Position Of Residue Research Articles

In silico study of inhibition activity of boceprevir drug against 2019-nCoV main protease.

Boceprevir drug is a ketoamide serine protease inhibitor with a linear peptidomimetic structure that exhibits inhibition activity against 2019-nCoV main protease. This paper reports electronic properties of boceprevir and its molecular docking as well as molecular dynamics simulation analysis with protein receptor. For this, the equilibrium structure of boceprevir has been obtained by DFT at B3LYP and ωB97XD levels with 6-311+G(d,p) basis set in gas and water mediums. HOMO-LUMO and absorption spectrum analysis have been used to evaluate the boceprevir's toxicity and photosensitivity, respectively. Molecular docking simulation has been performed to test the binding affinity of boceprevir with 2019-nCoV MPRO; which rendered a variety of desirable binding locations between the ligand and target protein's residue positions. The optimum binding location has been considered for molecular dynamics simulation. The findings have been addressed to clarify the boceprevir drug efficacy against the 2019-nCoV MPRO.

Engineered assemblies from isomeric pentapeptides augment dry eye treatment

Changing positions of amino acid residues in the peptide sequence alters the peptide’ s assembly behaviors, affording various nanostructures. However, it remains elusive that how subtle changes in the peptide sequence influence the in vivo bioactivity of peptide-based nanocarriers, further impacting the efficacy of the encapsulated drugs. We report here a class of isomeric pentapeptide amphiphiles that associate into filaments with different dimensions, which were further used as carriers of Diquafosol tetrasodium (DQS), for the treatment of dry eye disease. Our results suggest that subtle changes in peptide sequences resulted in dramatically different molecular packings and distinct morphologies, which were verified by molecular dynamics simulations. In vivo results show that the drug retention time could be prolonged by the peptidic nanostructures on the ocular surface but were highly morphological-dependent. The longer retention time promised better therapeutic efficacy. In terms of facile synthesis and good biocompatibility, we believe that these peptides could be used for eye disease treatments or other related areas.

An active machine learning discovery platform for membrane-disrupting and pore-forming peptides.

Membrane-disrupting and pore-forming peptides (PFPs) play a substantial role in bionanotechnology and can determine the life and death of cells. The control of chemical and ion transport through cell membranes is essential to maintaining concentration gradients. Likewise, the delivery of drugs and intracellular proteins aided by pore-forming agents is of interest in treating malfunctioning cells. Known PFPs tend to be up to 50 residues in length, which is commensurate with the thickness of a lipid bilayer. Accordingly, few short PFPs are known. Here we show that the discovery of PFPs can be accelerated via an active machine learning approach. The approach identified 71 potential PFPs from the 25.6 billion octapeptide sequence space; 13 sequences were tested experimentally, and all were found to have the predicted membrane-disrupting ability, with 1 forming highly stable pores. Experimental verification of the predicted pore-forming ability demonstrated that a range of short peptides can form pores in membranes, while the positioning and characteristics of residues that favour pore-forming behaviour were identified. This approach identified more ultrashort (8-residues, unmodified, non-cyclic) PFPs than previously known. We anticipate our findings and methodology will be useful in discovering new pore-forming and membrane-disrupting peptides for a range of applications from nanoreactors to therapeutics.

Vaccine-elicited and naturally elicited antibodies differ in their recognition of the HIV-1 fusion peptide.

Broadly neutralizing antibodies have been proposed as templates for HIV-1 vaccine design, but it has been unclear how similar vaccine-elicited antibodies are to their naturally elicited templates. To provide insight, here we compare the recognition of naturally elicited and vaccine-elicited antibodies targeting the HIV-1 fusion peptide, which comprises envelope (Env) residues 512-526, with the most common sequence being AVGIGAVFLGFLGAA. Naturally elicited antibodies bound peptides with substitutions to negatively charged amino acids at residue positions 517-520 substantially better than the most common sequence, despite these substitutions rarely appearing in HIV-1; by contrast, vaccine-elicited antibodies were less tolerant of sequence variation, with no substitution of residues 512-516 showing increased binding. Molecular dynamics analysis and cryo-EM structural analysis of the naturally elicited ACS202 antibody in complex with the HIV-1 Env trimer with an alanine 517 to glutamine substitution suggested enhanced binding to result from electrostatic interactions with positively charged antibody residues. Overall, vaccine-elicited antibodies appeared to be more fully optimized to bind the most common fusion peptide sequence, perhaps reflecting the immunization with fusion peptide of the vaccine-elicited antibodies.

