Domain Interface Research Articles

Study of Interaction Patterns Between Evolving Interfaces Due to Boiling Around Different Arrangements of Heated Cylinders

Abstract An effort has been made to numerically study the interactions between the vapor films generating from multiple heated cylinders placed in a saturated water pool with a view to understand the influence of neighboring interface on growth of vapor films. Lagrangian smoothed particle hydrodynamics (SPH) has been used for domain discretization and interface reconstruction. Unconstrained growth of vapor film around a cylinder is simulated first and the nature of vapor dynamics has been taken as a base case for the study of interaction patterns. Vapor growth has been understood through temporal phase contours, azimuthal thickness variation, and trajectory of the centroid of the vapor mass. It has been shown that the presence of cylinder in vertical neighborhood results in suppression of vapor film generating from the bottom cylinder, whereas, the vapor films generating from two cylinders placed in horizontal neighborhood experience a horizontal shift. Studies have also been made to observe multidirectional interactions of vapor films with heated cylinders placed in an inline array, vertically staggered and horizontally staggered arrangements. It has been found that the highest deviation from unconstrained growth occurs in case of the center cylinder in a horizontally staggered arrangement as compared to others. Mutual interaction coefficient and indices are proposed to judge the best possible arrangement in case of a requirement for placement of stacked cylinders.

Modeling and Structural Characterization of the Sweet Taste Receptor Heterodimer.

Sweet taste receptor, a heterodimer belonging to the class C G-protein coupled receptor (GPCR) family and composed of the T1R2 and T1R3 subunits, is responsible for the perception of natural sugars, sweet proteins, various d-amino acids, as well as artificial sweeteners. Despite the critical importance of the sweet receptor not only in mediating gustation but also in its role in the food industry, the architecture of the T1R2-T1R3 complex and the mechanism by which extracellular stimuli induce conformational changes that are propagated to the intracellular milieu, i.e., the signal transduction pathway, remain largely unknown. Here, we constructed and characterized a full-length structural model of the T1R2-T1R3 receptor, including both the transmembrane (TM) and extracellular (EC) domains of the heterodimer, using comparative modeling and extensive all-atom molecular dynamics simulations. Several heterodimer interfaces were first examined for the TM domain, and conformational changes occurring at the intracellular side and associated with the receptor's activation were characterized. From the analysis on the simulated data, putative allosteric binding sites for ligands, ions, and cholesterol were proposed. Also, insights into the protein interface of the TM domain upon activation are provided. The EC domain of the heterodimer, including both the Venus flytrap and cysteine-rich domains, was also investigated. Several important intersubunit interactions located at regions responsible for the receptor's proper function were observed, which resemble those recently identified in other class C GPCR members. Integration of the results from the TM and EC domains facilitates the generation of a full-length T1R2-T1R3 receptor. These findings along with the full-length structural model of the T1R2-T1R3 receptor provide a structural framework that may assist in understanding the mechanistic details associated with the receptor activation process for the sweet T1R2-T1R3 receptor as well as other members of the same family.

Molecular dynamics simulations of mechanical stress on oxidized membranes

Biomembranes are under constant attack of free radicals that may lead to lipid oxidation in conditions of oxidative stress. The products generated during lipid oxidation are responsible for structural and dynamical changes which may jeopardize the membrane function. For instance, the local rearrangements of oxidized lipid molecules may induce membrane rupture. In this study, we investigated the effects of mechanical stress on oxidized phospholipid bilayers (PLBs). Model bilayers were stretched until pore formation (or poration) using non-equilibrium molecular dynamics simulations. We studied single-component homogeneous membranes composed of lipid oxidation products, as well as two-component heterogeneous membranes with coexisting native and oxidized domains. In homogeneous membranes, the oxidation products with —OH and —OOH groups reduced the areal strain required for pore formation, whereas the oxidation product with O group behaved similarly to the native membrane. In heterogeneous membranes composed of oxidized and non-oxidized domains, we tested the hypothesis according to which poration may be facilitated at the domain interface region. However, results were inconclusive due to their large statistical variance and sensitivity to simulation setup parameters. We pointed out important technical issues that need to be considered in future simulations of mechanically-induced poration of heterogeneous membranes. This research is of interest for photodynamic therapy and plasma medicine, because ruptured and intact plasma membranes are experimentally considered hallmarks of necrotic and apoptotic cell death.

