Amino Acid Residues In Positions Research Articles

Chemiluminescence from recombinant spider silk under thermo-oxidative degradation

In this work, the oxidative degradation of recombinant spider silk in a solid state was studied based on chemiluminescence analysis. It was found that the chemiluminescence behavior of the recombinant spider silk closely resembled that of aliphatic polyamide: The emission developed without any induction time, and the maximum intensity was observed in the range of 480–520 nm. In combination with infrared spectroscopy, it was concluded that the oxidative degradation of the recombinant spider silk initiates mainly through the hydroperoxide formation at the Cα position of amino acid residues, and its unimolecular decomposition produces imide or α-keto amide groups together with the CL emission.

Structure-activity relationship study of tetrapeptide inhibitors of the Vascular Endothelial Growth Factor A binding to Neuropilin-1

Neuropilin-1 is considered as one of the key receptors responsible for signaling pathways involved in pathological angiogenesis necessary for tumor progression, therefore targeting of VEGF165 binding to NRP-1 could be a relevant strategy for antiangiogenic treatment. It was shown before that the VEGF165/NRP-1 interaction can be inhibited by short tetrapeptides with K/RXXR sequence.Here, we present a structure–activity relationship study of the systematic optimization of amino acid residues in positions 1–3 in the above tetrapeptides. All the 13 synthesized analogs possessed C-terminal arginine that is a necessary element for interaction with NRP-1. The obtained results of the inhibitory activity and modeling by molecular dynamics indicate that simultaneous interactions of the basic amino acid residues in position 1 and 4 (Arg) with Neuropilin-1 are crucial and their cooperation strongly affects the inhibitory activity. In addition, the binding strength is modulated by the flexibility of the peptide backbone (in the central part of the peptide), and the nature of the side chain of the amino acids at the second or third position. A dramatic decrease in the activity to the receptor is observed in flexible derivatives that are missing proline residues. The results described in this paper should prove useful for future studies aimed at establishing the best pharmacophore for inhibitors of VEGF165 binding to NRP-1.

A Suicide Mutation Affecting Proton Transfers to High-Valent Hemes Causes Inactivation of MauG during Catalysis.

In the absence of its substrate, the autoreduction of the high-valent bis-FeIV state of the hemes of MauG to the diferric state proceeds via a Compound I-like and then a Compound II-like intermediate. This process is coupled to oxidative damage to specific methionine residues and inactivation of MauG. The autoreduction of a P107V MauG variant, which is more prone to oxidative damage, proceeds directly from the bis-FeIV to the Compound II-like state with no detectable Compound I intermediate. Comparison of the crystal structures of native and P107V MauG reveals that this mutation alters the positions of amino acid residues in the heme site as well as the water network that delivers protons from the solvent to the hemes during their reduction. Kinetic, spectroscopic, and solvent kinetic isotope effect studies demonstrate that these changes in the heme site affect the protonation state of the ferryl heme and the relative efficiencies of two alternative pathways for the transfer of protons from solvent to the hemes. These changes enhance the rate of autoreduction of P107V MauG such that it competes with the catalytic reaction with substrate and causes the enzyme to inactivate itself during the steady-state reaction with H2O2 and its substrate. Thus, while this mutation has negligible effects on the initial steady-state kinetic parameters of MauG, it is a fatal mutation as it causes inactivation during catalysis.

Comparative Analysis of Immune Repertoires between Bactrian Camel's Conventional and Heavy-Chain Antibodies.