Reducing Self-Assembly by Increasing Net Charge: Effect on Biological Activity of Mastoparan C.

The ability of amphipathic peptides to arrange themselves in aqueous solutions, known as self-assembly, has been found to reduce the effectiveness of these peptides in interacting with cell membranes. Therefore, minimizing their tendency to self-assemble could be a potential strategy for enhancing the pharmacological properties of antimicrobial peptides (AMPs). To explore this idea, this study prepared a series of natural peptides mastoparan C (MPC) with increased net charge and hydrophilicity via alanine-to-lysine substitution and investigated the impact on the biological activity. The preliminary data suggested the influence of both the overall positive charge and the position of a lysine residue on the self-assembly of MPC and its derivatives. Besides, the analogue MPC-A5K,A8K displayed higher anticancer activity and comparable antimicrobial activity with significantly lower hemolysis than MPC. Hence, reducing self-assembly by expanding the cationic area could be a promising approach for developing potent and selective AMPs.

EPR Studies of Aβ42 Oligomers Indicate a Parallel In-Register β-Sheet Structure.

Aβ aggregation leads to the formation of both insoluble amyloid fibrils and soluble oligomers. Understanding the structures of Aβ oligomers is important for delineating the mechanism of Aβ aggregation and developing effective therapeutics. Here, we use site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to study Aβ42 oligomers prepared by using the protocol of Aβ-derived diffusible ligands. We obtained the EPR spectra of 37 Aβ42 oligomer samples, each spin-labeled at a unique residue position of the Aβ42 sequence. Analysis of the disordered EPR components shows that the N-terminal region has a lower local structural stability. Spin label mobility analysis reveals three structured segments at residues 9-11, 15-22, and 30-40. Intermolecular spin-spin interactions indicate a parallel in-register β-sheet structure, with residues 34-38 forming the structural core. Residues 16-21 also adopt the parallel in-register β-structure, albeit with weaker intermolecular packing. Our results suggest that there is a structural class of Aβ oligomers that adopt fibril-like conformations.

Insights into virulence: structure classification of the Vibrio parahaemolyticus RIMD mobilome.

The pandemic Vpar strain RIMD causes seafood-borne illness worldwide. Previous comparative genomic studies have revealed pathogenicity islands in RIMD that contribute to the success of the strain in infection. However, not all virulence determinants have been identified, and many of the proteins encoded in known pathogenicity islands are of unknown function. Based on the EOCD database, we used evolution-based classification of structure models for the RIMD proteome to improve our functional understanding of virulence determinants acquired by the pandemic strain. We further identify and classify previously unknown mobile protein domains as well as fast evolving residue positions in structure models that contribute to virulence and adaptation with respect to a pre-pandemic strain. Our work highlights key contributions of phage in mediating seafood born illness, suggesting this strain balances its avoidance of phage predators with its successful colonization of human hosts.

Chemical characterization, antioxidant and immunomodulatory activities of acetylated polysaccharides from Cyperus esculentus.

This research was designed to characterize the structure of Cyperus esculentus polysaccharide (CEP) and its acetylated one (ACEP), and then investigated the effects of acetylation on the changes in physicochemical properties, thermal stability, antioxidant and immunomodulatory activities. Results showed that CEP and ACEP were heteropolysaccharides consisting of glucose, mannose, arabinose and xylose. The main chain of CEP included α-1,4-Glcp residues with the branching points at the O-6 position of the α-1,6-Manp residues. Acetyl groups were substituted at the O-2 and O-6 positions of some glucose residues. Meanwhile, the acetylation remarkably improved the polysaccharides thermal stability, and the ACEP exhibited a greater antioxidant activity. Furthermore, CEP and ACEP were proved to protect RAW 264.7 cells against LPS-induced inflammation by improving cellular morphology and decreasing reactive oxygen species secretion. This study may highlight a new approach for developing a high value-added ingredient from C. esculentus for functional food industry.

