Toward α-1,3/4 fucosyltransferases targeted drug discovery: In silico uncovering of promising natural inhibitors of fucosyltransferase 6.

Sialyl Lewis X (sLex ) antigen is a fucosylated cell-surface glycan that is normally involved in cell-cell interactions. The enhanced expression of sLex on cell surface glycans, which is attributed to the upregulation of fucosyltransferase 6 (FUT6), has been implicated in facilitating metastasis in human colorectal, lung, prostate, and oral cancers. The role that the upregulated FUT6 plays in the progression of tumor to malignancy, with reduced survival rates, makes it a potential target for anticancer drugs. Unfortunately, the lack of experimental structures for FUT6 has hampered the design and development of its inhibitors. In this study, we used in silico techniques to identify potential FUT6 inhibitors. We first modeled the three-dimensional structure of human FUT6 using AlphaFold. Then, we screened the natural compound libraries from the COCONUT database to sort out potential natural products (NPs) with best affinity toward the FUT6 model. As a result of these simulations, we identified three NPs for which we predicted binding affinities and interaction patterns quite similar to those we calculated for two experimentally tested FUT6 inhibitors, that is, fucose mimetic-1 and a GDP-triazole derived compound. We also performed molecular dynamics (MD) simulations for the FUT6 complexes with identified NPs, to investigate their stability. Analysis of the MD simulations showed that the identified NPs establish stable contacts with FUT6 under dynamics conditions. On these grounds, the three screened compounds appear as promising natural alternatives to experimentally tested FUT6 synthetic inhibitors, with expected comparable binding affinity. This envisages good prospects for future experimental validation toward FUT6 inhibition.

D936Y and Other Mutations in the Fusion Core of the SARS-CoV-2 Spike Protein Heptad Repeat 1: Frequency, Geographical Distribution, and Structural Effect.

The crown of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is constituted by its spike (S) glycoprotein. S protein mediates the SARS-CoV-2 entry into the host cells. The "fusion core" of the heptad repeat 1 (HR1) on S plays a crucial role in the virus infectivity, as it is part of a key membrane fusion architecture. While SARS-CoV-2 was becoming a global threat, scientists have been accumulating data on the virus at an impressive pace, both in terms of genomic sequences and of three-dimensional structures. On 15 February 2021, from the SARS-CoV-2 genomic sequences in the GISAID resource, we collected 415,673 complete S protein sequences and identified all the mutations occurring in the HR1 fusion core. This is a 21-residue segment, which, in the post-fusion conformation of the protein, gives many strong interactions with the heptad repeat 2, bringing viral and cellular membranes in proximity for fusion. We investigated the frequency and structural effect of novel mutations accumulated over time in such a crucial region for the virus infectivity. Three mutations were quite frequent, occurring in over 0.1% of the total sequences. These were S929T, D936Y, and S949F, all in the N-terminal half of the HR1 fusion core segment and particularly spread in Europe and USA. The most frequent of them, D936Y, was present in 17% of sequences from Finland and 12% of sequences from Sweden. In the post-fusion conformation of the unmutated S protein, D936 is involved in an inter-monomer salt bridge with R1185. We investigated the effect of the D936Y mutation on the pre-fusion and post-fusion state of the protein by using molecular dynamics, showing how it especially affects the latter one.

Structural Insights in Mammalian Sialyltransferases and Fucosyltransferases: We Have Come a Long Way, but It Is Still a Long Way Down.

Mammalian cell surfaces are modified with complex arrays of glycans that play major roles in health and disease. Abnormal glycosylation is a hallmark of cancer; terminal sialic acid and fucose in particular have high levels in tumor cells, with positive implications for malignancy. Increased sialylation and fucosylation are due to the upregulation of a set of sialyltransferases (STs) and fucosyltransferases (FUTs), which are potential drug targets in cancer. In the past, several advances in glycostructural biology have been made with the determination of crystal structures of several important STs and FUTs in mammals. Additionally, how the independent evolution of STs and FUTs occurred with a limited set of global folds and the diverse modular ability of catalytic domains toward substrates has been elucidated. This review highlights advances in the understanding of the structural architecture, substrate binding interactions, and catalysis of STs and FUTs in mammals. While this general understanding is emerging, use of this information to design inhibitors of STs and FUTs will be helpful in providing further insights into their role in the manifestation of cancer and developing targeted therapeutics in cancer.