Compared to classical antibodies, camel heavy chain antibodies (HCAbs) are smaller in size due to lack of the light chain and the first constant domain of the heavy chain (CH1 region). The variable regions of HCAbs (VHHs) are more soluble and stable than that of conventional antibodies (VHs). Even with such simple structure, they are still functional in antigen binding. Although HCAbs have been extensively investigated over the past two decades, most efforts have been based upon low throughput sequence analysis, and there are only limited reports trying to analyze and describe the complete immune repertoire (IR) of camel HCAbs. Here we leveraged the high-throughput data generated by Next Generation Sequencing (NGS) of the variable domains of the antibody heavy chains from three Bactrian camels to conduct in-depth comparative analyses of the immunoglobulin repertoire. These include analyses of the complementary determining region 3 (CDR3) length and distribution, mutation rate, antibody characteristic amino acids, the distribution of the cysteine (Cys) codons, and the non-classical VHHs. We found that there is higher diversity in the CDR2 than in the other sub-regions, and there is a higher mutation rate in the VHHs than in the VHs (P < 0.05). In addition to substitutions at amino acid (AA) residue positions NO.49/50/52 between VH and VHH clones, we also observed other substitutions at the positions NO.40/54/57/96/101 that could lead to additional structural alterations. We also found that VH-derived VHH clones, referred to as non-classical VHH clones in this study, accounted for about 8% of all clones. Further, only 5%-10% clones had the Trp > Arg AA substitution at the first position of framework 4 for all types of clones. We present, for the first time, a relatively complete picture of the Bactrian camel antibody immune repertoire, including conventional antibody (Ab) and HCAbs, using PCR and in silico analysis based on high-throughput NGS data.

Alzheimer Disease Amyloid Beta Peptides: AFM and Voltammetric Characterization

Alzheimer’s disease (AD) is the most widespread form of dementia and was recently estimated to affect 44.4 million people worldwide. The main clinical manifestations of AD are loss of memory, cognitive skills, speech, and normative behaviour; however, accumulation of fibrillary amyloid beta (Aβ) peptides in plaques can occur and be observed decades before the AD symptoms. Native human amyloid beta (Aβ) peptides exist as short isoforms, soluble monomers, varying from 39 to 43 amino acids. In AD, dysregulation and overproduction of Aβ peptides occurs, leading to a cascade mechanism in which the peptides undergo morphological changes, from soluble, monomeric random coil or α-helix conformations, into aggregated β-sheet structures, a fibrillization process that involves numerous intermediate structures. While at the beginning of ’90 it was considered that the neurodegeneration in AD is caused by the deposition of Aβ peptide plaques (large Aβ aggregates) in the brain tissue, nowadays, what appeared to be primarily responsible for neurodegeneration are these transient heterogeneous small soluble oligomers and protofibrils, which are difficult to be investigated due to the fast aggregation rate of Aβ peptide. The time dependent structural modifications undergone by the synthetic human Aβ isoform peptides containing 40 and 42 amino acids, Aβ1-40 and Aβ1-42 peptides, i.e.the intermediary structures which appear during fibrilization, were studied by atomic force microscopy (AFM) at highly oriented pyrolytic graphite and differential pulse (DP) voltammetry at a glassy carbon electrode. The Aβ peptide solutions were incubated at room temperature, and in free chloride media, to limit the effect of temperature and salt concentration on the aggregation rate,. In order to fully understand how the electron transfer reaction was influenced by both the specific amino acid side-chain position and the fibrilization process, the Aβ1-40 and Aβ1-42 peptides aggregation was compared with the aggregation of four Aβ peptides control sequences that: (i) do not aggregate (inverse Aβ40-1 and Aβ42-1 and rat Aβ1-40Rat peptides), and (ii) lack specific electroactive amino acids (mutants Aβ1-40Phe10 and Aβ1-40Nle35peptides). The fibrilization process was investigated by AFM, via changes in the adsorption morphology, from initially random coiled structures, corresponding to the Aβ peptide monomers in random coil or in α-helix conformations, to aggregates, protofibrils and two types of fibrils, corresponding to the Aβ peptide in a β-sheet configuration. The hydrophobic carbon surface induced rapid changes in the Aβ peptides conformation, and differences between the adsorbed fibrils, formed at the carbon surface or in solution, were detected. The Aβ peptides fibrilization was electrochemically detected via the decrease of the Aβ peptide electroactive amino acids oxidation peak currents, in the DP voltammograms, that occurred in a time dependent manner. Structurally, the Aβ peptide sequences contained five electroactive amino acid residues, one tyrosine (Tyr10), three histidines (His6, His13 and His14) and one methionine (Met35). DP voltammograms showed that, according to their primary structure, the Aβ peptides undergo oxidation in one or two steps, the first electron transfer reaction corresponding to the Tyr10 residue oxidation and the second to His6, His13, His14 and Met35 residues. The highest contribution to the second oxidation peak current was from the His13 residue, followed by His14 and His6 residues, while the Met35residue presented the lowest oxidation peak current. The Aβ peptides electron transfer reaction depended on the sequence hydrophobicity, on the position of the electroactive amino acid residues in the sequence (the residues close to N-termini giving the highest oxidation peak currents), being also strongly influenced by the Aβ peptide secondary structure. The AFM and voltammetric results were consistent with the in vitro fibrilization mechanism, observed for different Aβ peptide sequences. The Aβ peptide monomers followed a nucleation process, leading to the formation of small nuclei. Once the nucleation process started, Aβ free monomers in solution were added to the nuclei and their dimensions grown forming various aggregate types, such as small oligomers and long fibrils with different morphologies. This polymerization reaction followed first-order kinetics, and is thermodynamically favored by the hydrophobic regions of Aβ peptides, as well as their β-sheet secondary structure. As fibrilization proceeded, the electroactive residues became more protected, and the transfer of electrons became more difficult.

