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41.
Leonardo Antnio Fernandes Anderson Albino Gomes Beatriz Gomes Guimares Maria de Lourdes Borba Magalhes Partha Ray Gustavo Felippe da Silva 《Protein science : a publication of the Protein Society》2022,31(6)
The binding of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein to the angiotensin‐converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high‐affinity and cost‐effective ACE2 mimetic ligands that disrupt this protein–protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2–5 kDa) and highly stable proteins containing solvent‐exposed alpha‐helix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha‐helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS‐CoV‐2 spike protein. The engineered proteins (h‐deface2, p‐deface2, and p‐deface2‐MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7°C), high‐affinity binding to the spike protein with apparent K d values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h‐deface2, p‐deface2, and p‐deface2‐MUT, respectively, and were used in a diagnostic assay that detected SARS‐CoV‐2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha‐helices in a constrained form for designing of high‐affinity ligands. 相似文献
42.
Oliveira MD Andrade CA Santos-Magalhães NS Coelho LC Teixeira JA Carneiro-da-Cunha MG Correia MT 《Letters in applied microbiology》2008,46(3):371-376
Aims: The aim of this work was to analyse the antimicrobial properties of a purified lectin from Eugenia uniflora L. seeds. Methods and Results: The E. uniflora lectin (EuniSL) was isolated from the seed extract and purified by ion‐exchange chromatography in DEAE‐Sephadex with a purification factor of 11·68. The purified lectin showed a single band on denaturing electrophoresis, with a molecular mass of 67 kDa. EuniSL agglutinated rabbit and human erythrocytes with a higher specificity for rabbit erythrocytes. The haemagglutination was not inhibited by the tested carbohydrates but glycoproteins exerted a strong inhibitory action. The lectin proved to be thermo resistant with the highest stability at pH 6·5 and divalent ions did not affect its activity. EuniSL demonstrated a remarkable nonselective antibacterial activity. EuniSL strongly inhibited the growth of Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella sp. with a minimum inhibitory concentration (MIC) of 1·5 μg ml?1, and moderately inhibited the growth of Bacillus subtilis, Streptococcus sp. and Escherichia coli with a MIC of 16·5 μg ml?1. Conclusions: EuniSL was found to be effective against bacteria. Significance and Impact of the Study: The strong antibacterial activity of the studied lectin indicates a high potential for clinical microbiology and therapeutic applications. 相似文献
43.
Saskia Vanderhaegen Marcus Fislage Katarzyna Domanska Wim Versées Els Pardon Vittorio Bellotti Jan Steyaert 《Protein science : a publication of the Protein Society》2013,22(10):1349-1357
To investigate early intermediates of β2‐microglobulin (β2m) amyloidogenesis, we solved the structure of β2m containing the amyloidogenic Pro32Gly mutation by X‐ray crystallography. One nanobody (Nb24) that efficiently blocks fibril elongation was used as a chaperone to co‐crystallize the Pro32Gly β2m monomer under physiological conditions. The complex of P32G β2m with Nb24 reveals a trans peptide bond at position 32 of this amyloidogenic variant, whereas Pro32 adopts the cis conformation in the wild‐type monomer, indicating that the cis to trans isomerization at Pro32 plays a critical role in the early onset of β2m amyloid formation. 相似文献
44.
Isabel C.M. Fensterseifer Mário R. Felício Eliane S.F. Alves Marlon H. Cardoso Marcelo D.T. Torres Carolina O. Matos Osmar N. Silva Timothy K. Lu Maurício V. Freire Natan C. Neves Sónia Gonçalves Luciano M. Lião Nuno C. Santos William F. Porto Cesar de la Fuente-Nunez Octavio L. Franco 《生物化学与生物物理学报:生物膜》2019,1861(7):1375-1387
Infections caused by Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa foremost among them, constitute a major worldwide health problem. Bioinformatics methodologies are being used to rationally design new antimicrobial peptides, a potential alternative for treating these infections. One of the algorithms used to develop antimicrobial peptides is the Joker, which was used to design the peptide PaDBS1R6. This study evaluates the antibacterial activities of PaDBS1R6 in vitro and in vivo, characterizes the peptide interaction to target membranes, and investigates the PaDBS1R6 structure in contact with mimetic vesicles. Moreover, we demonstrate that PaDBS1R6 exhibits selective antimicrobial activity against Gram-negative bacteria. In the presence of negatively charged and zwitterionic lipids the structural arrangement of PaDBS1R6 transits from random coil to α-helix, as characterized by circular dichroism. The tertiary structure of PaDBS1R6 was determined by NMR in zwitterionic dodecylphosphocholine (DPC) micelles. In conclusion, PaDBS1R6 is a candidate for the treatment of nosocomial infections caused by Gram-negative bacteria, as template for producing other antimicrobial agents. 相似文献
45.
46.