Near-White Light Emission from Lead(II) Metal-Organic Frameworks.

Reaction of bpy (bpy = 4,4'-bipyridine) with Pb(OAc)2·3H2O in DMF (DMF = dimethylformamide) afforded a metal-organic framework (MOF), [Pb2(µ-bpy)(µ-O2CCH3)2(µ-O2CCH3)2]·H2O (1). Reaction of bpy with Pb(O2CCF3)2 in a methanol and chloroform mixture furnished another MOF, [Pb(µ-bpy)(µ-O2CCF3)2]·1/2CHCl3 (2). However, the reaction of bpy with Pb(OAc)2·3H2O in the presence of trifluoroacetic acid in a similar reaction condition yielded a hydrogen-bonded zwitter-ionic complex of Pb(II), [Pb(bpy-H)2(O2CCF3)4] (3). All compounds have been characterized by single crystal X-ray crystallography, FT-IR, and 1H NMR spectroscopies. Compound 1 forms four heptacoordinated Pb(II) joined by (OCCH3)-O- linkages, resulting in a 3D noninterpenetrated MOF net with a four-connected uninodal sra (SrAl2) topology. However, in 2, tetra-connected Pb4(O2CCF3)8 cluster units are linked further through eight bpy ligands to furnish a doubly interpenetrated MOF with a new topology but having the very similar connectivity of 1, whereas 3 forms a zigzag hydrogen-bonded chain structure. The variation of carboxylate anions, pH of the reaction medium, and the ratio of the reactants profoundly affected the final topological structure of the compounds synthesized. The solid-state photoluminescence of 1-3 was investigated at room temperature. Interestingly 1, 2, and 3 achieved close to white light emission when excited at 329, 376, and 330 nm, respectively. The systematic understanding of the photophysical properties of analogous Pb-based compounds may open new perspectives for developing single-phase white-light-emitting materials using Pb(II) based MOFs.

Detecting Hachimoji DNA: An Eight-Building-Block Genetic System with MoS2 and Janus MoSSe Monolayers.

In the pursuit of personalized medicine, the development of efficient, cost-effective, and reliable DNA sequencing technology is crucial. Nanotechnology, particularly the exploration of two-dimensional materials, has opened different avenues for DNA nucleobase detection, owing to their impressive surface-to-volume ratio. This study employs density functional theory with van der Waals corrections to methodically scrutinize the adsorption behavior and electronic band structure properties of a DNA system composed of eight hachimoji nucleotide letters adsorbed on both MoS2 and MoSSe monolayers. Through a comprehensive conformational search, we pinpoint the most favorable adsorption sites, quantifying their adsorption energies and charge transfer properties. The analysis of electronic band structure unveils the emergence of flat bands in close proximity to the Fermi level post-adsorption, a departure from the pristine MoS2 and MoSSe monolayers. Furthermore, leveraging the nonequilibrium Green's function approach, we compute the current-voltage characteristics, providing valuable insights into the electronic transport properties of the system. All hachimoji bases exhibit physisorption with a horizontal orientation on both monolayers. Notably, base G demonstrates high sensitivity on both substrates. The obtained current-voltage (I-V) characteristics, both without and with base adsorption on MoS2 and the Se side of MoSSe, affirm excellent sensing performance. This research significantly advances our understanding of potential DNA sensing platforms and their electronic characteristics, thereby propelling the endeavor for personalized medicine through enhanced DNA sequencing technologies.