Interference of α-Synuclein Uptake by Monomeric β-Amyloid1-40 and Potential Core Acting Site of the Interference.

Increasing evidence suggests an important role of alpha-synuclein (α-Syn) in the pathogenesis of Parkinson's disease (PD). The inter-neuronal spread of α-Syn via exocytosis and endocytosis has been proposed as an explanation for the neuropathological findings of PD in sub-clinical and clinical phases. Therefore, interfering the uptake of α-Syn by neurons may be an important step in slowing or modifying the propagation of the disease. The purposes of our study were to investigate if the uptake of α-Syn fibrils can be specifically interfered with monomeric β-Amyloid1-40 (Aβ40) and to characterise the core acting site of interference. Using a radioisotope-labelled uptake assay, we found an 80% uptake reduction of α-Syn fibrils in neurons interfered with monomeric Aβ40, but not β-Amyloid1-42 (Aβ42) as compared to controls. This finding was further confirmed by enzyme-linked immunosorbent assay (ELISA) with α-Syn uptake reduced from about 80% (Aβ42) to about 20% (Aβ40) relative to controls. To define the region of Aβ40 peptide capable of the interference, we explored shorter peptides with less amino acid residues from both the C-terminus and N-terminus. We found that the interference effect was preserved if amino acid residue was trimmed to position 11 (from N-terminus) and 36 (from C-terminus), but dropped off significantly if residues were trimmed beyond these positions. We therefore deduced that the "core acting site" lies between amino acid residue positions 12-36. These findings suggest α-Syn uptake can be interfered with monomeric Aβ40 and that the core acting site of interference might lie between amino acid residue positions 12-36.

Editor's Highlight: Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS): A Web-Based Tool for Addressing the Challenges of Cross-Species Extrapolation of Chemical Toxicity.

Conservation of a molecular target across species can be used as a line-of-evidence to predict the likelihood of chemical susceptibility. The web-based Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS; https://seqapass.epa.gov/seqapass/) application was developed to simplify, streamline, and quantitatively assess protein sequence/structural similarity across taxonomic groups as a means to predict relative intrinsic susceptibility. The intent of the tool is to allow for evaluation of any potential protein target while remaining amenable to variable degrees of protein characterization, in the context of available information about the chemical/protein interaction and the molecular target itself. To accommodate this flexibility in the analysis, 3 levels of evaluation were developed. The first level of the SeqAPASS analysis compares primary amino acid sequences to a query sequence, calculating a metric for sequence similarity (including detection of orthologs); the second level evaluates sequence similarity within selected functional domains (eg, ligand-binding domain); and the third level of analysis compares individual amino acid residue positions of importance for protein conformation and/or interaction with the chemical upon binding. Each level of the SeqAPASS analysis provides additional evidence to apply toward rapid, screening-level assessments of probable cross species susceptibility. Such analyses can support prioritization of chemicals for further evaluation, selection of appropriate species for testing, extrapolation of empirical toxicity data, and/or assessment of the cross-species relevance of adverse outcome pathways. Three case studies are described herein to demonstrate application of the SeqAPASS tool: the first 2 focused on predictions of pollinator susceptibility to molt-accelerating compounds and neonicotinoid insecticides, and the third on evaluation of cross-species susceptibility to strobilurin fungicides. These analyses illustrate challenges in species extrapolation and demonstrate the broad utility of SeqAPASS for risk-based decision making and research.