SWAT‐LUT: A Desktop Graphical User Interface for Updating Land Use in SWAT

AbstractLong‐term simulations of agricultural watersheds have often been done assuming constant land use over time, but this is not a realistic assumption for many agricultural regions. This paper presents the soil and water assessment tool (SWAT)‐Landuse Update Tool (LUT), a standalone, user‐friendly desktop‐based tool for updating land use in the SWAT model that allows users to process multi‐year land use data. SWAT‐LUT is compatible with several SWAT model interfaces, provides users with several options to easily prepare and incorporate land use changes (LUCs) over a simulation period, and allows users to incorporate past or emerging land use categories. Incorporation of LUCs is expected to provide realistic model parameterization and scenario simulations. SWAT‐LUT is a public domain interface written in Python programming language. Two applications at the Fort Cobb Reservoir Experimental Watershed located in Oklahoma and pertinent results are provided to demonstrate its use. Incorporating LUCs related to implementation of recommended conservation practices over the years reduced discharge, evapotranspiration, sediment, total nitrogen, and total phosphorus loads by 59%, 9%, 68%, 53%, and 88%, respectively. The user’s manual is included in this article as Supporting Information. The SWAT‐LUT executable file and an example SWAT project with three land use rasters and the user’s manual are available at the United States Department of Agriculture‐Agricultural Research Service Grazinglands Research Laboratory website under Software. Editor’s note: This paper is part of the featured series on Optimizing Ogallala Aquifer Water Use to Sustain Food Systems. See the February 2019 issue for the introduction and background to the series.

Structure and chemistry of lysinoalanine crosslinking in the spirochaete flagella hook.

The flagellar hook protein FlgE from spirochaete bacteria self-catalyzes the formation of an unusual inter-subunit lysinoalanine (Lal) crosslink that is critical for cell motility. Unlike other known examples of Lal biosynthesis, conserved cysteine and lysine residues in FlgE spontaneously react to form Lal without the involvement of additional enzymes. Oligomerization of FlgE via its D0 and Dc domains drives assembly of the crosslinking site at the D1–D2 domain interface. Structures of the FlgED2 domain, dehydroalanine (DHA) intermediate, and Lal crosslinked FlgE subunits reveal successive snapshots of the reaction. Cys178 flips from a buried configuration to release hydrogen sulfide (H2S/HS−) and produce DHA. Interface residues provide hydrogen bonds to anchor the active site, facilitate β-elimination of Cys178, and polarize the peptide backbone to activate DHA for reaction with Lys165. Cysteine-reactive molecules accelerate DHA formation, whereas nucleophiles can intercept the DHA intermediate, thereby indicating a potential for Lal crosslink inhibitors to combat spirochaetal diseases.

Structural studies on the individual domains of human γS-crystallin and its G57W mutant unfolds mechanistic insights into childhood cataracts

Inter-domain interactions tune the exceptional stability of human γS-crystallin (γS-WT) in the eye lens, which lasts a lifetime with no protein turnover. Our recent NMR studies revealed the key role of G57W mutation in γS-WT, as the familial determinate of childhood cataracts. As the unusually exposed W57 is near the inter-domain interface, a recurring theme of this study is the upsetting of inter-domain contacts exposing hydrophobic patches, which may initiate aggregation at crystallin concentrations not so surprising in the eye lens. In this endeavour, to untangle the mechanistic pathways triggering aggregation in the cataract variant γS-G57W, we undertook high-resolution structural characterization of isolated domains vis-a-vis full length γS-crystallin. Here we report for the first time, thermodynamic and kinetic determinants of structural stability with their eccentric shifts under pathological stress employing sophisticated spectroscopy techniques. We propose that domain interface acts as an intrinsic stabilizer for the otherwise floppy N-terminal domain in γS-G57W than in γS-WT where it serves an extrinsic role. Our results present a residue resolved quantitative analysis for differential domain stabilities from non-linear temperature coefficients of 1HN chemical shifts using solution NMR spectroscopy. Consistent with the Ca2+-binding episode that lasted poorly for human lens crystallins, our results show that disease mutants attenuate it further and completely silence it in extreme cases. Overall, our study provides a compelling evidence for the diverse structural evolution of lens crystallins elucidating molecular details to apprehend lens opacification and suggests the scope of therapeutics in reducing the global trauma of cataracts.

Proline Residues in the Transmembrane/Extracellular Domain Interface Loops Have Different Behaviors in 5-HT3 and nACh Receptors.

