Displaying publications 1 - 20 of 79 in total

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  1. Fulltext NetSurfP-3.0: accurate and fast prediction of protein structural features by protein language models and deep learning
    Høie MH, Kiehl EN, Petersen B, Nielsen M, Winther O, Nielsen H, et al.
    Nucleic Acids Res, 2022 Jul 05;50(W1):W510-W515.
    PMID: 35648435 DOI: 10.1093/nar/gkac439
    Recent advances in machine learning and natural language processing have made it possible to profoundly advance our ability to accurately predict protein structures and their functions. While such improvements are significantly impacting the fields of biology and biotechnology at large, such methods have the downside of high demands in terms of computing power and runtime, hampering their applicability to large datasets. Here, we present NetSurfP-3.0, a tool for predicting solvent accessibility, secondary structure, structural disorder and backbone dihedral angles for each residue of an amino acid sequence. This NetSurfP update exploits recent advances in pre-trained protein language models to drastically improve the runtime of its predecessor by two orders of magnitude, while displaying similar prediction performance. We assessed the accuracy of NetSurfP-3.0 on several independent test datasets and found it to consistently produce state-of-the-art predictions for each of its output features, with a runtime that is up to to 600 times faster than the most commonly available methods performing the same tasks. The tool is freely available as a web server with a user-friendly interface to navigate the results, as well as a standalone downloadable package.
    Matched MeSH terms: Protein Structure, Secondary
  2. Fulltext Functional Insights into Silymarin as an Antiviral Agent against Enterovirus A71 (EV-A71)
    Lalani S, Masomian M, Poh CL
    Int J Mol Sci, 2021 Aug 15;22(16).
    PMID: 34445463 DOI: 10.3390/ijms22168757
    Enterovirus A71 (EV-A71) is a major neurovirulent agent capable of causing severe hand, foot and mouth disease (HFMD) associated with neurological complications and death. Currently, no FDA-approved antiviral is available for the treatment of EV-A71 infections. The flavonoid silymarin was shown to exert virucidal effects, but the binding site on the capsid was unknown. In this study, the ligand interacting site of silymarin was determined in silico and validated in vitro. Moreover, the potential of EV-A71 to develop resistance against silymarin was further evaluated. Molecular docking of silymarin with the capsid of EV-A71 indicated that silymarin binds to viral protein 1 (VP1) of EV-A71, specifically at the GH loop of VP1. The in vitro binding of silymarin with VP1 of EV-A71 was validated using recombinant VP1 through ELISA competitive binding assay. Continuous passaging of EV-A71 in the presence of silymarin resulted in the emergence of a mutant carrying a substitution of isoleucine by threonine (I97T) at position 97 of the BC loop of EV-A71. The mutation was speculated to overcome the inhibitory effects of silymarin. This study provides functional insights into the underlying mechanism of EV-A71 inhibition by silymarin, but warrants further in vivo evaluation before being developed as a potential therapeutic agent.
    Matched MeSH terms: Protein Structure, Secondary
  3. Cysteine-rich antimicrobial peptides from plants: The future of antimicrobial therapy
    Srivastava S, Dashora K, Ameta KL, Singh NP, El-Enshasy HA, Pagano MC, et al.
    Phytother Res, 2021 Jan;35(1):256-277.
    PMID: 32940412 DOI: 10.1002/ptr.6823
    There has been a spurt in the spread of microbial resistance to antibiotics due to indiscriminate use of antimicrobial agents in human medicine, agriculture, and animal husbandry. It has been realized that conventional antibiotic therapy would be less effective in the coming decades and more emphasis should be given for the development of novel antiinfective therapies. Cysteine rich peptides (CRPs) are broad-spectrum antimicrobial agents that modulate the innate immune system of different life forms such as bacteria, protozoans, fungi, plants, insects, and animals. These are also expressed in several plant tissues in response to invasion by pathogens, and play a crucial role in the regulation of plant growth and development. The present work explores the importance of CRPs as potent antimicrobial agents, which can supplement and/or replace the conventional antibiotics. Different plant parts of diverse plant species showed the presence of antimicrobial peptides (AMPs), which had significant structural and functional diversity. The plant-derived AMPs exhibited potent activity toward a range of plant and animal pathogens, protozoans, insects, and even against cancer cells. The cysteine-rich AMPs have opened new avenues for the use of plants as biofactories for the production of antimicrobials and can be considered as promising antimicrobial drugs in biotherapeutics.
