Solvent Exposure Research Articles

Computational protein structure elucidation from covalent labeling mass spectrometry data.

Mass spectrometry (MS)-based methods such as covalent labeling MS are increasingly used to obtain information about protein structure. However, in contrast to other high-resolution structure determination methods, this information is not sufficient to deduce all atom coordinates and can only inform on certain elements of structure, such as solvent exposure of individual residues or location of protein-protein interfaces. However, easy and reliable translation of this information into accurate structural protein and protein complex models remains particularly challenging, due to a lack of standardized and automated methods. Computational methods are needed to predict high-resolution protein structures from the mass spectrometry (MS) data. My group develops algorithms within the Rosetta software package that use mass spectrometry data to guide protein structure prediction. We have successfully implemented the use of MS covalent labeling data into the Rosetta software to predict protein and protein complex structure guided by the experimental data. We developed algorithms to predict protein structure from hydroxyl radical and diethylpyrocarbonate (DEPC) labeling data. To evaluate the effectiveness of our covalent-labeling guided structure prediction algorithms, we benchmarked their performance using proteins of known structure for which experimental covalent labeling data exists. For all benchmark proteins, better quality models were built when MS data was used in the scoring. For modeling with hydroxyl radical labeling data, we measured exposure of labeled residues, while in the case of DEPC labeling we further assessed the local hydrophobic microenvironment of serine, threonine, and tyrosine residues. We are now extending this work to use MS data in deep learning-based predictions of protein structure.

Effects of amino acid-functionalized silver nanoparticles on lysozyme amyloid fibrillogenesis

Amyloid fibrillogenesis of proteins have been considered as the major cause behind several degenerative diseases. Nanoparticles have been found to have great potential as a substrate for designing amyloid fibril inhibitors and/or modulators. This is mainly due to its ideal characteristics, such as small size, high surface area-to volume ratio, and modifiable surface. This study is aimed at examining the influence of amino acid-modified silver nanoparticles on amyloid fibril formation. We first synthesized and characterized the proline- and cysteine-functionalized silver nanoparticles (Pro-AgNP and Cys-AgNP). Next, the effects of functionalized silver nanoparticles on amyloid fibrillogenesis and structural changes of hen lysozyme were examined using various spectroscopic and biophysical tools. Our results showed that Pro-AgNP/Cys-AgNP dose-dependently suppressed lysozyme amyloid fibrillogenesis, whereas negligible fibril-inhibiting effect was observed in the bare AgNP or proline/cysteine. Moreover, reduced solvent exposure and α-to-β transition were detected in lysozyme upon the addition of Pro-AgNP/Cys-AgNP. Finally, the nature of interactions between lysozyme and Pro-AgNP/Cys-AgNP was further examined using fluorescence quenching and molecular docking. By altering the properties of nanoparticles to design suitable nanoprobes, we have shown that amyloid fibrillogenesis can be controlled.

Distribution and solvent exposure of Hsp70 chaperone binding sites across the Escherichia coli proteome.

Many proteins must interact with molecular chaperones to achieve their native state in the cell. Yet, how chaperone binding-site characteristics affect the folding process is poorly understood. The ubiquitous Hsp70 chaperone system prevents client-protein aggregation by holding unfolded conformations and by unfolding misfolded states. Hsp70 binding sites of client proteins comprise a nonpolar core surrounded by positively charged residues. However, a detailed analysis of Hsp70 binding sites on a proteome-wide scale is still lacking. Further, it is not known whether proteins undergo some degree of folding while chaperone bound. Here, we begin to address the above questions by identifying Hsp70 binding sites in 2258 Escherichia coli (E. coli) proteins. We find that most proteins bear at least one Hsp70 binding site and that the number of Hsp70 binding sites is directly proportional to protein size. Aggregation propensity upon release from the ribosome correlates with number of Hsp70 binding sites only in the case of large proteins. Interestingly, Hsp70 binding sites are more solvent-exposed than other nonpolar sites, in protein native states. Our findings show that the majority of E. coli proteins are systematically enabled to interact with Hsp70 even if this interaction only takes place during a fraction of the protein lifetime. In addition, our data suggest that some conformational sampling may take place within Hsp70-bound states, due to the solvent exposure of some chaperone binding sites in native proteins. In all, we propose that Hsp70-chaperone-binding traits have evolved to favor Hsp70-assisted protein folding devoid of aggregation.