Cys-loop receptors are important drug targets that are involved in signaling in the nervous system. The binding of neurotransmitters in the extracellular region of these receptors triggers an allosteric activation mechanism, the full details of which remain elusive, although structurally flexible loops in the interface between the extracellular region of Cys-loop receptors and the pore-forming transmembrane domain are known to play an important role. Here we explore the roles of three largely conserved Pro residues in two of these loops, the Cys-loop and M2-M3 loop, in 5-HT3A and α7 nACh receptors. The data from natural and noncanonical amino acid mutagenesis suggest that in both proteins a Pro is essential in the Cys-loop, probably because of its enhanced ability to form a cis peptide bond, although other factors are also involved. The important characteristics of Pros in the M2-M3 loop, however, differ in these two receptors: in the 5-HT3 receptor, the Pros can be replaced by some charged amino acids resulting in EC50s similar to those of wild-type receptors, while such substitutions in the nACh receptor ablate function. Ala substitution at one of these Pros also has different effects in the two receptors. Thus, our data show that even highly conserved residues can have distinct behaviors in related Cys-loop receptors.

Abstract 1854: Novel pateamine analogs to target the translation initiation factor eIF4A in chronic lymphocytic leukemia

Abstract The viability of chronic lymphocytic leukemia (CLL) is critically dependent upon staving off death by apoptosis, a hallmark of CLL pathophysiology. The overexpression of the Bcl-2 family proteins likely play a major role in the apoptosis blockade in CLL, and has been an effective target of CLL therapy. The recognition that Mcl-1, a major component of the anti-apoptotic response, is intrinsically short-lived and must be continually resynthesized suggested a novel therapeutic approach. Pateamine A (PatA), a macrolide marine natural product, inhibits cap-dependent translation by binding to the initiation factor eIF4A. We have previously reported the first total synthesis of PatA. Mechanistic studies suggested that binding of eIF4A to PatA caused the stalling of initiation complexes on mRNA, halting the translation initiation process. In this study, we demonstrated that a synthetic derivative of PatA, des-methyl des-amino PatA (DMDAPatA), blocked mRNA translation, reduced Mcl-1 protein and initiated apoptosis in CLL cells. This action was synergistic with the Bcl-2 antagonist ABT-199, by a mechanism to inhibit the two parallel arms of apoptosis control in CLL. However, avid binding to human plasma proteins limited DMDAPatA potency, precluding further development. To address this, we synthesized a new series of 27 PatA analogs with modifications on various regions of PatA, and screened their toxicity against the primary CLL cells. Any modifications on the side chain, or the rigid binding domain of the PatA macrolide ring led to complete loss of activity. Rather, introduction of an amino group at either the C2 or C3 positions of the flexible region of the macrocycle retained the activity and reduced plasma protein binding, likely through a lowered lipophilicity. We identified three new leads with potent inhibition of proteins synthesis and strong CLL cytotoxicity. They also exhibited greater selectivity towards CLL cells over normal lymphocytes comparing to the parental compound PatA. To gain structural insights into the interaction of the PatA analogs with eIF4A, a homology model of the human eIF4A1 was generated using the closed conformation of the eIF4A3 structure (PDB ID: 2HYI) as a template. The predicted PatA binding site is located at the interface of the N-terminal domain and the C-terminal domain, in between the RNA and ATP binding sites. In silico docking analysis of the PatA analogs to eIF4A correlated with their structure-activity relationships and suggested that these compounds may act by stabilizing the closed conformation of eIF4A. Thus, these novel PatA analogs hold promise for application to cancers within the appropriate biological context, such as CLL. Citation Format: Rong Chen, Mingzhao Zhu, Rajan R. Chaudhari, Omar Robles, Yuling Chen, Wesley Skillern, Qun Qin, William Wierda, Shuxing Zhang, Kenneth G. Hull, Daniel Romo, William Plunkett. Novel pateamine analogs to target the translation initiation factor eIF4A in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1854.