    Matched MeSH terms: Protein Structure, Secondary
  4. Mucoadhesive Low Molecular Chitosan Complexes to Protect rHuKGF from Proteolysis: In-vitro Characterization and FHs 74 Int Cell Proliferation Studies
    Tee YN, Kumar PV, Maki MAA, Elumalai M, Rahman SAKMEH, Cheah SC
    Curr Pharm Biotechnol, 2021;22(7):969-982.
    PMID: 33342408 DOI: 10.2174/1389201021666201218124450
    BACKGROUND: Recombinant Keratinocyte Growth Factor (rHuKGF) is a therapeutic protein used widely in oral mucositis after chemotherapy in various cancers, stimulating lung morphogenesis and gastrointestinal tract cell proliferation. In this research study, chitosan-rHuKGF polymeric complex was implemented to improve the stability of rHuKGF and used as rejuvenation therapy for the treatment of oral mucositis in cancer patients.

    OBJECTIVE: Complexation of rHuKGF with mucoadhesive low molecular weight chitosan to protect rHuKGF from proteolysis and investigate the effect of chitosan-rHuKGF complex on the proliferation rate of FHs 74 Int cells.

    METHODS: The interaction between chitosan and rHuKGF was studied by molecular docking. Malvern ZetaSizer Nano Zs and Fourier-Transform Infrared spectroscopy (FTIR) tests were carried out to characterize the chitosan-rHuKGF complex. In addition, SDS-PAGE was performed to investigate the interaction between chitosan-rHuKGF complex and pepsin. The effect of chitosan-rHuKGF complex on the proliferation rate of FHs 74 Int cells was studied by MTT assay.

    RESULTS: Chitosan-rHuKGF complex was formed through the hydrogen bonding proven by the docking studies. A stable chitosan-rHuKGF complex was formed at pH 4.5 and was protected from proteolysis and assessed by SDS PAGE. According to the MTT assay results, chitosan-rHuKGF complex increased the cell proliferation rate of FHs 74 Int cells.

    CONCLUSION: The developed complex improved the stability and the biological function of rHuKGF.

    Matched MeSH terms: Protein Structure, Secondary
  5. Fulltext Supramolecular Architectures of Nucleic Acid/Peptide Hybrids
    Higashi SL, Rozi N, Hanifah SA, Ikeda M
    Int J Mol Sci, 2020 Dec 12;21(24).
    PMID: 33322664 DOI: 10.3390/ijms21249458
    Supramolecular architectures that are built artificially from biomolecules, such as nucleic acids or peptides, with structural hierarchical orders ranging from the molecular to nano-scales have attracted increased attention in molecular science research fields. The engineering of nanostructures with such biomolecule-based supramolecular architectures could offer an opportunity for the development of biocompatible supramolecular (nano)materials. In this review, we highlighted a variety of supramolecular architectures that were assembled from both nucleic acids and peptides through the non-covalent interactions between them or the covalently conjugated molecular hybrids between them.
    Matched MeSH terms: Protein Structure, Secondary
  6. Structural basis for binding uronic acids by family 32 carbohydrate-binding modules
    Teh AH, Sim PF, Hisano T
    Biochem Biophys Res Commun, 2020 12 10;533(3):257-261.