Secreted Aspartyl Proteinases Targeted Multi-Epitope Vaccine Design for Candida dubliniensis Using Immunoinformatics.

Candida dubliniensis is an opportunistic pathogen associated with oral and invasive fungal infections in immune-compromised individuals. Furthermore, the emergence of C. dubliniensis antifungal drug resistance could exacerbate its treatment. Hence, in this study a multi-epitope vaccine candidate has been designed using an immunoinformatics approach by targeting C. dubliniensis secreted aspartyl proteinases (SAP) proteins. In silico tools have been utilized to predict epitopes and determine their allergic potential, antigenic potential, toxicity, and potential to elicit interleukin-2 (IL2), interleukin-4 (IL4), and IFN-γ. Using the computational tools, eight epitopes have been predicted that were then linked with adjuvants for final vaccine candidate development. Computational immune simulation has depicted that the immunogen designed emerges as a strong immunogenic candidate for a vaccine. Further, molecular docking and molecular dynamics simulation analyses revealed stable interactions between the vaccine candidate and the human toll-like receptor 5 (TLR5). Finally, immune simulations corroborated the promising candidature of the designed vaccine, thus calling for further in vivo investigation.

Biochemical and structural comparisons of non-nucleoside reverse transcriptase inhibitors against feline and human immunodeficiency viruses.

BACKGROUND: Feline immunodeficiency virus (FIV) causes an acquired immunodeficiency-like syndrome in cats. FIV is latent. No effective treatment has been developed for treatment the infected cats. The first and second generations non-nucleoside reverse transcriptase inhibitors (NNRTIs) for HIV treatment, nevirapine (NVP) and efavirenz (EFV), and rilpivirine (RPV), were used to investigate the potential of NNRTIs for treatment of FIV infection. OBJECTIVE: This study aims to use experimental and in silico approaches to investigate the potential of NNRTIs, NVP, EFV, and RPV, for inhibition of FIV reverse transcriptase (FIV-RT). METHODS: The FIV-RT and human immunodeficiency virus reverse transcriptase (HIV-RT) were expressed and purified using chromatography approaches. The purified proteins were used to determine the IC50 values with NVP, EFV, and RPV. Surface plasmon resonance (SPR) analysis was used to calculate the binding affinities of NNRTIs to HIV-RT and FIV-RT. The molecular docking and molecular dynamic simulations were used to demonstrate the mechanism of FIV-RT and HIV-RT with first and second generation NNRTI complexes. RESULTS: The IC50 values of NNRTIs NVP, EFV, and RPV against FIV-RT were in comparable ranges to HIV-RT. The SPR analysis showed that NVP, EFV, and RPV could bind to both enzymes. Computational calculation also supports that these NNRTIs can bind with both FIV-RT and HIV-RT. CONCLUSIONS: Our results suggest the first and second generation NNRTIs (NVP, EFV, and RPV) could inhibit both FIV-RT and HIV-RT.

Immunoinformatics-aided rational design of a multi-epitope vaccine targeting feline infectious peritonitis virus.

Feline infectious peritonitis (FIP) is a grave and frequently lethal ailment instigated by feline coronavirus (FCoV) in wild and domestic feline species. The spike (S) protein of FCoV assumes a critical function in viral ingress and infection, thereby presenting a promising avenue for the development of a vaccine. In this investigation, an immunoinformatics approach was employed to ascertain immunogenic epitopes within the S-protein of FIP and formulate an innovative vaccine candidate. By subjecting the amino acid sequence of the FIP S-protein to computational scrutiny, MHC-I binding T-cell epitopes were predicted, which were subsequently evaluated for their antigenicity, toxicity, and allergenicity through in silico tools. Our analyses yielded the identification of 11 potential epitopes capable of provoking a robust immune response against FIPV. Additionally, molecular docking analysis demonstrated the ability of these epitopes to bind with feline MHC class I molecules. Through the utilization of suitable linkers, these epitopes, along with adjuvants, were integrated to design a multi-epitope vaccine candidate. Furthermore, the stability of the interaction between the vaccine candidate and feline Toll-like receptor 4 (TLR4) was established via molecular docking and molecular dynamics simulation analyses. This suggests good prospects for future experimental validation to ascertain the efficacy of our vaccine candidate in inducing a protective immune response against FIP.

