 residues of the transmembrane domain (45-71) and 26 residues of the N- and C-termini of KCNE1 in lipid bilayered vesicles was analyzed in terms of nitroxide side-chain motion. Continuous wave-EPR spectral line shape analysis indicated the nitroxide spin label side-chains located in the KCNE1 TMD are less mobile when compared to the extracellular region of KCNE1. The EPR data also revealed that the C-terminus of KCNE1 is more mobile when compared to the N-terminus. EPR power saturation experiments were performed on 41 sites including 18 residues previously proposed to reside in the transmembrane domain (TMD) and 23 residues of the N- and C-termini to determine the topology of KCNE1 with respect to the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) lipid bilayers. The results indicated that the transmembrane domain is indeed buried within the membrane, spanning the width of the lipid bilayer. Power saturation data also revealed that the extracellular region of KCNE1 is solvent-exposed with some of the portions partially or weakly interacting with the membrane surface. These results are consistent with the previously published solution NMR structure of KCNE1 in micelles.

Investigating the interaction between peptides of the amphipathic helix of Hcf106 and the phospholipid bilayer by solid-state NMR spectroscopy.

The chloroplast twin arginine translocation (cpTat) system transports highly folded precursor proteins into the thylakoid lumen using the protonmotive force as its only energy source. Hcf106, as one of the core components of the cpTat system, is part of the precursor receptor complex and functions in the initial precursor-binding step. Hcf106 is predicted to contain a single amino terminal transmembrane domain followed by a Pro-Gly hinge, a predicted amphipathic α-helix (APH), and a loosely structured carboxy terminus. Hcf106 has been shown biochemically to insert spontaneously into thylakoid membranes. To better understand the membrane active capabilities of Hcf106, we used solid-state NMR spectroscopy to investigate those properties of the APH. In this study, synthesized peptides of the predicted Hcf106 APH (amino acids 28-65) were incorporated at increasing mol.% into 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and POPC/MGDG (monogalactosyldiacylglycerol; mole ratio 85:15) multilamellar vesicles (MLVs) to probe the peptide-lipid interaction. Solid-state (31)P NMR and (2)H NMR spectroscopic experiments revealed that the peptide perturbs the headgroup and the acyl chain regions of phospholipids as indicated by changes in spectral lineshape, chemical shift anisotropy (CSA) line width, and (2)H order SCD parameters. In addition, the comparison between POPC MLVs and POPC/MGDG MLVs indicated that the lipid bilayer composition affected peptide perturbation of the lipids, and such perturbation appeared to be more intense in a system more closely mimicking a thylakoid membrane.

Conformational dynamics of metallo-ß-lactamase CcrA during catalysis investigated by using DEER spectroscopy.

Previous crystallographic and mutagenesis studies have implicated the role of a position-conserved hairpin loop in the metallo-ß-lactamases in substrate binding and catalysis. In an effort to probe the motion of that loop during catalysis, rapid-freeze-quench double electron-electron resonance (RFQ-DEER) spectroscopy was used to interrogate metallo-ß-lactamase CcrA, which had a spin label at position 49 on the loop and spin labels (at positions 82, 126, or 233) 20-35 Å away from residue 49, during catalysis. At 10 ms after mixing, the DEER spectra show distance increases of 7, 10, and 13 Å between the spin label at position 49 and the spin labels at positions 82, 126, and 233, respectively. In contrast to previous hypotheses, these data suggest that the loop moves nearly 10 Å away from the metal center during catalysis and that the loop does not clamp down on the substrate during catalysis. This study demonstrates that loop motion during catalysis can be interrogated on the millisecond time scale.

Proceedings from the Turner Resource Network symposium: the crossroads of health care research and health care delivery.

Turner syndrome, a congenital condition that affects ∼1/2,500 births, results from absence or structural alteration of the second sex chromosome. There has been substantial effort by numerous clinical and genetic research groups to delineate the clinical, pathophysiological, cytogenetic, and molecular features of this multisystem condition. Questions about the molecular-genetic and biological basis of many of the clinical features remain unanswered, and health care providers and families seek improved care for affected individuals. The inaugural "Turner Resource Network (TRN) Symposium" brought together individuals with Turner syndrome and their families, advocacy group leaders, clinicians, basic scientists, physician-scientists, trainees and other stakeholders with interest in the well-being of individuals and families living with the condition. The goal of this symposium was to establish a structure for a TRN that will be a patient-powered organization involving those living with Turner syndrome, their families, clinicians, and scientists. The TRN will identify basic and clinical questions that might be answered with registries, clinical trials, or through bench research to promote and advocate for best practices and improved care for individuals with Turner syndrome. The symposium concluded with the consensus that two rationales justify the creation of a TRN: inadequate attention has been paid to the health and psychosocial issues facing girls and women who live with Turner syndrome; investigations into the susceptibility to common disorders such as cardiovascular or autoimmune diseases caused by sex chromosome deficiencies will increase understanding of disease susceptibilities in the general population.

Understanding the Mechanism of Action of Triazine-Phosphonate Derivatives as Flame Retardants for Cotton Fabric.

Countless hours of research and studies on triazine, phosphonate, and their combination have provided insightful information into their flame retardant properties on polymeric systems. However, a limited number of studies shed light on the mechanism of flame retardancy of their combination on cotton fabrics. The purpose of this research is to gain an understanding of the thermal degradation process of two triazine-phosphonate derivatives on cotton fabric. The investigation included the preparation of diethyl 4,6-dichloro-1,3,5-triazin-2-ylphosphonate (TPN1) and dimethyl (4,6-dichloro-1,3,5-triazin-2-yloxy) methyl phosphonate (TPN3), their application on fabric materials, and the studies of their thermal degradation mechanism. The studies examined chemical components in both solid and gas phases by using attenuated total reflection infrared (ATR-IR) spectroscopy, thermogravimetric analysis coupled with Fourier transform infrared (TGA-FTIR) spectroscopy, and 31P solid state nuclear magnetic resonance (31P solid state NMR), in addition to the computational studies of bond dissociation energy (BDE). Despite a few differences in their decomposition, TPN1 and TPN3 produce one common major product that is believed to help reduce the flammability of the fabric.

Structural investigation of the transmembrane domain of KCNE1 in proteoliposomes.

KCNE1 is a single-transmembrane protein of the KCNE family that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in KCNE1 have been linked to diseases such as long QT syndrome (LQTS), atrial fibrillation, sudden infant death syndrome, and deafness. The transmembrane domain (TMD) of KCNE1 plays a key role in mediating the physical association with KCNQ1 and in subsequent modulation of channel gating kinetics and conductance. However, the mechanisms associated with these roles for the TMD remain poorly understood, highlighting a need for experimental structural studies. A previous solution NMR study of KCNE1 in LMPG micelles revealed a curved transmembrane domain, a structural feature proposed to be critical to KCNE1 function. However, this curvature potentially reflects an artifact of working in detergent micelles. Double electron electron resonance (DEER) measurements were conducted on KCNE1 in LMPG micelles, POPC/POPG proteoliposomes, and POPC/POPG lipodisq nanoparticles to directly compare the structure of the TMD in a variety of different membrane environments. Experimentally derived DEER distances coupled with simulated annealing molecular dynamic simulations were used to probe the bilayer structure of the TMD of KCNE1. The results indicate that the structure is helical in proteoliposomes and is slightly curved, which is consistent with the previously determined solution NMR structure in micelles. The evident resilience of the curvature in the KCNE1 TMD leads us to hypothesize that the curvature is likely to be maintained upon binding of the protein to the KCNQ1 channel.