    PMID: 33010888 DOI: 10.1016/j.bbrc.2020.09.064
    The alginate lyase AlyQ from Persicobacter sp. CCB-QB2 is a three-domained enzyme with a carbohydrate-binding module (CBM) from family 32. The CBM32 domain, AlyQB, binds enzymatically cleaved but not intact alginate. Co-crystallisation of AlyQB with the cleaved alginate reveals that it binds to the 4,5-unsaturated mannuronic acid of the non-reducing end. The binding pocket contains a conserved R248 that interacts with the sugar's carboxyl group, as well as an invariant W303 that stacks against the unsaturated pyranose ring. Targeting specifically the non-reducing end is more efficient than the reducing end since the latter consists of a mixture of mannuronic acid and guluronic acid. AlyQB also seems unable to bind these two saturated sugars as they contain OH groups that will clash with the pocket. Docking analysis of YeCBM32, which binds oligogalacturonic acid, shows that the stacking of the pyranose ring is shifted in order to accommodate the sugar's axial C1-OH, and its R69 is accordingly elevated to bind the sugar's carboxyl group. Unlike AlyQB, YeCBM32's binding pocket is able to accommodate both saturated and unsaturated galacturonic acid.
    Matched MeSH terms: Protein Structure, Secondary
  7. G-quadruplex structures bind to EZ-Tn5 transposase
    Cree SL, Chua EW, Crowther J, Dobson RCJ, Kennedy MA
    Biochimie, 2020 Aug 14.
    PMID: 32805304 DOI: 10.1016/j.biochi.2020.07.022
    Next generation DNA sequencing and analysis of amplicons spanning the pharmacogene CYP2D6 suggested that the Nextera transposase used for fragmenting and providing sequencing priming sites displayed a targeting bias. This manifested as dramatically lower sequencing coverage at sites in the amplicon that appeared likely to form G-quadruplex structures. Since secondary DNA structures such as G-quadruplexes are abundant in the human genome, and are known to interact with many other proteins, we further investigated these sites of low coverage. Our investigation revealed that G-quadruplex structures are formed in vitro within the CYP2D6 pharmacogene at these sites, and G-quadruplexes can interact with the hyperactive Tn5 transposase (EZ-Tn5) with high affinity. These findings indicate that secondary DNA structures such as G-quadruplexes may represent preferential transposon integration sites and provide additional evidence for the role of G-quadruplex structures in transposition or viral integration processes.
    Matched MeSH terms: Protein Structure, Secondary
  8. Haloadaptation: insights from comparative modeling studies between halotolerant and non-halotolerant dehalogenases
    Edbeib MF, Aksoy HM, Kaya Y, Wahab RA, Huyop F
    J Biomol Struct Dyn, 2020 Aug;38(12):3452-3461.
    PMID: 31422756 DOI: 10.1080/07391102.2019.1657498
    Halophiles are extremophilic microorganisms that grow optimally at high salt concentrations by producing a myriad of equally halotolerant enzymes. Structural haloadaptation of these enzymes adept to thriving under high-salt environments, though are not fully understood. Herein, the study attempts an in silico investigation to identify and comprehend the evolutionary structural adaptation of a halotolerant dehalogenase, DehHX (GenBank accession number: KR297065) of the halotolerant Pseudomonas halophila, over its non-halotolerant counterpart, DehMX1 (GenBank accession number KY129692) produced by Pseudomonas aeruginosa. GC content of the halotolerant DehHX DNA sequence was distinctively higher (58.9%) than the non-halotolerant dehalogenases (55% average GC). Its acidic residues, Asp and Glu were 8.27% and 12.06%, respectively, compared to an average 5.5% Asp and 7% Glu, in the latter; but lower contents of basic and hydrophobic residues in the DehHX. The secondary structure of DehHX interestingly revealed a lower incidence of α-helix forming regions (29%) and a higher percentage of coils (57%), compared to 49% and 29% in the non-halotolerant homologues, respectively. Simulation models showed the DehHX is stable under a highly saline environment (25% w/v) by adopting a highly negative-charged surface with a concomitant weakly interacting hydrophobic core. The study thus, established that a halotolerant dehalogenase undergoes notable evolutionary structural changes related to GC content over its non-halotolerant counterpart, in order to adapt and thrive under highly saline environments.Communicated by Ramaswamy H. Sarma.
    Matched MeSH terms: Protein Structure, Secondary
  9. Discovery and Development of Novel Anti-fungal Peptides Against Foodspoiling Fungi
    Muhialdin BJ, Algboory HL, Mohammed NK, Kadum H, Hussin ASM, Saari N, et al.
    Curr Drug Discov Technol, 2020;17(4):553-561.