Molecular mechanism of ATP hydrolysis in F1-ATPase revealed by molecular simulations and single-molecule observations.

Enzymatic hydrolysis of nucleotide triphosphate (NTP) plays a pivotal role in protein functions. In spite of its biological significance, however, the chemistry of the hydrolysis catalysis remains obscure because of the complex nature of the reaction. Here we report a study of the molecular mechanism of hydrolysis of adenosine triphosphate (ATP) in F(1)-ATPase, an ATP-driven rotary motor protein. Molecular simulations predicted and single-molecule observation experiments verified that the rate-determining step (RDS) is proton transfer (PT) from the lytic water molecule, which is strongly activated by a metaphosphate generated by a preceding P(γ)-O(ß) bond dissociation (POD). Catalysis of the POD that triggers the chain activation of the PT is fulfilled by hydrogen bonds between Walker motif A and an arginine finger, which commonly exist in many NTPases. The reaction mechanism unveiled here indicates that the protein can regulate the enzymatic activity for the function in both the POD and PT steps despite the fact that the RDS is the PT step.

Immunoinformatics Aided Design and In-Vivo Validation of a Cross-Reactive Peptide Based Multi-Epitope Vaccine Targeting Multiple Serotypes of Dengue Virus.

Dengue virus (DENV) is an arboviral disease affecting more than 400 million people annually. Only a single vaccine formulation is available commercially and many others are still under clinical trials. Despite all the efforts in vaccine designing, the improvement in vaccine formulation against DENV is very much needed. In this study, we used a roboust immunoinformatics approach, targeting all the four serotypes of DENV to design a multi-epitope vaccine. A total of 13501 MHC II binding CD4+ epitope peptides were predicted from polyprotein sequences of four dengue virus serotypes. Among them, ten conserved epitope peptides that were interferon-inducing were selected and found to be conserved among all the four dengue serotypes. The vaccine was formulated using antigenic, non-toxic and conserved multi epitopes discovered in the in-silico study. Further, the molecular docking and molecular dynamics predicted stable interactions between predicted vaccine and immune receptor, TLR-5. Finally, one of the mapped epitope peptides was synthesized for the validation of antigenicity and antibody production ability where the in-vivo tests on rabbit model was conducted. Our in-vivo analysis clearly indicate that the imunogen designed in this study could stimulate the production of antibodies which further suggest that the vaccine designed possesses good immunogenicity.

Immunoinformatics-Aided Design of a Peptide Based Multiepitope Vaccine Targeting Glycoproteins and Membrane Proteins against Monkeypox Virus.

Monkeypox is a self-limiting zoonotic viral disease and causes smallpox-like symptoms. The disease has a case fatality ratio of 3-6% and, recently, a multi-country outbreak of the disease has occurred. The currently available vaccines that have provided immunization against monkeypox are classified as live attenuated vaccinia virus-based vaccines, which pose challenges of safety and efficacy in chronic infections. In this study, we have used an immunoinformatics-aided design of a multi-epitope vaccine (MEV) candidate by targeting monkeypox virus (MPXV) glycoproteins and membrane proteins. From these proteins, seven epitopes (two T-helper cell epitopes, four T-cytotoxic cell epitopes and one linear B cell epitopes) were finally selected and predicted as antigenic, non-allergic, interferon-γ activating and non-toxic. These epitopes were linked to adjuvants to design a non-allergic and antigenic candidate MPXV-MEV. Further, molecular docking and molecular dynamics simulations predicted stable interactions between predicted MEV and human receptor TLR5. Finally, the immune-simulation analysis showed that the candidate MPXV-MEV could elicit a human immune response. The results obtained from these in silico experiments are promising but require further validation through additional in vivo experiments.