Expression and characterization of EF-hand I loop mutants of aequorin replaced with other loop sequences of Ca2+-binding proteins: an approach to studying the EF-hand motif of proteins.

The binding properties of Ca(2+) to EF-hand I of aequorin (AQ) were characterized by replacing the loop sequence of EF-hand I (AQ[I]) with other known loop sequences of Ca(2+)-binding proteins, including photoproteins (aequorin, clytin-I, clytin-II and mitrocomin), Renilla luciferin-binding protein (RLBP) and calmodulin (CaM). For evaluation of the binding affinity of Ca(2+) to AQ[I] mutants, the half-decay time of the maximum intensity in the luminescence reaction triggered by Ca(2+) was used as an indicator and 22 kinds of AQ[I] mutants were expressed in Escherichia coli cells. AQ[I] mutants replaced with the EF-hand I and EF-hand III from photoproteins showed sufficient luminescence activity, but it was not shown by other EF-hands from RLBP and CaM. An AQ[I] mutant with a lysine or arginine residue at the second position of the non-conserved amino acid residue showed a slow-decay pattern of luminescence, indicating that the Ca(2+)-binding affinity to aequorin was reduced by a positive charge at the second position of the loop sequence. The specific loop sequence of the EF-hand I motif in aequorin caused the specific Ca(2+)-triggered luminescence pattern.

Computer analysis of protein functional sites projection on exon structure of genes in Metazoa

BackgroundStudy of the relationship between the structural and functional organization of proteins and their coding genes is necessary for an understanding of the evolution of molecular systems and can provide new knowledge for many applications for designing proteins with improved medical and biological properties. It is well known that the functional properties of proteins are determined by their functional sites. Functional sites are usually represented by a small number of amino acid residues that are distantly located from each other in the amino acid sequence. They are highly conserved within their functional group and vary significantly in structure between such groups. According to this facts analysis of the general properties of the structural organization of the functional sites at the protein level and, at the level of exon-intron structure of the coding gene is still an actual problem.ResultsOne approach to this analysis is the projection of amino acid residue positions of the functional sites along with the exon boundaries to the gene structure. In this paper, we examined the discontinuity of the functional sites in the exon-intron structure of genes and the distribution of lengths and phases of the functional site encoding exons in vertebrate genes. We have shown that the DNA fragments coding the functional sites were in the same exons, or in close exons. The observed tendency to cluster the exons that code functional sites which could be considered as the unit of protein evolution. We studied the characteristics of the structure of the exon boundaries that code, and do not code, functional sites in 11 Metazoa species. This is accompanied by a reduced frequency of intercodon gaps (phase 0) in exons encoding the amino acid residue functional site, which may be evidence of the existence of evolutionary limitations to the exon shuffling.ConclusionsThese results characterize the features of the coding exon-intron structure that affect the functionality of the encoded protein and allow a better understanding of the emergence of biological diversity.

Binding studies of hydroxylated Multi-Walled Carbon Nanotubes to hemoglobin, gamma globulin and transferrin

Biocompatibility of nanoparticles depends on their binding behavior with biomolecules. Herein, we have reported the interaction of three different biological macromolecules such as hemoglobin, gamma globulin and transferrin with hydroxyl group functionalized Multi-Walled Carbon Nanotubes (OH-MWCNTs). Multiple spectroscopic methods were utilized to identify the binding cum structural changes in biomolecules upon their interaction. Hyperchromic effect observed in the UV–visible spectra, and the quenching behavior from fluorescence emission evidences the existence of bio-nanotube complex formation. Synchronous and three-dimensional fluorescence spectra of biomolecules, in correspondence with Trp and Tyr residues showed the possible disturbance towards their aromatic micro-environment. Changes observed in the FTIR and FT-Raman amide bands, and amino acid residue position of biomolecules upon interaction with CNTs showed the possible effect towards their secondary structure. Further studies with CD spectroscopy indicated the loss of alpha-helical structures quantitatively. The study remains significant in evaluating the biosafety profile of functionalized MWCNTs for their in vivo biomedical applications.

Molecular identification and genetic diversity of open reading frame 7 field isolated porcine reproductive and respiratory syndrome in North Sumatera, Indonesia, in the period of 2008-2014.