Abstract 4826: Enhancement of PTEN activity via peptidomimetics

Abstract Compromised PTEN function is associated with multiple cancers. As per the continuum model, variable degree of PTEN inactivation drives distinct cancer phenotypes. Loss of PTEN activity, via genomic/non-genomic mechanisms, leads to enhanced oncogenic PI3K signaling. While kinase inhibitors have proved effective in the clinic, they are increasingly met with off-target effects and therapy-resistance due to compensatory feedback mechanisms. Although PTEN restoration therapy has proved promising in experimental models; implementation of these therapies in the clinic remains challenging. To fill this therapy gap, we have targeted endogenous PTEN for activation via peptidomimetics, mitigating PI3K signaling. Our selected peptidomimetics reduced cell proliferation, migration and cell cycle activity after treatment of non-small cell lung cancer cells expressing endogenous PTEN. Computational studies utilizing PTEN crystal structures revealed a binding site at the interface of the Phosphatase Domain (PD) and C2 Domain (C2D). Induced fit docking indicated that a unique functional group is responsible for peptidomimetic-mediated enhancement of PTEN activity. Molecular dynamics analysis revealed energetically favorable binding of our lead peptidomimetic at the PD/C2D interface, which allosterically modulated the conformation of the active site. Binding of our peptidomimetic altered the active site orientation of PIP3, increasing its binding affinity. In summary, we have developed the first-known direct PTEN activators and analyzed their effects in suppressing oncogenic activities in lung cancer. Refinement of our peptidomimetics, complemented with in vivo studies, will provide a novel clinical rationale to reduce doses of standard therapy and their associated toxicities, decreasing morbidity and mortality observed in the clinic. Citation Format: Emily Palumbo, Peng Teng, Prerna Malaney, Jacob Wilson, Fiona Kearns, Michael T. Kemp, Zhi Tian, Vladimir Uversky, Diane Allen-Gipson, Yu Chen, H. Lee Woodcock, Jianfeng Cai, Vrushank Dave. Enhancement of PTEN activity via peptidomimetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4826.

Gain-of-Function SHP2 E76Q Mutant Rescuing Autoinhibition Mechanism Associated with Juvenile Myelomonocytic Leukemia.

Juvenile myelomonocytic leukemia (JMML) is an invasive myeloproliferative neoplasm and is a childhood disease with very high clinical lethality. The SHP2 is encoded by the PTPN11 gene, which is a nonreceptor (pY)-phosphatase and mutation causes JMML. The structural hierarchy of SHP2 includes protein tyrosine phosphatase domain (PTP) and Src-homology 2 domain (N-SH2 and C-SH2). Somatic mutation (E76Q) in the interface of SH2-PTP domain is the most commonly identified mutation found in up to 35% of patients with JMML. The mechanism of this mutant associated with JMML is poorly understood. Here, molecular dynamics simulation was performed on wild-type and mutant (E76Q) of SHP2 to explore the precise impact of gain-of-function on PTP's activity. Consequently, such impact rescues the SHP2 protein from autoinhibition state through losing the interface interactions of Q256/F7 and S502/Q76 or weakening interactions of Q256/R4, Q510/G60, and Q506/A72 between N-SH2 and PTP domains. The consequences of these interactions further relieve the D'E loop away from the PTP catalytic site. The following study would provide a mechanistic insight for better understanding of how individual SHP2 mutations alter the PTP's activity at the atomic level.

Magnetoelectric effects and spin switching phenomena at the interface of chiral domains in spin-triplet superconductors

Spin-triplet superconductors with time-reversal symmetry breaking can naturally lead to chiral domain walls in their interior. We study the magnetic properties emerging at the interface of chiral domains with opposite winding by focusing on the effects of a superconducting phase drop across the wall and an applied electric gating. The local inversion symmetry breaking at the domain wall drives mixed singlet-triplet pairing configurations that allow a phase- and electric-controllable magnetization with resulting parallel or antiparallel orientations on the two sides of the domain wall. The magnetic switching is also generally accompanied by both spin and charge currents flowing along the edges, whose amplitudes depend on the achieved parallel or antiparallel magnetic phase. The specific magnetoelectric properties of chiral domains with zero or nonvanishing net magnetization near the wall may have several implications. They can be employed to detect the presence of unconventional pairing as well as to design information storage units based on spin-polarized states attached to topological defects.