Replacing thymine with a strongly pairing fifth Base: A combined quantum mechanics and molecular dynamics study.

The non-natural ethynylmethylpyridone C-nucleoside (W), a thymidine (T) analogue that can be incorporated in oligonucleotides by automated synthesis, has recently been reported to form a high fidelity base pair with adenosine (A) and to be well accommodated in B-DNA duplexes. The enhanced binding affinity for A of W, as compared to T, makes it an ideal modification for biotechnological applications, such as efficient probe hybridization for the parallel detection of multiple DNA strands. In order to complement the experimental study and rationalize the impact of the non-natural W nucleoside on the structure, stability and dynamics of DNA structures, we performed quantum mechanics (QM) calculations along with molecular dynamics (MD) simulations. Consistently with the experimental study, our QM calculations show that the A:W base pair has an increased stability as compared to the natural A:T pair, due to an additional CH-π interaction. Furthermore, we show that mispairing between W and guanine (G) causes a distortion in the planarity of the base pair, thus explaining the destabilization of DNA duplexes featuring a G:W pair. MD simulations show that incorporation of single or multiple consecutive A:W pairs in DNA duplexes causes minor changes to the intra- and inter-base geometrical parameters, while a moderate widening/shrinking of the major/minor groove of the duplexes is observed. QM calculations applied to selected stacks from the MD simulations also show an increased stacking energy for W, over T, with the neighboring bases.

Adsorption of Bovine Serum Albumin on Poly(vinylidene fluoride) Surfaces in the Presence of Ions: A Molecular Dynamics Simulation.

Adsorption of bovine serum albumin (BSA) on poly(vinylidene fluoride) (PVDF) surfaces in an aqueous environment was investigated in the presence and absence of excess ions using molecular dynamics simulations. The adsorption process involved diffusion of protein to the surface and dehydration of surface-protein interactions, followed by adsorption and denaturation. Although adsorption of BSA on PVDF surface was observed in the absence of excess ions, denaturation of BSA was not observed during the simulation (1 µs). Basic and acidic amino acids of BSA were found to be directly interacting with PVDF surface. Simulation in a 0.1 M NaCl solution showed delayed adsorption of BSA on PVDF surfaces in the presence of excess ions, with BSA not observed in close proximity to PVDF surface within 700 ns. Adsorption of Cl- on PVDF surface increased its negative charge, which repelled negatively charged BSA, thereby delaying the adsorption process. These results will be helpful for understanding membrane fouling phenomena in polymeric membranes, and fundamental advancements in these areas will lead to a new generation of membrane materials with improved antifouling properties and reduced energy demands.

Fouling-Resistant and Self-Cleaning Aliphatic Polyketone Membrane for Sustainable Oil-Water Emulsion Separation.

The cost-effective treatment of emulsified oily wastewater discharged by many industries and human societies is a great challenge. Herein, based on an aliphatic polyketone (PK) polymer with a good membrane formation ability and an intrinsic intermediate hydrophilicity, a new class of reduced PK (rPK) membranes combining an all hydrophilic and electrically neutral surface chemistry comprising ketone and hydroxyl groups, and a fibril-like morphology featuring re-entrant structure, was facilely prepared by phase separation and following fast surface reduction. The synergetic cooperation of surface chemistry and surface geometry endowed the prepared membranes with excellent superhydrophilicity, underwater superoleophobicity, and underoil superhydrophilicity, in addition to antiprotein-adhesion property. Thus, fouling-resistant and self-cleaning filtrations of challenging oil-in-water emulsions containing adhesive oil, surfactant, high salinity, and proteins were effortlessly realized with high flux (up to ∼50 000 L m-2 h-1 bar-1), slow and reversible flux decline, and low oil permeate (<20 ppm). In contrast, a commercial superhydrophilic microporous membrane made of mixed cellulose ester suffered severe fouling gradually or immediately when carrying out the emulsion filtrations due to its less than ideal surface properties. It is believed that this class of membranes with desirable superwettability, high flux, and preparation simplicity can be a potential new benchmark for high performance and large-scale oil-water separation in complex environments.