Aim:Molecular identification and genetic diversity of open reading frame 7 (ORF7) of field isolated porcine reproductive and respiratory syndrome virus (PRRSV) in North Sumatera, Indonesia, in the period of 2008-2014.Materials and Methods:A total of 47 PRRSV samples were collected from the death case of pigs. The samples were collected from different districts in the period of 2008-2014 from North Sumatera province. Two pairs of primer were designed to amplify ORF7 of Type 1 and 2 PRRSV based on the sequence of reference viruses VR2332 and Lelystad. Viral RNAs were extracted from samples using PureLink™ micro-to-Midi total RNA purification system (Invitrogen). To amplify the ORF7 of PRRSV, the synthesis cDNA and DNA amplification were performed by reverse transcription polymerase chain reaction (RT-PCR) and nested PCR method. Then the DNA sequencing of PCR products and phylogenetic analysis were accomplished by molecular evolutionary genetics analysis version 6.0 software program.Results:RT-: PCR and nested PCR used in this study had successfully detected of 18 samples positive PRRS virus with the amplification products at 703bp and 508bp, respectively. Sequencing of the ORF7 shows that 18 PRRS viruses isolated from North Sumatera belonged to North American (NA). JXA1 Like and classic NA type viruses. Several mutations were detected, particularly in the area of nuclear localization signal (NLS1) and in NLS2. In the local viruses, which were related closed to JXA1 virus; there are two differences in amino acids in position 12 and 43 of ORF7. Our tested viruses showed that the amino acid positions 12 and 43 are Asparagine and Arginine, while the reference virus (VR2332, Lelystad, and JXA1) occupied both by Lysine. Based on differences in two amino acids at position 12 and 43 showed that viruses from North Sumatera has its own uniqueness and related closed to highly pathogenic PRRS (HP-PRRS) virus (JXA1).Conclusion:The results demonstrated that North Sumatera type PRRS virus has caused PRRS outbreaks in pig in North Sumatera between 2008 and 2014. The JAX1 like viruses had unique amino acid residue in position 12 and 43 of asparagine and lysine, and these were genetic determinants of North Sumatera viruses compared to other PRRS viruses.

Characterization of aluminum resistant Anoxybacillus sp. SK 3-4 isolated from a hot spring.

The Anoxybacillus sp. SK 3-4, previously isolated from a hot spring, was screened for its heavy metals resistance (Al(3+), Mn(2+), Cu(2+), Co(2+), Zn(2+), and Ni(2+)) and the strain was found to be most resistant to aluminum. Significant growth of the strain was observed when it was grown in medium containing aluminum (200 mg L(-1)-800 mg L(-1)) with relative growth rates ranging between 77% and 100%. A gene encoding the aluminum resistance protein (accession number: WP_021095658.1) was found in genome of strain SK 3-4, which revealed high sequence identity (>95%) to its homologues from Anoxybacillus species. Sequence comparisons with two functionally characterized aluminum resistance proteins, namely G2alt and ALU1-P, showed 97% and 81% of sequence identity, respectively. Four putative metal binding sites were detected in SK 3-4 aluminum resistance protein and G2alt at same amino acid residue positions of 186, 195, 198, and 201. Strain SK 3-4 was found to be able to remove aluminum from aqueous solution. This study demonstrated that Anoxybacillus sp. SK 3-4 could be applied in the treatment of aluminum contaminated wastewater.