Kinetic Stability of Long-Lived Human Lens γ-Crystallins and Their Isolated Double Greek Key Domains

The γ-crystallins of the eye lens nucleus are among the longest-lived proteins in the human body. Synthesized in utero, they must remain folded and soluble throughout adulthood to maintain lens transparency and avoid cataracts. γD- and γS-crystallin are two major monomeric crystallins of the human lens. γD-crystallin is concentrated in the oldest lens fiber cells, the lens nucleus, whereas γS-crystallin is concentrated in the younger cells of the lens cortex. The kinetic stability parameters of these two-domain proteins and their isolated domains were determined and compared. Kinetic unfolding experiments monitored by fluorescence spectroscopy in varying concentrations of guanidinium chloride were used to extrapolate unfolding rate constants and half-lives of the crystallins in the absence of the denaturant. Consistent with their long lifespans in the lens, extrapolated half-lives for the initial unfolding step were on the timescale of years. Both proteins’ isolated N-terminal domains were less kinetically stable than their respective C-terminal domains at denaturant concentrations predicted to disrupt the domain interface, but at low denaturant concentrations, the relative kinetic stabilities were reversed. Cataract-associated aggregation has been shown to proceed from partially unfolded intermediates in these proteins; their extreme kinetic stability likely evolved to protect the lens from the initiation of aggregation reactions. Our findings indicate that the domain interface is the source of significant kinetic stability. The gene duplication and fusion event that produced the modern two-domain architecture of vertebrate lens crystallins may be the origin of their high kinetic as well as thermodynamic stability.

Conformational coupling by trans-phosphorylation in calcium calmodulin dependent kinase II.

The calcium calmodulin-dependent protein kinase II (CaMKII) is a dodecameric holoenzyme important for encoding memory. Its activation, triggered by binding of calcium-calmodulin, persists autonomously after calmodulin dissociation. One (receiver) kinase captures and subsequently phosphorylates the regulatory domain peptide of a donor kinase forming a chained dimer as the first stage of autonomous activation. Protein dynamics simulations examined the conformational changes triggered by dimer formation and phosphorylation, aimed to provide a molecular rationale for human mutations that result in learning disabilities. Ensembles generated from X-ray crystal structures were characterized by network centrality and community analysis. Mutual information related collective motions to local fragment dynamics encoded with a structural alphabet. Implicit solvent tCONCOORD conformational ensembles revealed the dynamic architecture of inactive kinase domains was co-opted in the activated dimer but the network hub shifted from the nucleotide binding cleft to the captured peptide. Explicit solvent molecular dynamics (MD) showed nucleotide and substrate binding determinants formed coupled nodes in long-range signal relays between regulatory peptides in the dimer. Strain in the extended captured peptide was balanced by reduced flexibility of the receiver kinase C-lobe core. The relays were organized around a hydrophobic patch between the captured peptide and a key binding helix. The human mutations aligned along the relays. Thus, these mutations could disrupt the allosteric network alternatively, or in addition, to altered binding affinities. Non-binding protein sectors distant from the binding sites mediated the allosteric signalling; providing possible targets for inhibitor design. Phosphorylation of the peptide modulated the dielectric of its binding pocket to strengthen the patch, non-binding sectors, domain interface and temporal correlations between parallel relays. These results provide the molecular details underlying the reported positive kinase cooperativity to enrich the discussion on how autonomous activation by phosphorylation leads to long-term behavioural effects.

Crystal Structures of the Full-Length Murine and Human Gasdermin D Reveal Mechanisms of Autoinhibition, Lipid Binding, and Oligomerization

Gasdermin D (GSDMD) is an effector molecule forpyroptosis downstream of canonical and noncanonical inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases triggers the oligomerization and lipid binding by its N-terminal domain, which assembles membrane pores, whereas its C-terminal domain binds the N-terminal domain to inhibit pyroptosis. Despite recent progress in our understanding of the structure and function of the murine gasdermin A3 (mGSDMA3), the molecular mechanisms of GSDMD activation and regulation remain poorly characterized. Here, we report the crystal structures of the full-length murine and human GSDMDs, which reveal the architecture of the GSDMD N-terminal domains and demonstrate distinct and common features of autoinhibition among gasdermin family members utilizing their β1-β2 loops. Disruption of the intramolecular domain interface enhanced pyroptosis, whereas mutations at the predicted lipid-binding or oligomerization surface reduced cytolysis. Our study provides a framework for understanding the autoinhibition, lipid binding, and oligomerization of GSDMD by using overlapping interfaces.

Spermidine Synthase (SPDS) Undergoes Concerted Structural Rearrangements Upon Ligand Binding - A Case Study of the Two SPDS Isoforms From Arabidopsis thaliana.