Electrostatic interactions - key determinants of the metal selectivity in La3+ and Ca2+ binding proteins.

Nearly half of all known proteins contain metal co-factors. In the course of evolution two dozen metal cations (mostly monovalent and divalent species) have been selected to participate in processes of vital importance for living organisms. Trivalent metal cations have also been selected, although to a lesser extent as compared with their mono- and divalent counterparts. Notably, factors governing the metal selectivity in trivalent metal centers in proteins are less well understood than those in the respective divalent metal centers. Thus, the source of high La3+/Ca2+ selectivity in lanthanum-binding proteins, as compared with that of calcium-binding proteins (i.e., calmodulin), is still shrouded in mystery. The well-calibrated thermochemical calculations, performed here, reveal the dominating role of electrostatic interactions in shaping the metal selectivity in La3+-binding centers. The calculations also disclose other (second-order) determinants of metal selectivity in these systems, such as the rigidity and extent of solvent exposure of the binding site. All these factors are also implicated in shaping the metal selectivity in Ca2+-binding proteins.

Organic solvent exposure for the chronic kidney disease: updated systematic review with meta-analysis.

Studies on the relationship between organic solvent exposure and chronic kidney disease (CKD) have presented inconsistent results. Definition of CKD has changed in 2012, and other cohort studies have been newly published. Therefore, this study aimed to newly confirm the relationship between organic solvent exposure and CKD through an updated meta-analysis including additional studies. This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. The search was conducted on January 2, 2023 using Embase and MEDLINE databases. Case-control and cohort studies on the relationship between organic solvent exposure and CKD were included. Two authors independently reviewed full-text. Of 5,109 studies identified, a total of 19 studies (control studies: 14 and cohort studies: 5) were finally included in our meta-analysis. The pooled risk of CKD in the organic solvent exposed group was 2.44 (1.72-3.47). The risk of a low-level exposure group was 1.07 (0.77-1.49). The total risk of a high-level exposure group was 2.44 (1.19-5.00). The risk of glomerulonephritis was 2.69 (1.18-6.11). The risk was 1.46 (1.29-1.64) for worsening of renal function. The pooled risk was 2.41 (1.57-3.70) in case-control studies and 2.51 (1.34-4.70) in cohort studies. The risk of subgroup classified as 'good' by the Newcastle Ottawa scale score was 1.93 (1.43-2.61). This study confirmed that the risk of CKD was significantly increased in workers exposed to mixed organic solvents. Further research is needed to determine the exact mechanisms and thresholds. Surveillance for kidney damage in the group exposed to high levels of organic solvents should be conducted. PROSPERO Identifier: CRD42022306521.

Endometrial hyperplasia features in conditions of organic solvents exposure

Objective: to establish clinical and molecular features of endometrial hyperplasia in conditions of organic solvents exposure in patients with obesity. Materials and methods: postmenopausal women with endometrial hyperplasia (morphological criteria) were included to observational cross-sectional study. The main group included 140 subjects with occupational organic solvents exposure, aged 57,5 (46;64) years, of whom 70 had obesity and 70 had normal weight. Comparison group — 140 women without of occupational health risks, aged 56,5 (46;65) years, 68 with and 72 without obesity. Investigated workers were painters in machine building industry with length of service above 10 years, in post-contact period lasted for 1-5 years were 29 (20.7%) of participants. Organic solvents concentrations at the workplace’s areas were 1.5-5.5 times above occupational exposure limits. Linear and logistic regression were used to explore relationships. Level of significance p<0.05. Results: workers exposed to organic solvents had maximal percent of atypical endometrial hyperplasia: 34.3% in obesity subjects and 18.6% in those without obesity, in the comparison group 10.3% in subjects with and 6.9% in those without obesity, p=0.004. Subgroup with toxicant exposure with obesity characterized by maximal expression of Ki-67 (55,9±2,51, 51,4±4,95, 41,5±3,29 and 30,1±2,18 scores respectively), higher rate of uterine bleeding, M-echo thickness, blood interleukin 1β concentration. Atypical endometrial hyperplasia was associated with length of service (OR 1.80), maximal short-term exposure concentration of toluene (OR 2.51), waist circumference (OR 1.68), Ki-67 (OR 2,01), estrogen receptors on epithelium (OR 1.92). Conclusions: organic solvents exposure in postmenopausal women with endometrial hyperplasia contributes to development of atypical hyperplasia, systemic inflammation, Ki-67 expression, higher grades in subjects with obesity.