    PMID: 31309892 DOI: 10.2174/1570163816666190715120038
    BACKGROUND: Despite the extensive research carried out to develop natural antifungal preservatives for food applications, there are very limited antifungal agents available to inhibit the growth of spoilage fungi in processed foods. Scope and Approach: Therefore, this review summarizes the discovery and development of antifungal peptides using lactic acid bacteria fermentation to prevent food spoilage by fungi. The focus of this review will be on the identification of antifungal peptides, potential sources, the possible modes of action and properties of peptides considered to inhibit the growth of spoilage fungi. Key Findings and Conclusions: Antifungal peptides generated by certain lactic acid bacteria strains have a high potential for applications in a broad range of foods. The mechanism of peptides antifungal activity is related to their properties such as low molecular weight, concentration and secondary structure. The antifungal peptides were proposed to be used as bio-preservatives to reduce and/or replace chemical preservatives.
    Matched MeSH terms: Protein Structure, Secondary
  10. Molecular characterization of Schistosoma mansoni tegument annexins and comparative analysis of antibody responses following parasite infection
    Leow CY, Willis C, Leow CH, Hofmann A, Jones M
    Mol Biochem Parasitol, 2019 12;234:111231.
    PMID: 31628972 DOI: 10.1016/j.molbiopara.2019.111231
    Schistosomes are parasitic blood flukes that infect approximately 250 million people worldwide. The disease known as schistosomiasis, is the second most significant tropical parasitic disease after malaria. Praziquantel is the only effective drug currently licensed for schistosomiasis and there are concerns about resistance to the drug. There has been much effort to develop vaccines against schistosomiasis to produce long-term protection in endemic regions. Surface-associated proteins, and in particular, those expressed in the body wall, or tegument, have been proposed as potential vaccine targets. Of these, annexins are thought to be of integral importance for the stability of this apical membrane system. Here, we present the structural and immunobiochemical characterization of four homologous annexins namely annexin B30, annexin B5a, annexin B7a and annexin B5b from S. mansoni. Bioinformatics analysis showed that there was no signal peptide predicted for any annexin in this study. Further analysis showed that each of all four annexin protein possesses a primary structure consisting of a short but variable N-terminal region and a long C-terminal core containing four homologous annexin repeats (I-IV), which contain five alpha-helices. The life cycle expression profile of each annexin was assessed using quantitative PCR. The results showed that the overall transcript levels of the each of four homologous annexins were relatively low in the egg stage, but increased gradually after the transition of cercariae (the invasive schistosome larvae) to schistosomula (the post-invasive larvae). Circular dichroism (CD) demonstrated that rAnnexin B30, rAnnexin B5a and rAnnexin 7a were folded, showing a secondary structure content rich in alpha-helices. The membrane binding affinity was enhanced when rAnnexin B30, rAnnexin B5a and rAnnexin 7a was incubated in the presence of Ca2+. All annexin members evaluated in this study were immunolocalized to the tegument, with immunoreactivity also occurring in cells and in muscle of adult parasites. All four recombinant annexins were immunoreactive and they were recognized by the sera of mice infected with S. mansoni. In conclusion, the overall results present the molecular characterization of annexin B30, annexin B5a, annexin B7a and annexin B5b from S. mansoni in host-parasite interactions and strongly suggest that the molecules could be useful candidates for vaccine or diagnostic development.
    Matched MeSH terms: Protein Structure, Secondary
  11. Investigation on Secondary Structure Alterations of Protein Drugs as an Indicator of Their Biological Activity Upon Thermal Exposure
    Zeeshan F, Tabbassum M, Kesharwani P
    Protein J, 2019 10;38(5):551-564.