Spermidine synthases (SPDSs) catalyze the production of the linear triamine, spermidine, from putrescine. They utilize decarboxylated S-adenosylmethionine (dc-SAM), a universal cofactor of aminopropyltransferases, as a donor of the aminopropyl moiety. In this work, we describe crystal structures of two SPDS isoforms from Arabidopsis thaliana (AtSPDS1 and AtSPDS2). AtSPDS1 and AtSPDS2 are dimeric enzymes that share the fold of the polyamine biosynthesis proteins. Subunits of both isoforms present the characteristic two-domain structure. Smaller, N-terminal domain is built of the two β-sheets, while the C-terminal domain has a Rossmann fold-like topology. The catalytic cleft composed of two main compartments, the dc-SAM binding site and the polyamine groove, is created independently in each AtSPDS subunits at the domain interface. We also provide the structural details about the dc-SAM binding mode and the inhibition of SPDS by a potent competitive inhibitor, cyclohexylamine (CHA). CHA occupies the polyamine binding site of AtSPDS where it is bound at the bottom of the active site with the amine group placed analogously to the substrate. The crystallographic snapshots show in detail the structural rearrangements of AtSPDS1 and AtSPDS2 that are required to stabilize ligands within the active site. The concerted movements are observed in both compartments of the catalytic cleft, where three major parts significantly change their conformation. These are (i) the neighborhood of the glycine-rich region where aminopropyl moiety of dc-SAM is bound, (ii) the very flexible gate region with helix η6, which interacts with both, the adenine moiety of dc-SAM and the bound polyamine or inhibitor, and (iii) the N-terminal β-hairpin, that limits the putrescine binding grove at the bottom of the catalytic site.

(Invited) Transient Electron Spin Polarization Imaging of Photoinduced Interfacial Charge Separation Geometries in Organic Photovoltaic Cell

The organic photovoltaic (OPV) devises have recently attracted much attention as the next generation thin-film solar cells that can be low-cost, flexible and light compared to the conventional silicon solar cells. The spin coating method from mixed solutions consisting of conjugate polymers as the electron donors (D) and fullerene derivatives as the acceptors (A) are employed to produce the solid photoactive layer of the OPV cells. Recent progresses in the organic materials and in device structures have enabled the remarkably efficient OPV cells as high as 12 % in the light energy conversion efficiency, which is however still lower than that in the silicon solar cells. In the organic photoactive layers, it is well known that the molecules tend to be self-organized to form the phase segregation by the polymers and by the fullerene derivatives to generate the bulk heterojunction (BHJ) by the domain interfaces. The BHJ thin-films composed of poly(3-alkylthiophene) (P3AT) and [6,6]-C61-butyric acid methyl ester (PC60BM) have been regarded as a standard and representative material of the photoactive layer in the OPV devices. Extensive studies have been performed to investigate the physical and photochemical properties of the OPV-related materials. The ultrafast photoinduced charge-injection dynamics has been clarified by the light-excitations of poly(3-hexylthiophene-2,5-diyl) (P3HT) domains in the P3HT:PCBM blend films. Recent studies have demonstrated that the photoinduced, contact charge-transfer (CT) states are initially generated at the D/A domain-interfaces and play significant roles on the photocurrent generations in the P3AT:PCBM blends. Thus, the electrons and holes need to escape from the Coulomb attraction which energy is supposed to be several hundreds of meV in the CT state. To elucidate why and how the photo-carriers escape from the CT binding, it is important to directly observe not only the separation distances between the electron and the hole but also the relative molecular positions and orientations of the charged species just after the interfacial charge conductions. As for such separated electron-hole pairs, the electronic coupling matrix elements (V CR) are key interaction to understand how the energy-wasting charge-recombination (CR) processes are inhibited. Connection between the geometry and the V CRin the CS state will also lead to unveiling 1) the initial photoconduction pathways in the molecular orbitals and 2) the electronic conduction characteristics relating to the trap-depth at the D/A domain interface.[1] The molecular motion in the organic semiconductors is another key to the efficient dissociation. The molecular libration in the blend should enhance the entropy of the dissociated CS state as compared to that of the bound CT state. [2] The time-resolved electron paramagnetic resonance (TREPR) spectroscopy is a powerful tool to detect unpaired spins and to investigate their electronic structures, geometries and dynamics.[1,2] In our previous studies, the geometries and the V CRvalues were reported for the interfacial CS states in the BHJ blend films at cryogenic temperature.[1] These distant CS states separated by c.a. 2 nm were hypothesized to be shallow traps that are weakly bound by the electrostatic attraction between delocalized charges. However, the properties of the electronic states and of the charge motions are unknown in those separated charges. In the present study, we have developed a three-dimensional mapping tool of the “electron spin polarization imaging method” obtained by quantum mechanical analysis of the TREPR signals to clarify precise charge geometries in the distant CS states.[3] We herein show directional charge-separation dynamics and motions at the domain interfaces in an organic solar cell composed of ITO(150 nm)/PEDOT:PSS(~40 nm)/P3HT:PCBM (~160 nm)/LiF(0.6 nm)/Al(100 nm) by using the spin polarization imaging map. [1] Kobori, Y.; Miura, J. Phys. Chem. Lett. 2015, 6, 113-123. [2] Miura, T.; Tao, R.; Shibata, S.; Umeyama, T.; Tachikawa, T.; Imahori, H.; Kobori, Y. J. Am. Chem. Soc. 2016, 138, 5879-5885. [3] Hasegawa, M.; Nagashima, H.; Minobe, R.; Tachikawa, T.; Mino, H.; Kobori, Y. J. Phys. Chem. Lett. 2017, 8, 1179-1184.