OR33: CLINICALLY USEFUL TOOL FOR COMPARING THEORETICAL AND EVIDENCE BASED HLA EPITOPES

Aim Theoretical HLA epitopes (THEps) were defined structurally by three-dimensional molecular modeling and amino acid sequence differences whereas evidence based HLA epitopes (EBHEps) considers the ability of a putative HLA epitope (HEps) to induce antibody. Such HEps are clinically relevant if the antibody is produced in humans on exposure to alloantigen. The aim of this study is to provide a cross reference tool for comparison of structurally defined HEps and EBHEps that may be of use to laboratories interested in defining patient sera by epitope reactivity. Method A comparative analysis of THEps from the International Registry of Antibody-Defined HLA Epitopes ( http://epregistry.com.br ), and evidence based Class I and Class II HLA epitopes from publications. The analysis was arranged in a cross referenced tabular format. Results We found 67 EBHEps that corresponded to theoretical Class I HEps epitope. Of the 67 theoretical Class I HEps, 7 were reactive with mouse monoclonal antibodies only and 60/67 epitopes were reactive with human allosera. Interestingly, 6 of the 60 human allosera - reactive epitopes were present on HLA-A and B, 2/60 were present on HLA-A, B and C, and 3/60 human reactive epitopes were present on HLA-B and C. We also found 55 EBHEps that corresponded to theoretical DR epitopes and16 evidence based DQ epitopes that corresponded to theoretical epitopes. Based on this data, tables were created to cross reference each of the THEps with the EBHEps thereby facilitating analysis of the patient’s antibody data. Conclusion The nomenclature of the theoretical epitopes ( http://epregistry.com.br ) is based on the position of the polymorphic amino acid residue in numerical order in the polypeptide chain followed by the single letter amino acid code (in alphabetical order). Evidence based epitopes had been published as a list in consecutive numerical order unrelated to the polymorphic amino acid position. One of the major goals of the HLA epitope registry is to identify clinically relevant HLA epitopes that have been verified experimentally with human allosera. The charts developed in our study allow for the convenient cross correlation between evidence based epitopes and theoretical epitopes that will be updated according to new evidence of human allosera reactivity. A.J. Norin: Speaker’s Bureau; Company/Organization; Immuncor - Lifecodes. Scientific/Medical Advisor; Company/Organization; ICON CL.

Formation Constants of Copper(II) Complexes with Tripeptides Containing Glu, Gly, and His: Potentiometric Measurements and Modeling by Generalized Multiplicative Analysis of Variance

We report a systematic study of the effects of types and positions of amino acid residues of tripeptides on the formation constants logβ, acid dissociation constants pKa, and the copper coordination modes of the copper(II) complexes with 27 tripeptides formed from the amino acids glutamic acid, glycine, and histidine. logβ values were calculated from pH titrations with l mmol L(-1):1 mmol L(-1) solutions of the metal and ligand and previously reported ligand pKa values. Generalized multiplicative analysis of variance (GEMANOVA) was used to model the logβ values of the saturated, most protonated, monoprotonated, logβ(CuL) - logβ(HL), and pKa of the amide group. The resulting model of the saturated copper species has a two-term model describing an interaction between the central and the C-terminal residues plus a smaller, main effect of the N-terminal residue. The model supports the conclusion that two copper coordination modes exist depending on the absence or presence of His at the central position, giving species in which copper is coordinated via two or three fused chelate rings, respectively. The GEMANOVA model for pKamide, which is the same as that for the saturated complex, showed that Gly-Gly-His has the lowest pKamide values among the 27 tripeptides. Visible spectroscopy indicated the formation of metal-ligand dimers for tripeptides His-His-Gly and His-His-Glu, but not for His-His-His, and the formation of multiple ligand bis compexes CuL2 and Cu(HL)2 for tripeptides (Glu/Gly)-His-(Glu/Gly) and His-(Glu/Gly)-(Glu/Gly), respectively.

Determination of Residues Responsible for Substrate and Product Specificity of Solanum habrochaites Short-Chain cis-Prenyltransferases

Isoprenoids are diverse compounds that have their biosynthetic origin in the initial condensation of isopentenyl diphosphate and dimethylallyl diphosphate to form C10 prenyl diphosphates that can be elongated by the addition of subsequent isopentenyl diphosphate units. These reactions are catalyzed by either cis-prenyltransferases (CPTs) or trans-prenyltransferases. The synthesis of volatile terpenes in plants typically proceeds through either geranyl diphosphate (C10) or trans-farnesyl diphosphate (C15), to yield monoterpenes and sesquiterpenes, respectively. However, terpene biosynthesis in glandular trichomes of tomato (Solanum lycopersicum) and related wild relatives also occurs via the cis-substrates neryl diphosphate (NPP) and 2Z,6Z-farnesyl diphosphate (Z,Z-FPP). NPP and Z,Z-FPP are synthesized by neryl diphosphate synthase1 (NDPS1) and Z,Z-farnesyl diphosphate synthase (zFPS), which are encoded by the orthologous CPT1 locus in tomato and Solanum habrochaites, respectively. In this study, comparative sequence analysis of NDPS1 and zFPS enzymes from S. habrochaites accessions that synthesize either monoterpenes or sesquiterpenes was performed to identify amino acid residues that correlate with the ability to synthesize NPP or Z,Z-FPP. Subsequent structural modeling, coupled with site-directed mutagenesis, highlighted the importance of four amino acids located within conserved domain II of CPT enzymes that form part of the second α-helix, for determining substrate and product specificity of these enzymes. In particular, the relative positioning of aromatic amino acid residues at positions 100 and 107 determines the ability of these enzymes to synthesize NPP or Z,Z-FPP. This study provides insight into the biochemical evolution of terpene biosynthesis in the glandular trichomes of Solanum species.