    PMID: 31054037 DOI: 10.1007/s10930-019-09837-4
    Protein drugs are important therapeutic agents however; they may degrade during formulation processing. The objective of this study was to investigate the correlation between secondary structure alterations and the retentions of biological activity of protein upon the application of thermal stress. Catalase, horseradish peroxidase and α- chymotrypsin were employed as model proteins. Each protein was heated in a solid and solution state at a temperature of 70 °C for 1 h. Attenuated total reflectance Fourier transform infrared spectroscopy, size-exclusion chromatography and biological activity assay were performed. Results showed that heat-exposure of protein solids at 70 °C caused minimum changes in secondary structure and biological activity was almost retained. However, thermal exposure of protein aqueous solution induced significant changes in the secondary structure indicated by area overlap values and caused considerable reduction in the biological activity. The changes in secondary structures were found to be in full alignment with the loss of biological activity for both protein solids as well as aqueous solutions. Catalase lost entire biological activity upon heating in the solution state. In conclusion, the findings of the present study indicate a direct correlation between protein secondary structure alterations and the retention of biological activity which can be taken into account during the development and delivery of protein drugs formulations.
    Matched MeSH terms: Protein Structure, Secondary
  12. Fulltext In silico evidence of de novo interactions between ribosomal and Epstein - Barr virus proteins
    Sim EU, Talwar SP
    BMC Mol Cell Biol, 2019 08 15;20(1):34.
    PMID: 31416416 DOI: 10.1186/s12860-019-0219-y
    BACKGROUND: Association of Epstein-Barr virus (EBV) encoded latent gene products with host ribosomal proteins (RPs) has not been fully explored, despite their involvement in the aetiology of several human cancers. To gain an insight into their plausible interactions, we employed a computational approach that encompasses structural alignment, gene ontology analysis, pathway analysis, and molecular docking.

    RESULTS: In this study, the alignment analysis based on structural similarity allows the prediction of 48 potential interactions between 27 human RPs and the EBV proteins EBNA1, LMP1, LMP2A, and LMP2B. Gene ontology analysis of the putative protein-protein interactions (PPIs) reveals their probable involvement in RNA binding, ribosome biogenesis, metabolic and biosynthetic processes, and gene regulation. Pathway analysis shows their possible participation in viral infection strategies (viral translation), as well as oncogenesis (Wnt and EGFR signalling pathways). Finally, our molecular docking assay predicts the functional interactions of EBNA1 with four RPs individually: EBNA1-eS10, EBNA1-eS25, EBNA1-uL10 and EBNA1-uL11.

    CONCLUSION: These interactions have never been revealed previously via either experimental or in silico approach. We envisage that the calculated interactions between the ribosomal and EBV proteins herein would provide a hypothetical model for future experimental studies on the functional relationship between ribosomal proteins and EBV infection.

    Matched MeSH terms: Protein Structure, Secondary
  13. Effects of formulation development methods on the stability of model protein pharmaceuticals embedded in solid lipid matrices
    Tabbassum M, Zeeshan F
    Pharm Dev Technol, 2019 Jun;24(5):649-662.
    PMID: 30474456 DOI: 10.1080/10837450.2018.1551902
    This study was conducted to investigate the influence of formulation development methods on the stability (secondary structure, aggregation, and biological activity) of protein drugs embedded in lipid matrices. Catalase, horseradish peroxidase, and α-chymotrypsin were employed as model proteins, while Precirol® AT05 (glyceryl palmitostearate) was used as lipid matrix. Protein-loaded lipid matrices were prepared using melting and mixing and wet granulation methods. Attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy, size exclusion chromatography (SEC) and biological activity analyses were performed. ATR FT-IR analysis indicated significant interference of the lipid with the protein amide-I band, which was eliminated using spectral subtraction. Wet granulation method induced more changes in protein secondary structure compared to melting and mixing method. SEC analysis gave evidence of protein aggregation for catalase upon adopting the wet granulation method. The biological activity of catalase was found to reduce significantly than other two proteins upon using wet granulation method, which might be ascribed to both secondary structure alterations and the formation of aggregates. Horseradish peroxidase and α-chymotrypsin did not form any soluble aggregates. In conclusion, melting and mixing method emerged as a better incorporation method compared to wet granulation because of better stability shown by the formulated proteins.
    Matched MeSH terms: Protein Structure, Secondary
  14. NetSurfP-2.0: Improved prediction of protein structural features by integrated deep learning
    Klausen MS, Jespersen MC, Nielsen H, Jensen KK, Jurtz VI, Sønderby CK, et al.
    Proteins, 2019 06;87(6):520-527.