A Critical Review of Lion Research in South Africa: The Impact of Researcher Perspective, Research Mode, and Power Structures on Outcome Bias and Implementation Gaps

Understanding the state of research, and its effectiveness, in a predominantly Life Sciences sphere requires an assessment of knowledge growth dynamics, and the associated scientific and bibliometric impacts. We aim to create and evaluate, in a systematic review process, a macro-structure of the science generated in lion research in South Africa (SA) from 1990 to 2018. First, we classified the evidence architecture of lion research data extracted from the Web of Science Core Collection. Then, we identified prominent features that the datasets reveal in terms of authorship and ownership, as defined by first author affiliation, geographical location. Fifteen sub-disciplines were identified to characterize the topics. From 2000 onwards, multidisciplinary and interdisciplinary contributions started to emerge, catering for research problems defined at the interface of the academic-practitioner domains. These included social and economics components, and were aligned with conservation framings that seek to evaluate conservation within market-based versus people-based approaches. Study areas were concentrated within SA (61.8%) and the remainder was either conducted in the rest of Africa (22.9%), or in various combinations of geographical focus. Author affiliation indicated that 63.1 % of first authors had a South African affiliation. The rest of Africa was poorly represented at 2.4%. The majority (57.1%) of the first authors was male, but from 2014 to 2018, female researchers outnumbered males; however, male first authors continued to be cited more frequently. Furthermore, we provide a systemic analysis of the way in which research contributes to lion conservation. Overall, three voices dominate this area. Firstly, Mode 1 research has been driving research output in a ‘vicious circle’, motivated by researchers’ quest for accumulating academic rewards. Secondly, the citation impact shows a gender disparity against the recognition of female researchers. Lastly, a power imbalance against authors from the rest of Africa became apparent, whereby their role is mainly shaped towards being team contributors. This research shows that effective conservation requires appropriate knowledge to be generated, and this to be effectively translated into practical applications, with all perspectives providing the opportunity for balanced contribution and influence. Imbalances such as revealed are here likely to prevail more broadly.

PVB/ATO Nanocomposites for Glass Coating Applications: Effects of Nanoparticles on the PVB Matrix

Films made of poly(vinyl butyral) (PVB) and antimony-doped tin oxide (ATO) nanoparticles (NPs), both uncoated and surface-modified with an alkoxysilane, were prepared by solution casting at filler volume fractions ranging from 0.08% to 4.5%. The films were characterized by standard techniques including transmission electron microscopy, thermogravimetric analysis and differential scanning calorimetry (DSC). In the polymeric matrix, the primary NPs (diameter ~10 nm) aggregate exhibiting different morphologies depending on the presence of the surface coating. Coated ATO NPs form spherical particles (with a diameter of 300–500 nm), whereas more elongated fractal structures (with a thickness of ~250 nm and length of tens of micrometers) are formed by uncoated NPs. The fraction of the polymer interacting with the NPs is always negligible. In agreement with this finding, DSC data did not reveal any rigid interface and 1H time domain nuclear magnetic resonance (NMR) and fast field-cycling NMR did not show significant differences in polymer dynamics among the different samples. The ultraviolet-visible-near infrared (UV-Vis-NIR) transmittance of the films decreased compared to pure PVB, especially in the NIR range. The solar direct transmittance and the light transmittance were extracted from the spectra according to CEN EN 410/2011 in order to test the performance of our films as plastic layers in laminated glass for glazing.