Type 2B von Willebrand disease mutations differentially perturb autoinhibition of the A1 domain

AbstractType 2B von Willebrand disease (VWD) is an inherited bleeding disorder in which a subset of point mutations in the von Willebrand factor (VWF) A1 domain and recently identified autoinhibitory module (AIM) cause spontaneous binding to glycoprotein Ibα (GPIbα) on the platelet surface. All reported type 2B VWD mutations share this enhanced binding; however, type 2B VWD manifests as variable bleeding complications and platelet levels in patients, depending on the underlying mutation. Understanding how these mutations localizing to a similar region can result in such disparate patient outcomes is essential for detailing our understanding of VWF regulatory and activation mechanisms. In this study, we produced recombinant glycosylated AIM-A1 fragments bearing type 2B VWD mutations and examined how each mutation affects the A1 domain’s thermodynamic stability, conformational dynamics, and biomechanical regulation of the AIM. We found that the A1 domain with mutations associated with severe bleeding occupy a higher affinity state correlating with enhanced flexibility in the secondary GPIbα-binding sites. Conversely, mutation P1266L, associated with normal platelet levels, has similar proportions of high-affinity molecules to wild-type (WT) but shares regions of solvent accessibility with both WT and other type 2B VWD mutations. V1316M exhibited exceptional instability and solvent exposure compared with all variants. Lastly, examination of the mechanical stability of each variant revealed variable AIM unfolding. Together, these studies illustrate that the heterogeneity among type 2B VWD mutations is evident in AIM-A1 fragments.

Maternal occupational exposure to organic solvents and intrauterine growth in the ELFE cohort

BackgroundIn developed countries, about 15% of women are occupationally exposed to solvents. Associations between this maternal occupational exposure and intrauterine fetal growth are inconsistent, but almost no existing study has investigated this relation by solvent family (oxygenated, petroleum, and chlorinated), although they may affect fetal growth differently. ObjectivesTo investigate the relations between maternal occupational solvent exposure, by solvent family, and the risk of neonates born small for gestational age (SGA), or with low birthweight, or with small head circumference (HC). MethodsAmong the 18,040 women enrolled in the Elfe rather than included in the Elfe birth cohort, we included 13,026 women who worked during pregnancy (72% of the cohort). Information about maternal occupations and industrial activities during pregnancy was collected by questionnaire at the maternity ward, and completed at 2-month when necessary. Using Matgéné job-exposure matrices, we assessed maternal occupational exposure to solvents. Logistic and multiple linear regressions were used to assess the association between maternal occupational solvent exposure and SGA status, birth weight, and HC. Analyses were conducted for exposure during pregnancy and also stratified by the trimester that pregnancy leave began. ResultsWe observed a higher risk of SGA newborns among mothers occupationally exposed during pregnancy to petroleum solvents (ORadjusted = 1.26; 95%CI: 1.01 to 1.57). Among women working until the third trimester of pregnancy, we observed a higher risk of SGA newborns to those occupationally exposed to oxygenated solvents (ORadjusted = 1.75; 95%CI: 1.11 to 2.75), a significantly lower birthweight for infants of mothers exposed to petroleum solvents (βadjusted = −47.37 g; −89.33 to −5.42), and a lower HC among newborns of those occupationally exposed to oxygenated solvents (βadjusted = −0.28; −0.49 to −0.07) and to chlorinated solvents (βadjusted = −0.29; −0.53 to −0.05). DiscussionOur results suggest that maternal occupational solvent exposure may influence fetal growth, especially exposure into the third trimester of pregnancy.