    PMID: 30785653 DOI: 10.1002/prot.25674
    The ability to predict local structural features of a protein from the primary sequence is of paramount importance for unraveling its function in absence of experimental structural information. Two main factors affect the utility of potential prediction tools: their accuracy must enable extraction of reliable structural information on the proteins of interest, and their runtime must be low to keep pace with sequencing data being generated at a constantly increasing speed. Here, we present NetSurfP-2.0, a novel tool that can predict the most important local structural features with unprecedented accuracy and runtime. NetSurfP-2.0 is sequence-based and uses an architecture composed of convolutional and long short-term memory neural networks trained on solved protein structures. Using a single integrated model, NetSurfP-2.0 predicts solvent accessibility, secondary structure, structural disorder, and backbone dihedral angles for each residue of the input sequences. We assessed the accuracy of NetSurfP-2.0 on several independent test datasets and found it to consistently produce state-of-the-art predictions for each of its output features. We observe a correlation of 80% between predictions and experimental data for solvent accessibility, and a precision of 85% on secondary structure 3-class predictions. In addition to improved accuracy, the processing time has been optimized to allow predicting more than 1000 proteins in less than 2 hours, and complete proteomes in less than 1 day.
    Matched MeSH terms: Protein Structure, Secondary
  15. Unravelling protein -organic solvent interaction of organic solvent tolerant elastase from Pseudomonas aeruginosa strain K crystal structure
    Said ZSAM, Arifi FAM, Salleh AB, Rahman RNZRA, Leow ATC, Latip W, et al.
    Int J Biol Macromol, 2019 Apr 15;127:575-584.
    PMID: 30658145 DOI: 10.1016/j.ijbiomac.2019.01.056
    The utilization of organic solvents as reaction media for enzymatic reactions provides numerous industrially attractive advantages. However, an adaptation of enzyme towards organic solvent is unpredictable and not fully understood because of limited information on the organic solvent tolerant enzymes. To understand how the enzyme can adapt to the organic solvent environment, structural and computational approaches were employed. A recombinant elastase from Pseudomonas aeruginosa strain K was an organic solvent tolerant zinc metalloprotease was successfully crystallized and diffracted up to 1.39 Å. Crystal structure of elastase from strain K showed the typical, canonical alpha-beta hydrolase fold consisting of 10-helices (118 residues), 10- β-strands (38 residues) and 142 residues were formed other secondary structure such as loop and coil to whole structure. The elastase from Pseusomonas aeruginosa strain K possess His-140, His-144 and Glu-164 served as a ligand for zinc ion. The conserved catalytic triad was composed of Glu-141, Tyr-155 and His-223. Three-dimensional structure features such as calcium-binding and presence of disulphide-bridge contribute to the stabilizing the elastase structure. Molecular dynamic (MD) simulation of elastase revealed that, amino acid residues located at the surface area and disulphide bridge in Cys-30 to Cys-58 were responsible for enzyme stability in organic solvents.
    Matched MeSH terms: Protein Structure, Secondary
  16. Conventional versus microwave assisted synthesis, molecular docking and enzyme inhibitory activities of new 3,4,5-trisubstituted-1,2,4-triazole analogues
    Virk NA, Rehman A, Abbasi MA, Siddiqui SZ, Rashid U, Iqbal J, et al.
    Pak J Pharm Sci, 2018 Jul;31(4(Supplementary)):1501-1510.
    PMID: 30058542
    N-(Substituted)-5-(1-(4-methoxyphenylsulfonyl)piperidin-4-yl)-4H-1,2,4-triazol-3-ylthio) acetamide were synthesized by following conventional as well as microwave assisted protocol through five consecutive steps under the impact of various reaction conditions to control the reaction time and the yield of product. Starting from 4-methoxybenzenesulfonyl chloride and ethyl isonipecotate, product 3 was obtained which was converted into product 4 by treating with hydrazine hydrate. In step 3, the product 4 was refluxed with methyl isothiocyanate and KOH to yield compound 5 which was finally treated with variety of N-substituted acetamides to yield an array of different new compounds (8a-k). These synthesized compounds were evaluated for their inhibition potential against bovine carbonic anhydrase (bCA-II), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. Compound 8g demonstrated good activity against bCA-II, AChE and BChE with IC50 values of 8.69 ± 0.38 μM, 11.87±0.19 μM and 26.01±0.55 μM respectively. SAR studies assisted with molecular docking were carried out to explore the mode of binding of the compounds against the studied enzymes.