Dual Superlyophobic Aliphatic Polyketone Membranes for Highly Efficient Emulsified Oil-Water Separation: Performance and Mechanism.

Efficient treatment of difficult emulsified oil-water wastes is a global challenge. Membranes exhibiting unusual dual superlyophobicity (combined underwater superoleophobicity and underoil superhydrophobicity) are intriguing to realize high-efficiency separation of both oil-in-water and water-in-oil emulsions. For the first time, a robust polymeric membrane demonstrating dual superlyophobicity to common apolar oils was facilely fabricated via a simple one-step phase separation process using an aliphatic polyketone (PK) polymer, thanks to a conjunction of intermediate hydrophilicity and re-entrant fibril-like texture upon the prepared PK membrane. Further chemical modification to improve surface hydrophilicity slightly can enable dual superlyophobicity to both apolar and polar oils. It is found that a nonwetting composite state of oil against water or water against oil was obtainable on the membrane surfaces only when the probe liquids possess an equilibrium contact angle (θow or θwo) larger than the critical re-entrant angle of the textured surfaces (73°), which can explain the existences of dual superlyophobicity and also the nonwetting to fully wetting transitions. A simple design chart was developed to map out the operational windows of material hydrophilicity and re-entrant geometry, that is, a possible zone, to help in the rational design of similar interfacial systems from various materials. Switchable filtrations of oil-in-water and water-in-oil nanoemulsions were achieved readily with both high flux and high rejection. The simplicity and scalability of the membrane preparation process and the well-elucidated underlying mechanisms illuminate the great application potential of the PK-based superwetting membranes.

Interaction of arginine, lysine, and guanidine with surface residues of lysozyme: implication to protein stability.

Additives are widely used to suppress aggregation of therapeutic proteins. However, the molecular mechanisms of effect of additives to stabilize proteins are still unclear. To understand this, we herein perform molecular dynamics simulations of lysozyme in the presence of three commonly used additives: arginine, lysine, and guanidine. These additives have different effects on stability of proteins and have different structures with some similarities; arginine and lysine have aliphatic side chain, while arginine has a guanidinium group. We analyze atomic contact frequencies to study the interactions of the additives with individual residues of lysozyme. Contact coefficient, quantified from contact frequencies, is helpful in analyzing the interactions with the guanidine groups as well as aliphatic side chains of arginine and lysine. Strong preference for contacts to the additives (over water) is seen for the acidic followed by polar and the aromatic residues. Further analysis suggests that the hydration layer around the protein surface is depleted more in the presence of arginine, followed by lysine and guanidine. Molecular dynamics simulations also reveal that the internal dynamics of protein, as indicated by the lifetimes of the hydrogen bonds within the protein, changes depending on the additives. Particularly, we note that the side-chain hydrogen-bonding patterns within the protein differ with the additives, with several side-chain hydrogen bonds missing in the presence of guanidine. These results collectively indicate that the aliphatic chain of arginine and lysine plays a critical role in the stabilization of the protein.

A graph theoretical approach for analysis of protein flexibility change at protein complex formation.