Structural Remodeling Mechanism of the Toxic Amyloid Fibrillary Mediated by Epigallocatechin-3-gallate.

Numerous therapeutic agents and strategies were designed targeting the therapies of Alzheimer's disease, but many have been suspended due to their severe clinical side effects (such as encephalopathy) on patients. The attractiveness for small molecules with good biocompatibility is therefore restarted. Epigallocatechin-3-gallate (EGCG), extracted from green tea, is expected to be a promising small-molecule drug candidate, which can remodel the structure of preformed β-sheet-rich oligomers/fibrils and then effectively interfere with neurodegenerative processes. However, as the structure of non-fibrillary aggregates cannot be directly characterized, the atomic details of the underlying inhibitory and destructive mechanisms still remain elusive to date. Here, all-atom molecular dynamics simulations and experiments were carried out to elucidate the EGCG-induced remodeling mechanism of amyloid β (Aβ) fibrils. We showed that EGCG was indeed an effective Aβ fibril inhibitor. EGCG was capable of mediating conformational rearrangement of Aβ1-42 fibrils (from a β-sheet to a random coil structure) and triggering the disintegration of fibrils in a dose-dependent manner. EGCG redirected the structure of Aβ by breaking the β-sheet structure and hydrogen bonds between peptide chains within the Aβ protofibrils, especially the parallel β-strand (L17VFFAEDVGS26). Moreover, reduced solvent exposure and multisite binding patterns all tended to induce the conformation conversion of Aβ17-42 pentameric protofibrils, destroying pre-formed fibrils and inhibiting continued fibril growth. Detailed data analysis revealed that structural features of EGCG with abundant benzene ring and phenolic hydroxyl moieties preferentially interact with the parallel β-strands to effectually hinder the interaction of the interpeptide chain and the growth of the ordered β-sheet structure. Furthermore, experimental studies confirmed that EGCG was able to disaggregate the preformed fibrils and alter the protein structure. This study will enable a deeper understanding of fundamental principles for design of structural-based inhibitors.

Molecular Insights into the Calcium Binding in Troponin C through a Molecular Dynamics Study

Calcium-binding proteins play critical roles in various biological processes such as signal transduction, cell growth, and transcription factor regulation. Ion binding and target binding of Ca2+-binding proteins are highly related. Therefore, understanding the ion binding mechanism will benefit the relevant inhibitor design toward the Ca2+-binding proteins. The EF-hand is the typical ion binding motif in Ca2+-binding proteins. Previous studies indicate that the ion binding affinity of the EF-hand increases with the peptide length, but this mechanism has not been fully understood. Herein, using molecular dynamics simulations, thermodynamic integration calculations, and molecular mechanics Poisson-Boltzmann surface area analysis, we systematically investigated four Ca2+-binding peptides containing the EF-hand loop in site III of rabbit skeletal troponin C. These four peptides have 13, 21, 26, and 34 residues. Our simulations reproduced the observed trend that the ion binding affinity increases with the peptide length. Our results implied that the E-helix motif preceding the EF-hand loop, likely the Phe99 residue in particular, plays a significant role in this regulation. The E-helix has a significant impact on the backbone and side-chain conformations of the Asp103 residue, rigidifying important hydrogen bonds in the EF-hand and decreasing the solvent exposure of the Ca2+ ion, hence leading to more favorable Ca2+ binding in longer peptides. The present study provides molecular insights into the ion binding in the EF-hand and establishes an important step toward elucidating the responses of Ca2+-binding proteins toward the ion and target availability.