    Matched MeSH terms: Protein Structure, Secondary
  17. Structure Prediction of a Novel Exo-β-1,3-Glucanase: Insights into the Cold Adaptation of Psychrophilic Yeast Glaciozyma antarctica PI12
    Mohammadi S, Parvizpour S, Razmara J, Abu Bakar FD, Illias RM, Mahadi NM, et al.
    Interdiscip Sci, 2018 Mar;10(1):157-168.
    PMID: 27475956 DOI: 10.1007/s12539-016-0180-9
    We report a detailed structural analysis of the psychrophilic exo-β-1,3-glucanase (GaExg55) from Glaciozyma antarctica PI12. This study elucidates the structural basis of exo-1,3-β-1,3-glucanase from this psychrophilic yeast. The structural prediction of GaExg55 remains a challenge because of its low sequence identity (37 %). A 3D model was constructed for GaExg55. Threading approach was employed to determine a suitable template and generate optimal target-template alignment for establishing the model using MODELLER9v15. The primary sequence analysis of GaExg55 with other mesophilic exo-1,3-β-glucanases indicated that an increased flexibility conferred to the enzyme by a set of amino acids substitutions in the surface and loop regions of GaExg55, thereby facilitating its structure to cold adaptation. A comparison of GaExg55 with other mesophilic exo-β-1,3-glucanases proposed that the catalytic activity and structural flexibility at cold environment were attained through a reduced amount of hydrogen bonds and salt bridges, as well as an increased exposure of the hydrophobic side chains to the solvent. A molecular dynamics simulation was also performed using GROMACS software to evaluate the stability of the GaExg55 structure at varying low temperatures. The simulation result confirmed the above findings for cold adaptation of the psychrophilic GaExg55. Furthermore, the structural analysis of GaExg55 with large catalytic cleft and wide active site pocket confirmed the high activity of GaExg55 to hydrolyze polysaccharide substrates.
    Matched MeSH terms: Protein Structure, Secondary
  18. Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectroscopy as an Analytical Method to Investigate the Secondary Structure of a Model Protein Embedded in Solid Lipid Matrices
    Zeeshan F, Tabbassum M, Jorgensen L, Medlicott NJ
    Appl Spectrosc, 2018 Feb;72(2):268-279.
    PMID: 29022355 DOI: 10.1177/0003702817739908
    Protein drugs may encounter conformational perturbations during the formulation processing of lipid-based solid dosage forms. In aqueous protein solutions, attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy can investigate these conformational changes following the subtraction of spectral interference of solvent with protein amide I bands. However, in solid dosage forms, the possible spectral contribution of lipid carriers to protein amide I band may be an obstacle to determine conformational alterations. The objective of this study was to develop an ATR FT-IR spectroscopic method for the analysis of protein secondary structure embedded in solid lipid matrices. Bovine serum albumin (BSA) was chosen as a model protein, while Precirol AT05 (glycerol palmitostearate, melting point 58 ℃) was employed as the model lipid matrix. Bovine serum albumin was incorporated into lipid using physical mixing, melting and mixing, or wet granulation mixing methods. Attenuated total reflection FT-IR spectroscopy and size exclusion chromatography (SEC) were performed for the analysis of BSA secondary structure and its dissolution in aqueous media, respectively. The results showed significant interference of Precirol ATO5 with BSA amide I band which was subtracted up to 90% w/w lipid content to analyze BSA secondary structure. In addition, ATR FT-IR spectroscopy also detected thermally denatured BSA solid alone and in the presence of lipid matrix indicating its suitability for the detection of denatured protein solids in lipid matrices. Despite being in the solid state, conformational changes occurred to BSA upon incorporation into solid lipid matrices. However, the extent of these conformational alterations was found to be dependent on the mixing method employed as indicated by area overlap calculations. For instance, the melting and mixing method imparted negligible effect on BSA secondary structure, whereas the wet granulation mixing method promoted more changes. Size exclusion chromatography analysis depicted the complete dissolution of BSA in the aqueous media employed in the wet granulation method. In conclusion, an ATR FT-IR spectroscopic method was successfully developed to investigate BSA secondary structure in solid lipid matrices following the subtraction of lipid spectral interference. The ATR FT-IR spectroscopy could further be applied to investigate the secondary structure perturbations of therapeutic proteins during their formulation development.