A Potential Specificity Code for B. subtilis SsbA and SsbB Quaternary Structures

Bacterial single‐stranded (ss) DNA‐binding proteins (SSBs) bind and protect ssDNA intermediates formed during genome maintenance reactions and help to recruit DNA replication and repair proteins to their sites of action through interactions mediated by their C‐terminal tails. While SSB has been studied extensively in E. coli, much less is known about the paralogous SSBs, SsbA and SsbB, that are found in many gram‐positive bacteria such as B. subtilis. Similarly to E. coli SSB, B. subtilis SsbA and SsbB both bind ssDNA through an oligosaccharide/oligonucleotide binding (OB) domain and function as homo‐tetramers. However, SsbB lacks the critical C‐terminal tail responsible for protein‐protein interactions, leading to differences in functionality. Although the OB domains of B. subtilis SsbA and SsbB are 63% identitical to one another, native gel analysis indicates that mixtures of SsbA and SsbB do not exchange monomers, but instead maintain strictly homo‐tetrameric quaternary structures. A recent crystal structure of B. subtilis SsbA revealed a unique hydrogen‐bonding interface between SsbA monomers that was not observed in the SsbB structure. We hypothesize that these interface differences could prevent SsbA/SsbB hetero‐tetramer formation. To test this hypothesis, we engineered a B. subtilis SsbA interface variant that change a key OB domain interface residue to the corresponding residue in SsbB (Asp74Ser). This variant failed to allow hetero‐tetramer formation. However, since Asp74 is only one residue in a more complex interaction network, we are now testing our hypothesis using a double variant of SsbA (Asp74Ser, Arg4Gln) that better mimics the SsbB interface network for mixed SsbA/SsbB tetramer formation. If a variant that is capable of forming mixed SsbA/SsbB tetramers is discovered, we plan to use it to test the effects of mixed‐tetramer formation in vivo and to further probe the functions of SSBs in gram‐positive bacteria.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Spatial Organization of a cAMP/Ca2+‐Regulated Signaling Complex: A Structural Study of an AKAP79‐Scaffolded Complex Containing Type IIβ PKA and Calcineurin

cAMP‐dependent protein kinase (PKA) regulates many cell processes including development, differentiation, and memory. In its resting state, PKA is a tetrameric holoenzyme consisting of a regulatory (R) subunit dimer and two catalytic (C) subunits. Four isoforms of the R‐subunit exist (RIα, RIβ, RIIα, and RIIβ). Although each isoform varies in sequence and quaternary structure, all share a common domain organization. The strength, duration, and specificity of PKA activity within the cell are mostly dependent upon isoform‐specific subcellular localization. The D/D domain interface of the R‐ subunits serves as a docking site for A Kinase Anchoring Proteins (AKAPs) that target PKA to specific cellular locations. In cardiac myocytes and neurons, PKA is localized to Cav1.2 L‐type calcium channels by AKAP79 and up‐regulates calcium influx by phosphorylating the channel's cytosolic C‐terminal tail. AKAP79 also binds the Ca2+/calmodulin‐dependent protein phosphatase type 2B (PP2B, or calcineurin) at a site very close to that of PKA binding. Given the close proximity of the calcineurin and PKA binding sites on AKAP79, calcineurin has been suggested to play roles in the regulation of calcium channels, along with regulation of PKA activity itself. Using a variety of biochemical and biophysical techniques, we have isolated and characterized a stable complex of the C‐terminal tail of AKAP79, RIIβ holoenzyme, and calciuneurin (“ARC” for short). We show a stoichiometry of 1:1:1 (AKAP79ct: RIIβ2‐C2 :CNt) using native mass spectroscopy, and SAXS/SANS techniques confirm that this is a compact complex, suggesting a close interaction between PKA and calcineurin when bound to AKAP79. Altogether, we suggest beyond its function of localizing PKA and CN in close spatial proximity to the Cav1.2 L‐type calcium channel, the ARC signalosome fosters extended protein‐protein interactions and regulations that suggest tight spatiotemporal control. Support for this work was provided through NIH GM034921.Support or Funding InformationJames Hall is an IRACDA fellow and was supported by NIGMS/NIH award K12GM068524. This work was also supported by a grant from the National Institutes of Health GM034921 to S.S. Taylor. This Research used resources at the High Flux Isotope Reactor and Spallation Neutron Source, which are DOE Office of Science User Facilities operated by Oak Ridge National Laboratory. This work utilized SasView software, originally developed by the DANSE project under NSF award DMR‐0520547.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.