Activation Mechanism of RhoA Caused by Constitutively Activating Mutations G14V and Q63L

RhoA, a member of Rho GTPases, regulates myriad cellular processes. Abnormal expression of RhoA has been implicated in various diseases, including cancers, developmental disorders and bacterial infections. RhoA mutations G14V and Q63L have been reported to constitutively activate RhoA. To figure out the mechanisms, in total, 1.8 μs molecular dynamics (MD) simulations were performed here on RhoAWT and mutants G14V and Q63L in GTP-bound forms, followed by dynamic analysis. Both mutations were found to affect the conformational dynamics of RhoA switch regions, especially switch I, shifting the whole ensemble from the wild type's open inactive state to different active-like states, where T37 and Mg2+ played important roles. In RhoAG14V, both switches underwent thorough state transition, whereas in RhoAQ63L, only switch I was sustained in a much more closed conformation with additional hydrophobic interactions introduced by L63. Moreover, significantly decreased solvent exposure of the GTP-binding site was observed in both mutants with the surrounding hydrophobic regions expanded, which furnished access to water molecules required for hydrolysis more difficult and thereby impaired GTP hydrolysis. These structural and dynamic differences first suggested the potential activation mechanism of RhoAG14V and RhoAQ63L. Together, our findings complemented the understanding of RhoA activation at the atomic level and can be utilized in the development of novel therapies for RhoA-related diseases.

Concerted enhanced-sampling simulations to elucidate the helix-fibril transition pathway of intrinsically disordered α-Synuclein

Fibril formation of α-synuclein is linked with Parkinson's disease. The intrinsically disordered nature of α-syn provides extensive conformational plasticity and becomes difficult to characterize its transition pathway from native monomeric to disease-associated fibril form. We implemented different simulation methods such as steered dynamics-umbrella sampling, and replica-exchange and conventional MD simulations to access various conformational states of α-syn. Nineteen distinct intermediate structures were identified by free energy landscape and cluster analysis. They were then sorted based on secondary structure and solvent exposure of fibril-core residues to illustrate the fibril dissociation pathway. The analysis showed that following the initial dissociation of the polypeptide chain from the fibril, α-syn might form either compact-conformations by long-range interactions or extended-conformations stabilized by local interactions. This leads α-syn to adapt two different pathways. The secondary structure, solvation, contact distance, interaction energies and backbone dihedrals of thirty-two selected residues were analyzed for all the 19 intermediates. The results suggested that formation of β-turns, reorganization of salt bridges, and dihedral changes in the hydrophobic regions are the major driving forces for helix-fibril transition. Structural features of the intermediates also correlated with the earlier experimental and computational studies. The study provides critical information on the fibrillation pathway of α-syn.

Solvent-Induced Degradation of Electrochemically Exfoliated Vanadium Selenide Visualized by Electron Microscopy.

Recently discovered two-dimensional ferromagnetic materials (2DFMs) have rapidly gained much interest in the fields of spintronics and computing, where they may prove powerful tools for miniaturizing devices such as magnetic tunnel junctions and spin-transfer torque memory bits. In addition, heterojunctions and twisted bilayer stacks of such materials may yield exotic spin textures. However, preparation of such devices is complicated by the air sensitivity of many 2DFMs. Here, we report details on the preparation of few-to-monolayer flakes of vanadium selenide (VSe2) using electrochemical exfoliation in propylene carbonate. We also present a detailed study of the effects of air on the structure and magnetic properties of bare and passivated VSe2 after different concentrations of surface passivation treatment. We characterized the microstructure of holes in the VSe2 flakes and the formation of new compounds arising from air exposure, solvent exposure during the exfoliating process, and deliberate electron beam irradiation (sculpting). We sculpt VSe2 flakes while retaining the 1T-VSe2 lattice structure, opening the door for top-down patterned high-resolution 2DFM nanostructures. Additionally, investigation of the magnetic response of nanosheets using magnetic force microscopy (MFM) showed that the oxidation-induced damage only affects the surface fields locally and does not quench large-scale magnetic signal. The findings of this study pave the way toward practical incorporation of 2D ferromagnetic materials in nanoelectronics.