    Matched MeSH terms: Protein Structure, Secondary/physiology*
  19. Investigation on Secondary Structure Perturbations of Proteins Embedded in Solid Lipid Matrices as a Novel Indicator of their Biological Activity upon In Vitro Release
    Zeeshan F, Tabbassum M, Jorgensen L, Medlicott NJ
    AAPS PharmSciTech, 2018 Feb;19(2):769-782.
    PMID: 29134579 DOI: 10.1208/s12249-017-0883-1
    Protein biologics are prone to conformational changes during formulation development. Limited methods are available for conformational analysis of proteins in solid state and in the presences of formulation excipients. The aim of this study was to investigate the secondary structures of proteins encased in solid lipid matrices as a novel indicator of their stability upon in vitro release. Model proteins namely catalase and lysozyme were incorporated into lipid namely Precirol® AT05 (glycerol palmitostearate, melting point 58°C) at 30% w/w loading using melting and mixing and wet granulation methods. Attenuated total reflectance (ATR-FTIR) spectroscopy, size-exclusion chromatography (SEC) and biological activity analyses were performed. The information about secondary structure was acquired using second derivative analysis of amide-I band (1600-1700 cm-1). ATR analysis demonstrated interference of lipid spectrum with protein amide-I band which was subsequently subtracted to allow the analysis of the secondary structure. ATR spectra amide-I bands showed shifts peak band positions compared to native protein for matrices prepared using wet granulation. SEC analysis gave evidence of protein aggregation for catalase which was increased using wet granulation. The biological activity of catalase was statistically different from that of control and was affected by the incorporation method and was found to be in alignment with ATR spectral changes and extent of aggregation. In conclusion, ATR spectroscopy could analyze protein secondary structure in lipid matrices provided lipid interference was minimized. The ATR spectral changes and formation of aggregates can indicate the loss in biological activity of protein released from solid lipid matrices.
    Matched MeSH terms: Protein Structure, Secondary*
  20. Biophysical and computational characterization of vandetanib-lysozyme interaction
    Kabir MZ, Hamzah NAB, Ghani H, Mohamad SB, Alias Z, Tayyab S
    Spectrochim Acta A Mol Biomol Spectrosc, 2018 Jan 15;189:485-494.
    PMID: 28843881 DOI: 10.1016/j.saa.2017.08.051
    Interaction of an anticancer drug, vandetanib (VDB) with a ligand transporter, lysozyme (LYZ) was explored using multispectroscopic techniques, such as fluorescence, absorption and circular dichroism along with computational analysis. Fluorescence data and absorption results confirmed VDB-LYZ complexation. VDB-induced quenching was characterized as static quenching based on inverse correlation of KSV with temperature as well as kq values. The complex was characterized by the weak binding constant (Ka=4.96-3.14×103M-1). Thermodynamic data (ΔS=+12.82Jmol-1K-1; ΔH=-16.73kJmol-1) of VDB-LYZ interaction revealed participation of hydrophobic and van der Waals forces along with hydrogen bonds in VDB-LYZ complexation. Microenvironmental perturbations around tryptophan and tyrosine residues as well as secondary and tertiary structural alterations in LYZ upon addition of VDB were evident from the 3-D fluorescence, far- and near-UV CD spectral analyses, respectively. Interestingly, addition of VDB to LYZ significantly increased protein's thermostability. Molecular docking results suggested the location of VDB binding site near the LYZ active site while molecular dynamics simulation results suggested stability of VDB-LYZ complex. Presence of Mg2+, Ba2+ and Zn2+ was found to interfere with VDB-LYZ interaction.
    Matched MeSH terms: Protein Structure, Secondary
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