New alignment method for remote protein sequences by the direct use of pairwise sequence correlations and substitutions.

Understanding protein sequences and how they relate to the functions of proteins is extremely important. One of the most basic operations in bioinformatics is sequence alignment and usually the first things learned from these are which positions are the most conserved and often these are critical parts of the structure, such as enzyme active site residues. In addition, the contact pairs in a protein usually correspond closely to the correlations between residue positions in the multiple sequence alignment, and these usually change in a systematic and coordinated way, if one position changes then the other member of the pair also changes to compensate. In the present work, these correlated pairs are taken as anchor points for a new type of sequence alignment. The main advantage of the method here is its combining the remote homolog detection from our method PROST with pairwise sequence substitutions in the rigorous method from Kleinjung et al. We show a few examples of some resulting sequence alignments, and how they can lead to improvements in alignments for function, even for a disordered protein.

Risk reduction of tank explosion based on passivation of unusable propellant residues

To reduce the risk of explosion of propellant tanks of expended spacecraft and launch vehicles with liquid rocket engines in orbit, as well as in case of emergency situation, for example, loss of orientation, the Inter-Agency Space Debris Coordination Committee recommends passivation measures, including the discharge of residual liquid propellant and pressurant gas. In ANSYS-Fluent program complex possible initial positions of liquid propellant residues in a spherical tank at its rotation under conditions of low gravitational fields are determined. The values of liquid propellant residues depending on their initial position in the spherical tank at opening of the drain line for discharge of gas–liquid mixture into the ambient space are determined. The concept of formation of two-phase flows of liquid propellant on the example of the spherical tank at tangential entry of compressed gas is offered. The relationship between the number of gas inlet points and the effectiveness of the developed method (expressed as the ratio of the mass of expelled liquid propellant to the mass of gas expended) is demonstrated. For instance, the use of 2 gas inlet points achieves an efficiency of up to 30 %, while employing 3 gas inlet points increases it to 89 %.

Revisiting the influence of pH on 1JCαH and chemical shifts of glycine and alanine short oligopeptides.

The pH dependence of several NMR parameters of glycine and alanine short oligopeptides has been reported previously in different studies. Here we have thoroughly examined, summarized and demonstrated the dependences of 1H, 13C and 15N chemical shifts and protonation states of amino acids using two-dimensional NMR experiments. Nevertheless, 1JCαH one bond spin-spin coupling constants are more informative and convenient for determination of the position and protonation state of glycine and alanine residue in the oligopeptide chain. In particular, for various oligopeptides (up to six residues), it was shown that the pH dependence of 1JCαH of N-terminal glycine and alanine residues is larger than that of C-terminal groups, and in backbone residues, it is not influenced by pH and only slightly depends on the position of the amino acid residue in the chain.

Characterization of bovine immunodeficiency virus genes and proteins

The goal of the study was determining the effect of natural selection on the bovine immunodeficiency virus (BIV) through the identification of polymorphisms by comparing a number of genes, open reading frames and proteins and analyzing the effect of nonsynonymous substitutions in the Vif protein of BIV isolates on the Vif conformational parameters. The selection pressure on the genes was estimated by Tajima test, as well as the determination of the ratio of nonsynonymous substitutions (Ka) to synonymous (Ks) Ka/Ks. Ka and Ks coefficients, their ratio (Ka/Ks) were calculated on the basis of alignments of amino acid and nucleotide sequences of the vif gene of BIV isolates. The nonsynonymous (dN) to synonymous (dS) substitution rate ratio per nucleotide site dN/dS was calculated to estimate the effect of selection on the vif gene by SLAC method on Datamonkey server. The number of hydrogen bonds, α-helices, β-sheets, β-turns for the protein tertiary structures of proteins was determined by I-TASSER server. Genes, open reading frames (ORFs) and the U3 regulatory region of BIV genome which are under the influence of selection have been identified. Gag, pol, s, vif genes, ORF W and ORF Y are under the negative (purifying) selection pressure. Env gene and the U3 regulatory region are under positive selection. Shannon entropy analysis (peaks that are specific to the positions of amino acid residues), interpreted as polymorphisms, revealed 16, 8, and 4 nonsynonymous substitutions for Pol, Gag, and Vif BIV proteins, respectively. Significant changes in the number of hydrogen bonds, α-helices, β-layers, β-turns were determined by the analysis of the effect of four nonsynonymous substitutions in the Vif protein on the conformational parameters of Vif of two BIV isolates. The multidirectional effect of selection on BIV genes is shown