Incorporation of D2O-Induced Fluorine Chemical Shift Perturbations into Ensemble-Structure Characterization of the ERalpha Disordered Region.

Structural characterization of intrinsically disordered proteins (IDPs) requires a concerted effort between experiments and computations by accounting for their conformational heterogeneity. Given the diversity of experimental tools providing local and global structural information, constructing an experimental restraint-satisfying structural ensemble remains challenging. Here, we use the disordered N-terminal domain (NTD) of the estrogen receptor alpha (ERalpha) as a model system to combine existing small-angle X-ray scattering (SAXS) and hydroxyl radical protein footprinting (HRPF) data and newly acquired solvent accessibility data via D2O-induced fluorine chemical shifting (DFCS) measurements. A new set of DFCS data for the solvent exposure of a set of 12 amino acid positions were added to complement previously acquired HRPF measurements for the solvent exposure of the other 16 nonoverlapping amino acids, thereby improving the NTD ensemble characterization considerably. We also found that while choosing an initial ensemble of structures generated from a different atomic-level force field or sampling/modeling method can lead to distinct contact maps even when the same sets of experimental measurements were used for ensemble-fitting, comparative analyses from these initial ensembles reveal commonly recurring structural features in their ensemble-averaged contact map. Specifically, nonlocal or long-range transient interactions were found consistently between the N-terminal segments and the central region, sufficient to mediate the conformational ensemble and regulate how the NTD interacts with its coactivator proteins.

Stability and expression of SARS-CoV-2 spike-protein mutations.

Protein fold stability likely plays a role in SARS-CoV-2 S-protein evolution, together with ACE2 binding and antibody evasion. While few thermodynamic stability data are available for S-protein mutants, many systematic experimental data exist for their expression. In this paper, we explore whether such expression levels relate to the thermodynamic stability of the mutants. We studied mutation-induced SARS-CoV-2 S-protein fold stability, as computed by three very distinct methods and eight different protein structures to account for method- and structure-dependencies. For all methods and structures used (24 comparisons), computed stability changes correlate significantly (99% confidence level) with experimental yeast expression from the literature, such that higher expression is associated with relatively higher fold stability. Also significant, albeit weaker, correlations were seen between stability and ACE2 binding effects. The effect of thermodynamic fold stability may be direct or a correlate of amino acid or site properties, notably the solvent exposure of the site. Correlation between computed stability and experimental expression and ACE2 binding suggests that functional properties of the SARS-CoV-2 S-protein mutant space are largely determined by a few simple features, due to underlying correlations. Our study lends promise to the development of computational tools that may ideally aid in understanding and predicting SARS-CoV-2 S-protein evolution.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11010-022-04588-w.

(Invited) Bipolar Membranes with a 3D Junction of Interlocking Electrospun Fibers