Sequence and structure based models of HIV-1 protease and reverse transcriptase drug resistance.

BackgroundSuccessful management of chronic human immunodeficiency virus type 1 (HIV-1) infection with a cocktail of antiretroviral medications can be negatively affected by the presence of drug resistant mutations in the viral targets. These targets include the HIV-1 protease (PR) and reverse transcriptase (RT) proteins, for which a number of inhibitors are available on the market and routinely prescribed. Protein mutational patterns are associated with varying degrees of resistance to their respective inhibitors, with extremes that can range from continued susceptibility to cross-resistance across all drugs.ResultsHere we implement statistical learning algorithms to develop structure- and sequence-based models for systematically predicting the effects of mutations in the PR and RT proteins on resistance to each of eight and eleven inhibitors, respectively. Employing a four-body statistical potential, mutant proteins are represented as feature vectors whose components quantify relative environmental perturbations at amino acid residue positions in the respective target structures upon mutation. Two approaches are implemented in developing sequence-based models, based on use of either relative frequencies or counts of n-grams, to generate vectors for representing mutant proteins. To the best of our knowledge, this is the first reported study on structure- and sequence-based predictive models of HIV-1 PR and RT drug resistance developed by implementing a four-body statistical potential and n-grams, respectively, to generate mutant attribute vectors. Performance of the learning methods is evaluated on the basis of tenfold cross-validation, using previously assayed and publicly available in vitro data relating mutational patterns in the targets to quantified inhibitor susceptibility changes.ConclusionOverall performance results are competitive with those of a previously published study utilizing a sequence-based strategy, while our structure- and sequence-based models provide orthogonal and complementary prediction methodologies, respectively. In a novel application, we describe a technique for identifying every possible pair of RT inhibitors as either potentially effective together as part of a cocktail, or a combination that is to be avoided.

Cypher/ZASP Is a Novel A-kinase Anchoring Protein

PKA signaling is important for the post-translational modification of proteins, especially those in cardiomyocytes involved in cardiac excitation-contraction coupling. PKA activity is spatially and temporally regulated through compartmentalization by protein kinase A anchoring proteins. Cypher/ZASP, a member of PDZ-LIM domain protein family, is a cytoskeletal protein that forms multiprotein complexes at sarcomeric Z-lines. It has been demonstrated that Cypher/ZASP plays a pivotal structural role in the structural integrity of sarcomeres, and several of its mutations are associated with myopathies including dilated cardiomyopathy. Here we show that Cypher/ZASP, interacting specifically with the type II regulatory subunit RIIα of PKA, acted as a typical protein kinase A anchoring protein in cardiomyocytes. In addition, we show that Cypher/ZASP itself was phosphorylated at Ser(265) and Ser(296) by PKA. Furthermore, the PDZ domain of Cypher/ZASP interacted with the L-type calcium channel through its C-terminal PDZ binding motif. Expression of Cypher/ZASP facilitated PKA-mediated phosphorylation of the L-type calcium channel in vitro. Additionally, the phosphorylation of the L-type calcium channel at Ser(1928) induced by isoproterenol was impaired in neonatal Cypher/ZASP-null cardiomyocytes. Moreover, Cypher/ZASP interacted with the Ser/Thr phosphatase calcineurin, which is a phosphatase for the L-type calcium channel. Taken together, our data strongly suggest that Cypher/ZASP not only plays a structural role for the sarcomeric integrity, but is also an important sarcomeric signaling scaffold in regulating the phosphorylation of channels or contractile proteins.