Compendium of Chromatographic Behavior of Post-translationally and Chemically Modified Peptides in Bottom-Up Proteomic Experiments.

We have systematically evaluated the chromatographic behavior of post-translationally/chemically modified peptides using data spanning over 70 of the most relevant modifications. These retention properties were measured for standard bottom-up proteomic settings (fully porous C18 separation media, 0.1% formic acid as ion-pairing modifier) using collections of modified/nonmodified peptide pairs. These pairs were generated by spontaneous degradation, chemical or enzymatic treatment, analysis of synthetic peptides, or the cotranslational incorporation of noncanonical proline analogues. In addition, these measurements were validated using external data acquired for synthetic peptides and enzymatically induced citrullination. Working in units of hydrophobicity index (HI, % ACN) and evaluating the average retention shifts (ΔHI) represent the simplest approach to describe the effect of modifications from a didactic point of view. Plotting HI values for modified (y-axis) vs nonmodified (x-axis) counterparts generates unique slope and intercept values for each modification defined by the chemistry of the modifying moiety: its hydrophobicity, size, pKa of ionizable groups, and position of the altered residue. These composition-dependent correlations can be used for coarse incorporation of PTMs into models for prediction of peptide retention. More accurate predictions would require the development of specific sequence-dependent algorithms to predict ΔHI values.

Transcriptomic investigation of the molecular mechanisms underlying resistance to the neonicotinoid thiamethoxam and the pyrethroid lambda-cyhalothrin in Euschistus heros (Hemiptera: Pentatomidae).

Laboratory-selected resistant strains of Euschistus heros to thiamethoxam (NEO) and lambda-cyhalothrin (PYR) were recently reported in Brazil. However, the mechanisms conferring resistance to these insecticides in E. heros remain unresolved. We utilized comparative transcriptome profiling and single nucleotide polymorphism (SNP) calling of susceptible and resistant strains of E. heros to investigate the molecular mechanism(s) underlying resistance. The E. heros transcriptome was assembled, generating 91 673 transcripts with a mean length of 720 bp and N50 of 1795 bp. Comparative gene expression analysis between the susceptible (SUS) and NEO strains identified 215 significantly differentially expressed (DE) transcripts. DE transcripts associated with the xenobiotic metabolism were all up-regulated in the NEO strain. The comparative analysis of the SUS and PYR strains identified 204 DE transcripts, including an esterase (esterase FE4), a glutathione-S-transferase, an ABC transporter (ABCC1) and aquaporins that were up-regulated in the PYR strain. We identified 9588 and 15 043 nonsynonymous SNPs in the PYR and NEO strains. One of the SNPs (D70N) detected in the NEO strain occurs in a subunit (α5) of the nAChRs, the target site of neonicotinoid insecticides. Nevertheless, this residue position in α5 is not conserved among insects. Neonicotinoid and pyrethroid resistance in laboratory-selected E. heros is associated with a potential metabolic resistance mechanism by the overexpression of proteins commonly involved in the three phases of xenobiotic metabolism. Together these findings provide insight into the potential basis of resistance in E. heros and will inform the development and implementation of resistance management strategies against this important pest. © 2023 Society of Chemical Industry.

Sensory-Guided Isolation, Identification, and Active Site Calculation of Novel Umami Peptides from Ethanol Precipitation Fractions of Fermented Grain Wine (Huangjiu).