Bipolar membranes (BPMs), typically laminated layers of anion-exchange and cation-exchange polymers, have the unique capability of splitting water at a potential near 0.83 V. Such membranes are used in electrodialysis membrane separation processes. They also have applications in water electrolyzers, CO2 electrolysis cells, and self-humidifying fuel cells. We report here on recent developments regarding BPMs with a high interfacial area, 3D nanofiber junction.Membranes were prepared by first creating a bipolar junction layer, by the simultaneous electrospinning of anion-exchange and cation-exchange polymers, with the addition of catalyst nanoparticles to facilitate water splitting. Solvent vapor exposure and hot-pressing closed all interfiber pores, to create a dense film of interpenetrating and interlocking nanofibers of positively and negatively charged polymers. In one fabrication method, dense films of solution cast anion and cation-exchange polymers were hot-pressed onto the opposing surfaces of the junction layer to create a tri-layer BPM. Membranes were also fabricated by electrospinning all three layers of the BPM: first spinning anion-exchange fibers, followed by dual fiber spinning with catalyst spraying, and then spinning only cation-exchange fibers. Solvent exposure and hot-pressing closed all interfiber voids.BPMs were made with a junction layer 12-15 mm thick, where the total membrane thickness was 50-80 mm. Membranes were prepared with both hydrocarbon and fluoropolymer ionomers, e.g., sulfonated poly(ether ether ketone) or perfluorosulfonic acid as the cation-exchange polymer and either quaternized poly(phenylene oxide), AEMIONä (an imidazolium-based polymer sold by Ionomr Innovations, Inc.), or PiperION (a poly(aryl piperidinium) from Versogen) as the anion-exchange polymer. A variety of different junction layer catalysts powders were examined, including Al(OH)3 and graphene oxide. Membranes were evaluated in an H-cell for the collection of steady-state current-voltage data and in a flow cell for long-term constant current water splitting (electrolysis) operation, typically with an aqueous Na2SO4 electrolyte. The 3D junction membranes performed exceptionally well: (i) the water splitting potential was low (near the expected value of 0.83 V), (ii) the transmembrane voltage drop was small at high currents (e.g., a voltage drop of 1.1 V up to 1.1 A/cm2), and (iii) the extended bipolar reaction zone for water splitting with interlocking fibers allowed for high current density operation with no evidence of membrane degradation.In this talk, the effect of membrane composition (the type/amount of anion and cation exchange polymers and choice of catalyst) and structure (the thickness of the layers) on short-term and long-term membrane performance will be discussed. Results will be presented for operating the bipolar membrane in water splitting and water generation modes.

Study of Parental Occupational Exposures to Solvents and Risk of Autism Spectrum Disorder in Offspring

BACKGROUND AND AIM: Solvents can accumulate in fat and later metabolize during pregnancy. Additionally, these toxicants could lead to germline mutations. We investigated associations between multiple parental occupational solvent exposures and odds of autism spectrum disorder (ASD) diagnosis in offspring. METHODS: Using population-based data from the Danish National Patient Registry, we identified a subset of ASD cases from 1982 to 2016 and 1:100 sex and birth-year matched controls obtained from the Central Population Register. We determined parental occupation history from age 16 years through 6 months post-childbirth from the Danish Pension Fund and estimated cumulative benzene, methylcyclohexane, toluene, trichloroethylene, perchloroethylene, and 1,1,1-trichloroethane exposure for mothers and fathers based on job exposure matrices (JEMs). Conditional logistic regression was used to estimate adjusted odds ratios (aOR) and 95% confidence intervals (CI) for exposures one year prior to conception and during pregnancy. RESULTS: In analysis of data from 1898 people with ASD and 141,702 without ASD, there were no observed associations between maternal solvent exposures during pregnancy and risk of ASD in offspring. However, analysis for maternal benzene exposure one year prior to conception revealed higher adjusted odds for those with any exposure (aOR=1.55; 95% CI 1.02, 2.37). Additionally, although there were no significant associations between paternal solvent exposures during pregnancy and ASD risk, there were significantly increased odds of ASD in offspring whose fathers were exposed to benzene (aOR=3.07; 95% CI 1.72, 5.46) and toluene (aOR=1.66; 95% CI 1.05, 2.62) one year before conception. CONCLUSIONS: Although previous studies have suggested parental solvent exposure during pregnancy can contribute to pathogenesis of ASD, our findings suggest that risk may be increased through exposures prior to conception. However, unavoidable non-differential misclassification from the use of JEMs may have masked truly increased risk during pregnancy.

Residue-Specific Impact of EDTA and Methionine on Protein Oxidation in Biotherapeutics Formulations Using an Integrated Biotherapeutics Drug Product Development Workflow