Sodium channel SCN8A (Nav1.6): properties and de novo mutations in epileptic encephalopathy and intellectual disability

The sodium channel Nav1.6, encoded by the gene SCN8A, is one of the major voltage-gated channels in human brain. The sequences of sodium channels have been highly conserved during evolution, and minor changes in biophysical properties can have a major impact in vivo. Insight into the role of Nav1.6 has come from analysis of spontaneous and induced mutations of mouse Scn8a during the past 18 years. Only within the past year has the role of SCN8A in human disease become apparent from whole exome and genome sequences of patients with sporadic disease. Unique features of Nav1.6 include its contribution to persistent current, resurgent current, repetitive neuronal firing, and subcellular localization at the axon initial segment (AIS) and nodes of Ranvier. Loss of Nav1.6 activity results in reduced neuronal excitability, while gain-of-function mutations can increase neuronal excitability. Mouse Scn8a (med) mutants exhibit movement disorders including ataxia, tremor and dystonia. Thus far, more than ten human de novo mutations have been identified in patients with two types of disorders, epileptic encephalopathy and intellectual disability. We review these human mutations as well as the unique features of Nav1.6 that contribute to its role in determining neuronal excitability in vivo. A supplemental figure illustrating the positions of amino acid residues within the four domains and 24 transmembrane segments of Nav1.6 is provided to facilitate the location of novel mutations within the channel protein.

Monoclonal antibody-based ELISA for detection of antibodies against H5 avian influenza viruses

Diagnostic and containment measures are essential for the management of avian influenza. In this study, a monoclonal antibody (MAb)-based competitive ELISA for detecting antibodies against H5 avian influenza viruses was developed and validated. Twenty-five anti-H5 MAbs were characterised using competitive, indirect and sandwich ELISAs, immunofluorescence, Western blotting and virus neutralisation and haemagglutination inhibition assays. One MAb (5D8) with wide intra-subtype cross-reactivity was selected and characterised using escape mutant selection. Epitope analysis showed that this MAb recognises a conformational epitope comprising amino acid residues in positions 170, 235 and 240 located in the receptor binding domain. The diagnostic performance of the test was evaluated by ROC analysis using a panel of 950 known sera collected from different avian species, including chickens, turkeys, ducks, pheasants, wild Anseriformes and ostriches. The competitive ELISA had excellent diagnostic performance and discriminatory power with high Se and Sp values (Se: 99.6-95% CI 98.0-100; Sp: 99.4-95% CI 98.5-99.8). In addition to its excellent diagnostic performance, properties of the competitive ELISA, such as high feasibility of testing sera without pre-treatment and potential for automation and instrument-mediated detection, make it ideal for screening samples, confirming positive HI assay results or analysing samples that are difficult to test using the HI assay.

The Radical Ion Chemistry of S-Nitrosylated Peptides

The radical ion chemistry of a suite of S-nitrosopeptides has been investigated. Doubly and triply-protonated ions of peptides NYCGLPGEYWLGNDK, NYCGLPGEYWLGNDR, NYCGLPGERWLGNDR, NACGAPGEKWAGNDK, NYCGLPGEKYLGNDK, NYGLPGCEKWYGNDK and NYGLPGEKWYGCNDK were subjected to electron capture dissociation (ECD), and collision-induced dissociation (CID). The peptide sequences were selected such that the effect of the site of S-nitrosylation, the nature and position of the basic amino acid residues, and the nature of the other amino acid side chains, could be interrogated. The ECD mass spectra were dominated by a peak corresponding to loss of (•)NO from the charge-reduced precursor, which can be explained by a modified Utah-Washington mechanism. Some backbone fragmentation in which the nitrosyl modification was preserved was also observed in the ECD of some peptides. Molecular dynamics simulations of peptide ion structure suggest that the ECD behavior was dependent on the surface accessibility of the protonated residue. CID of the S-nitrosylated peptides resulted in homolysis of the S-N bond to form a long-lived radical with loss of (•)NO. The radical peptide ions were isolated and subjected to ECD and CID. ECD of the radical peptide ions provided an interesting comparison to ECD of the unmodified peptides. The dominant process was electron capture without further dissociation (ECnoD). CID of the radical peptide ions resulted in cysteine, leucine, and asparagine side chain losses, and radical-induced backbone fragmentation at tryptophan, tyrosine, and asparagine residues, in addition to charge-directed backbone fragmentation.