Huangjiu is rich in low-molecular-weight peptides and has an umami taste. In order for its umami peptides to be discovered, huangjiu was subjected to ultrafiltration, ethanol precipitation, and macroporous resin purification processes. The target fractions were gathered according to sensory evaluation. Subsequently, we used peptidomics to identify the sum of 4158 peptides in most umami fractions. Finally, six novel umami peptides (DTYNPR, TYNPR, SYNPR, RFRQGD, NFHHGD, and FHHGD) and five umami-enhancing peptides (TYNPR, SYNPR, NFHHGD, FHHGD, and TVDGPSH) were filtered via virtual screening, molecular docking, and sensory verification. Moreover, the structure-activity relationship was discussed using computational approaches. Docking analysis showed that all umami peptides tend to bind with T1R1 through hydrogen bonds and hydrophobic forces, which involve key residues HIS71, ASP147, ARG151, TYR220, SER276, and ALA302. The active site calculation revealed that the positions of the key umami residues D and R in the terminal may cause taste differences in identified peptides.

SUMOylation of the ubiquitin ligase component KEAP1 at K39 upregulates NRF2 and its target function in lung cancer cell proliferation

Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) is important for the expression of genes associated with oxidative stress. The levels of NRF2 are controlled by Kelch-like ECH-associated protein 1 (KEAP1)-dependent degradation. Although oxidative stress is known to suppress KEAP1 activity to stabilize the levels of NRF2, the mechanism for this control is unclear. Here, we identify that KEAP1 is modified by SUMO1 at the lysine residue position 39 (K39). Arginine replacement of this lysine (K39R) in KEAP1 did not affect its stability, subcellular localization, or dimerization but promoted the formation of the Cullin 3 ubiquitin ligase and increased NRF2 ubiquitination. This was accompanied by decreased NRF2 expression. Gene reporter assays showed that the transcription of antioxidant response elements was heightened in KEAP1-WT cells compared to cells expressing the KEAP1-K39R SUMO1 substrate mutant. Consistent with this, chromatin immunoprecipitation assays revealed higher NRF2 binding to the promoter regions of antioxidant genes in cells expressing the KEAP1-WT compared to the KEAP1-K39R mutant protein in H1299 lung cancer cell. The significance of this suppression of KEAP1 activity by its SUMOylation was tested in a subcutaneous tumor model of H1299 lung cancer cell lines that differentially expressed the WT and K39R KEAP1 constructs. This model showed that mutating the SUMOylation site on KEAP1 altered the production of reactive oxygen species and suppressed tumor growth. Taken together, our study recognizes that NRF2-dependent redox control is regulated by the SUMOylation of KEAP1. These findings identify a potential new therapeutic option to counteract oxidative stress.

Rapid Determination of the Topology of Oligomeric α-Helical Membrane Proteins by Water- and Lipid-Edited Methyl NMR.

Single-span oligomeric α-helical transmembrane proteins are common in virus ion channels, which are targets of antiviral drugs. Knowledge about the high-resolution structures of these oligomeric α-helical bundles is so far scarce. Structure determination of these membrane proteins by solid-state NMR traditionally requires resolving and assigning protein chemical shifts and measuring many interhelical distances, which are time-consuming. To accelerate experimental structure determination, here we introduce a simple solid-state NMR approach that uses magnetization transfer from water and lipid protons to the protein. By detecting the water- and lipid-transferred intensities of the high-sensitivity methyl 13C signals of Leu, Val, and Ile residues, which are highly enriched in these membrane proteins, we can derive models of the topology of these homo-oligomeric helical bundles. The topology is specified by the positions of amino acid residues in heptad repeats and the orientations of residues relative to the channel pore, lipids, and the helical interface. We demonstrate this water- and lipid-edited methyl NMR approach on the envelope (E) protein of SARS-CoV-2, the causative agent of the COVID-19 pandemic. We show that water-edited and lipid-edited 2D 13C-13C correlation spectra can be measured with sufficient sensitivity. Even without resolving multiple residues of the same type in the NMR spectra, we can obtain the helical bundle topology. We apply these experiments to the structurally unknown E proteins of the MERS coronavirus and the human coronavirus NL63. The resulting structural topologies show interesting differences in the positions of the aromatic residues in these three E proteins, suggesting that these viroporins may have different mechanisms of activation and ion conduction.