Hitherto analyses of protein complexes are frequently confined to the changes in the interface of the protein subunits undergoing interaction, while the holistic picture of the protein monomers' structure transformation, or the pervasive rigidity adopted by the newly formed complex are most often than not improperly evaluated in spite of the multiple and deep insights that they can yield about the interaction process itself at the molecular level, or at the higher level of genomic functional analyses for which relevant systems biological information can be obtained. To address this aspect of protein-protein interaction we propose in this work a newly developed algorithm that is based on graph theoretical instances and makes possible the evaluation of the changes in the flexibility of the interacting molecules and the rigidity adopted at complex formation. Since one can also figure out the opposite process, i.e. that in which the complex decomposes into its constituent subunits, each of which may accomplish another vital role in the organism, the methodology proposed here is also able to address such problem. The algorithm we propose performs a rigidity and/or flexibility evaluation of every node (atom) on the network constituted by the entire set of intra and inter-molecular inter-atomic interactions. Comparison of flexible or rigid molecular regions or domains within the complex with those in the respective isolated monomers leads to quantification of the loss (or gain) in the number of degrees of freedom at complex formation and their effects on protein complex formation mechanisms. This index is also valuable in the identification of collective motions within the protein that may play a critical role in the process of complex formation, and the influences they may have in the behavior and function of the complex (as well as the subunits constituting it) within the organism. Furthermore, the methodology, embedded in protein docking algorithms allows the development of a framework for categorizing and ranking decoys output by broadly used grid scoring type algorithms, one of which is the system for protein-protein interaction system MIAX that has been under continuous development in recent years.

Effects of water concentration on the free volume of amino acid ionic liquids investigated by molecular dynamics simulations.

Amino acid ionic liquids (AAILs) are gaining attention because of their potential in CO2 capture technology. Molecular dynamics simulations of AAILs tetramethylammonium glycinate ([N1111][Gly]), tetrabutylammonium glycinate ([N4444][Gly]), and 1,1,1-trimethylhydrazinium glycinate ([aN111][Gly]) and their corresponding mixtures with water were performed to investigate the effect of water concentration on the cation-anion interactions. The water content significantly influenced the free volume (FV) and fractional free volume (FFV) of the AAILs that varied with the hydrophobic and hydrophilic nature of the ion pairs. Under dry conditions, the FFV increased with increasing cation molecular sizes, indicative of proportional adsorption of any inert gases, such as N2, as consistent with experimental observations. Furthermore, the polarity of the cation played an important role in FFV and hence the diffusion of the AAILs. Density functional theory calculations suggested that hydrophilic [aN111][Gly] featured stronger interactions in the presence of water, whereas the hydrophobic IL showed weaker interactions. The carboxylate group of glycinate displayed stronger interactions with water than the cation. The computational study provided qualitative insight into the role of FV of the AAILs on CO2 and N2 absorption and suggests that [aN111][Gly] has CO2 adsorption capacity in the presence of water superior to that of other studied AAILs.

A Saccharomyces cerevisiae assay system to investigate ligand/AdipoR1 interactions that lead to cellular signaling.

Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides' ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to important mammalian adiponectin-AdipoR1 signaling pathways. This system should facilitate the development of therapeutic inventions targeting adiponectin and/or AdipoR physiology.

Arginine-aromatic interactions and their effects on arginine-induced solubilization of aromatic solutes and suppression of protein aggregation.

We examine the interaction of aromatic residues of proteins with arginine, an additive commonly used to suppress protein aggregation, using experiments and molecular dynamics simulations. An aromatic-rich peptide, FFYTP (a segment of insulin), and lysozyme and insulin are used as model systems. Mass spectrometry shows that arginine increases the solubility of FFYTP by binding to the peptide, with the simulations revealing the predominant association of arginine to be with the aromatic residues. The calculations further show a positive preferential interaction coefficient, Γ(XP), contrary to conventional thinking that positive Γ(XP)'s indicate aggregation rather than suppression of aggregation. Simulations with lysozyme and insulin also show arginine's preference for aromatic residues, in addition to acidic residues. We use these observations and earlier results reported by us and others to discuss the possible implications of arginine's interactions with aromatic residues on the solubilization of aromatic moieties and proteins. Our results also highlight the fact that explanations based purely on Γ(XP), which measures average affinity of an additive to a protein, could obscure or misinterpret the underlying molecular mechanisms behind additive-induced suppression of protein aggregation.