Sudano MJ Paschoal DM Rascado Tda S Magalhães LC Crocomo LF de Lima-Neto JF Landim-Alvarenga Fda C 《Theriogenology》2011,75(7):1211-1220
The objective was to evaluate supplementation of fetal calf serum (FCS) and phenazine ethosulfate (PES), a metabolic regulator that inhibits fatty acid synthesis, in culture media during in vitro production (IVP) of bovine embryos. Taking oocyte fertilization (n = 4,320) as Day 0, four concentrations of FCS (0, 2.5, 5, and 10%) and three periods of exposure to PES (without addition—Control; after 60 h—PES Day 2.5 of embryo culture; and after 96 h—PES Day 4) were evaluated. Increasing FCS concentration in the culture media enhanced lipid accumulation (P < 0.05), increased apoptosis in fresh (2.5%: 19.1 ± 1.8 vs 10%: 28.4 ± 2.3, P < 0.05; mean ± SEM) and vitrified (2.5%: 42.8 ± 2.7 vs 10%: 69.2 ± 3.4, P < 0.05) blastocysts, and reduced blastocoele re-expansion after vitrification (2.5%: 81.6 ± 2.5 vs 10%: 67.3 ± 3.5, P < 0.05). The addition of PES in culture media, either from Days 2.5 or 4, reduced lipid accumulation (P < 0.05) and increased blastocoele re-expansion after vitrification (Control: 72.0 ± 3.0 vs PES Day 2.5: 79.9 ± 2.8 or PES Day 4: 86.2 ± 2.4, P < 0.05). However, just the use of PES from D4 reduced apoptosis in vitrified blastocysts (Control: 52.0 ± 3.0 vs PES Day 4: 39.2 ± 2.4, P < 0.05). Independent of FCS withdrawal or PES addition to culture media, the in vivo control group had lesser lipid accumulation, a lower apoptosis rate, and greater cryotolerance (P < 0.05). The increased lipid content was moderately correlated with apoptosis in vitrified blastocysts (r = 0.64, P = 0.01). In contrast, the increased apoptosis in fresh blastocysts was strongly correlated with apoptosis in vitrified blastocysts (r = 0.94, P < 0.0001). Therefore, using only 2.5% FCS and the addition of PES from Day 4, increased the survival of IVP embryos after vitrification. Moreover, embryo quality, represented by the fresh apoptosis rate, was better than lipid content for predicting embryo survival after vitrification. 相似文献
47.
48.
Andrade RF Rocha-Neto IC Santos LB de Santana CN Diniz MV Lobão TP Goés-Neto A Pinho ST El-Hani CN 《PLoS computational biology》2011,7(5):e1001131
This paper proposes a new method to identify communities in generally weighted complex networks and apply it to phylogenetic analysis. In this case, weights correspond to the similarity indexes among protein sequences, which can be used for network construction so that the network structure can be analyzed to recover phylogenetically useful information from its properties. The analyses discussed here are mainly based on the modular character of protein similarity networks, explored through the Newman-Girvan algorithm, with the help of the neighborhood matrix . The most relevant networks are found when the network topology changes abruptly revealing distinct modules related to the sets of organisms to which the proteins belong. Sound biological information can be retrieved by the computational routines used in the network approach, without using biological assumptions other than those incorporated by BLAST. Usually, all the main bacterial phyla and, in some cases, also some bacterial classes corresponded totally (100%) or to a great extent (>70%) to the modules. We checked for internal consistency in the obtained results, and we scored close to 84% of matches for community pertinence when comparisons between the results were performed. To illustrate how to use the network-based method, we employed data for enzymes involved in the chitin metabolic pathway that are present in more than 100 organisms from an original data set containing 1,695 organisms, downloaded from GenBank on May 19, 2007. A preliminary comparison between the outcomes of the network-based method and the results of methods based on Bayesian, distance, likelihood, and parsimony criteria suggests that the former is as reliable as these commonly used methods. We conclude that the network-based method can be used as a powerful tool for retrieving modularity information from weighted networks, which is useful for phylogenetic analysis. 相似文献
49.
50.
Rita Rocha Celso M. Teixeira-Duarte João M.P. Jorge João Henrique Morais-Cabral 《Journal of structural biology》2019,205(3):34-43
RCK (regulating conductance of K+) domains are common regulatory domains that control the activity of eukaryotic and prokaryotic K+ channels and transporters. In bacteria these domains play roles in osmoregulation, regulation of turgor and membrane potential and in pH homeostasis. Whole-genome sequencing unveiled RCK gene redundancy, however the biological role of this redundancy is not well understood. In Bacillus subtilis, there are two closely related RCK domain proteins (KtrA and KtrC) that regulate the activity of the Ktr cation channels. KtrA has been well characterized but little is known about KtrC. We have characterized the structural and biochemical proprieties of KtrC and conclude that KtrC binds ATP and ADP, just like KtrA. However, in solution KtrC exist in a dynamic equilibrium between octamers and non-octameric species that is dependent on the bound ligand, with ATP destabilizing the octameric ring relative to ADP. Accordingly, KtrC-ADP crystal structures reveal closed octameric rings similar to those in KtrA, while KtrC-ATP adopts an open assembly with RCK domains forming a super-helix. In addition, both KtrC-ATP and -ADP octamers are stabilized by the signaling molecule cyclic-di-AMP, which binds to KtrC with high affinity. In contrast, c-di-AMP binds with 100-fold lower affinity to KtrA. Despite these differences we show with an E. coli complementation assay that KtrC and KtrA are interchangeable and able to form functional transporters with both KtrB and KtrD. The distinctive properties of KtrC, in particular ligand-dependent assembly/disassembly, suggest that this protein has a specific physiological role that is distinct from KtrA. 相似文献