Genotyped functional screening of soluble Fab clones enables in-depth analysis of mutation effects

Monoclonal antibodies (mAbs) and their fragments are widely used in therapeutics, diagnostics and basic research. Although display methods such as phage display offer high-throughput, affinities of individual antibodies need to be accurately measured in soluble format. We have developed a screening platform capable of providing genotyped functional data from a total of 9216 soluble, individual antigen binding fragment (Fab) clones by employing next-generation sequencing (NGS) with hierarchical indexing. Full-length, paired variable domain sequences (VL–VH) are linked to functional screening data, enabling in-depth analysis of mutation effects. The platform was applied to four phage display-selected scFv/Fab screening projects and one site-saturation VH affinity maturation project. Genotyped functional screening simultaneously enabled the identification of affinity improving mutations in the VH domain of Fab 49A3 recognizing Dengue virus non-structural protein 1 (NS1) serotype 2 and informed on VH residue positions which cannot be changed from wild-type without decreasing the affinity. Genotype-based identification revealed to us the extent of intraclonal signal variance inherent to single point screening data, a phenomenon often overlooked in the field. Moreover, genotyped screening eliminated the redundant selection of identical genotypes for further study and provided a new analysis tool to evaluate the success of phage display selections and remaining clonal diversity in the screened repertoires.

Structure determination and analysis of titin A-band fibronectin type III domains provides insights for disease-linked variants and protein oligomerisation

Titin is the largest protein found in nature and spans half a sarcomere in vertebrate striated muscle. The protein has multiple functions, including in the organisation of the thick filament and acting as a molecular spring during the muscle contraction cycle. Missense variants in titin have been linked to both cardiac and skeletal myopathies. Titin is primarily composed of tandem repeats of immunoglobulin and fibronectin type III (Fn3) domains in a variety of repeat patterns; however, the vast majority of these domains have not had their high-resolution structure determined experimentally. Here, we present the crystal structures of seven wild type titin Fn3 domains and two harbouring rare missense variants reported in hypertrophic cardiomyopathy (HCM) patients. All domains present the typical Fn3 fold, with the domains harbouring variants reported in HCM patients retaining the wild-type conformation. The effect on domain folding and stability were assessed for five rare missense variants found in HCM patients: four caused thermal destabilization of between 7 and 13 °C and one prevented the folding of its domain. The structures also allowed us to locate the positions of residues whose mutations have been linked to congenital myopathies and rationalise how they convey their deleterious effects. We find no evidence of physiological homodimer formation, excluding one hypothesised mechanism as to how titin variants could exert pathological effects.

Interaction of DNA Methyltransferase Dnmt3a with Phosphorus Analogs of S-Adenosylmethionine and S-Adenosylhomocysteine

Enzymatic methyltransferase reactions are of crucial importance for cell metabolism. S-Adenosyl-L-methionine (AdoMet) is a main donor of the methyl group. DNA, RNA, proteins, and low-molecular-weight compounds are substrates of methyltransferases. In mammals, DNA methyltransferase Dnmt3a de novo methylates the C5 position of cytosine residues in CpG sequences in DNA. The methylation pattern is one of the factors that determine the epigenetic regulation of gene expression. Here, interactions with the catalytic domain of Dnmt3a was for the first time studied for phosphonous and phosphonic analogs of AdoMet and S-adenosyl-L-homocysteine (AdoHcy), in which the carboxyl group was substituted for respective phosphorus-containing group. These AdoMet analogs were shown to be substrates of Dnmt3a, and the methylation efficiency was only halved as compared with that of natural AdoMet. Both phosphorus-containing analogs of AdoHcy, which is a natural methyltransferase inhibitor, showed similar inhibitory activities toward Dnmt3a and were approximately four times less active than AdoHcy. The finding that the phosphonous and phosphonic analogs are similar in activity was quite unexpected because the geometry and charge of their phosphorus-containing groups differ substantially. The phosphorus-containing analogs of AdoMet and AdoHcy are discussed as promising tools for investigation of methyltransferases.