The rational design and selection of formulation composition to meet molecule-specific and product-specific needs are critical for biotherapeutics development to ensure physical and chemical stability. This work, based on three antibody-based (mAb) proteins (mAbA, mAbB, and mAbC), evaluates residue-specific impact of EDTA and methionine on protein oxidation, using an integrated biotherapeutics drug product development workflow. This workflow includes statistical experimental design, high-throughput experimental automation and execution, structure-based in silico modeling, inferential statistical analysis, and enhanced interactive data visualization of large datasets. This oxidation study evaluates the impact of formulation parameters including pH, protein concentration, and the presence of polysorbate 80 on the oxidation of specific conserved and variable residues of mAbs A, B, and C in the presence of stressors (iron, peroxide) and/or protectants (EDTA, L-methionine). Residue-specific analysis by automated high-throughput peptide mapping demonstrates differential residue-specific effects of EDTA and methionine in protecting against oxidation, highlighting the need for molecule-specific and product-specific selection of these excipients during formulation development. Computational modeling based on a homology model and the two-shell water coordination method (WCN) was employed to gain mechanistic understanding of residue-specific oxidation susceptibility of methionine residues. The computational determinants of local solvent exposure of methionine residues showed good correlation of WCN with experimentally determined oxidation for corresponding residues. The rapid generation of high-resolution data, statistical data analysis and interactive visualization of the high-throughput residue-level data containing ∼200 unique formulations facilitate residue-specific, molecule-specific and product-specific oxidation (global and local) assessment for oxidation protectants during early development for mAbs and related mAb-based modalities.

ATP-site inhibitors induce unique conformations of the acute myeloid leukemia-associated Src-family kinase, Fgr

The Src-family kinase Fgr is expressed primarily in myeloid hematopoietic cells and contributes to myeloid leukemia. Here, we present X-ray crystal structures of Fgr bound to the ATP-site inhibitors A-419259 and TL02-59, which show promise as anti-leukemic agents. A-419259 induces a closed Fgr conformation, with the SH3 and SH2 domains engaging the SH2-kinase linker and C-terminal tail, respectively. In the Fgr:A-419259 complex, the activation loop of one monomer inserts into the active site of the other, providing a snapshot of trans-autophosphorylation. By contrast, TL02-59 binding induced SH2 domain displacement from the C-terminal tail and SH3 domain release from the linker. Solution studies using HDX MS were consistent with the crystal structures, with A-419259 reducing and TL02-59 enhancing solvent exposure of the SH3 domain. These structures demonstrate that allosteric connections between the kinase and regulatory domains of Src-family kinases are regulated by the ligand bound to the active site.

Presentation of HIV and Mtb derived peptides by HLA-E

Unconventional, Mamu-E-restricted CD8+ T cell responses directed against a diverse range of epitopes have been implicated as immune correlates of sterile immune protection in rhesus CMV-vectored SIV vaccination. In light of these findings, we have re-explored the peptide binding specificity of the human ortholog, HLA-E, from the view of developing a parallel HIV vaccination strategy in humans. Given the extraordinary breadth of vaccine-elicited, Mamu-E-restricted responses, we first optimised and developed two highly sensitive peptide binding affinity assays to determine whether broad epitope sampling also extends to HLA-E. We found extensive overlap in the binding repertoires of the human and rhesus orthologs, and the majority of SIV-derived Mamu-E-restricted epitopes tested also bound HLA-E. We also demonstrated binding for a number of HIV and Mtb derived epitopes identified from rhesus macaques vaccinated with rhCMV-HIV Gag immunogens or from active tuberculosis infection in humans, respectively. As previous crystal structures of HLA-E were exclusively solved in complex with canonical MHC-I signal peptides, we next investigated the structural basis underlying pathogen-derived peptide binding. We obtained a panel of crystal structures of HLA-E in complex with different epitopes including a number from Mtb in addition to the HIV counterpart to the vaccine-identified SIV Gag ‘supertope’69. Strikingly, despite the presence of canonical primary anchor residues, the HIV pep- tide adopted an unusual motif within the peptide binding groove, generating a solvent exposure profile that deviated from canonical signal peptide motifs, with potential implications for T cell receptor recognition and immunogenicity. Further, structural and mutagenesis analyses illustrated a greater tolerance for hydrophobic and polar residues in the primary pockets than previously anticipated. In conclusion, our combined structural and peptide binding data suggest a previously underestimated breadth of the HLA-E peptidome and support the potential for broadly targeted HLA-E-restricted HIV vaccine development.