Characterization of Secondary Structure and Thermal Stability by Biophysical Methods of the D-alanyl,D-alanine Ligase B Protein from Escherichia coli

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Autor(es): dc.contributorUniversidade Estadual de Maringá (UEM)-
Autor(es): dc.contributorUniversidade Estadual Paulista (UNESP)-
Autor(es): dc.contributorUniversidade Federal do Rio de Janeiro (UFRJ)-
Autor(es): dc.contributorThe University of Warwick-
Autor(es): dc.creatorJúnior, José Renato Pattaro-
Autor(es): dc.creatorCaruso, Ícaro Putinhon-
Autor(es): dc.creatorde Sá, Jéssica Maróstica-
Autor(es): dc.creatorMezalira, Taniara Suelen-
Autor(es): dc.creatorLima, Diego de Souza-
Autor(es): dc.creatorPilau, Eduardo Jorge-
Autor(es): dc.creatorRoper, David-
Autor(es): dc.creatorFernandez, Maria Aparecida-
Autor(es): dc.creatorSeixas, Flavio Augusto Vicente-
Data de aceite: dc.date.accessioned2025-08-21T20:57:20Z-
Data de disponibilização: dc.date.available2025-08-21T20:57:20Z-
Data de envio: dc.date.issued2023-03-01-
Data de envio: dc.date.issued2023-03-01-
Data de envio: dc.date.issued2021-12-31-
Fonte completa do material: dc.identifierhttp://dx.doi.org/10.2174/0929866529666220405104446-
Fonte completa do material: dc.identifierhttp://hdl.handle.net/11449/241190-
Fonte: dc.identifier.urihttp://educapes.capes.gov.br/handle/11449/241190-
Descrição: dc.descriptionBackground: Peptidoglycan (PG) is a key structural component of the bacterial cell wall and interruption of its biosynthesis is a validated target for antimicrobials. Of the enzymes involved in PG biosynthesis, D-alanyl,D-alanine ligase B (DdlB) is responsible for the condensation of two alanines, forming D-Ala-D-Ala, which is required for subsequent extracellular transpeptidase crosslinking of the mature peptidoglycan polymer. Objective: We aimed at the biophysical characterization of recombinant Escherichia coli DdlB (EcDdlB), considering parameters of melting temperature (Tm), calorimetry and Van’t Hoff enthalpy changes of denaturation (U∆Hcal and ∆HU vH ), as well as characterization of elements of secondary structure at three different pHs. Methods: DdlB was overexpressed in E. coli BL21 and purified by affinity chromatography. Thermal stability and structural characteristics of the purified enzyme were analyzed by circular dichroism (CD), differential scanning calorimetry and fluorescence spectroscopy. Results: The stability of EcDdlB increased with proximity to its pI of 5.0, reaching the maximum at pH 5.4 with Tm and U∆HvH of 52.68 ºC and 484 kJ.mol-1, respectively. Deconvolutions of the CD spectra at 20 ºC showed a majority percentage of α-helix at pH 5.4 and 9.4, whereas for pH 7.4, an equal contribution of β-structures and α-helices was calculated. Thermal denaturation process of EcDdlB proved to be irreversible with an increase in β-structures that can contribute to the formation of protein aggregates. Conclusion: Such results will be useful for energy minimization of structural models aimed at virtual screening simulations, providing useful information in the search for drugs that inhibit peptidoglycan synthesis.-
Descrição: dc.descriptionConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)-
Descrição: dc.descriptionCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)-
Descrição: dc.descriptionDepartament of Technology Universidade Estadual de Maringá, PR-
Descrição: dc.descriptionDepartment of Physics Instituto de Biociências Letras e Ciências Exatas – Universidade Estadual Paulista “Júlio de Mesquita Filho”, SP-
Descrição: dc.descriptionNational Center for Nuclear Magnetic Resonance of Macromolecules Institute of Medical Biochemistry and National Center for Structure Biology and Bioimaging (CENABIO) Universidade Federal do Rio de Janeiro, RJ-
Descrição: dc.descriptionDepartament of Chemistry Universidade Estadual de Maringá, PR-
Descrição: dc.descriptionSchool of Life Sciences The University of Warwick-
Descrição: dc.descriptionDepartament of Biotechnology Genetics and Cell Biology Universidade Estadual de Maringá, PR-
Descrição: dc.descriptionDepartment of Physics Instituto de Biociências Letras e Ciências Exatas – Universidade Estadual Paulista “Júlio de Mesquita Filho”, SP-
Descrição: dc.descriptionCAPES: 001-
Formato: dc.format448-459-
Idioma: dc.languageen-
Relação: dc.relationProtein and Peptide Letters-
???dc.source???: dc.sourceScopus-
Palavras-chave: dc.subjectbacterial cell wall-
Palavras-chave: dc.subjectcircular dichroism-
Palavras-chave: dc.subjectD-alanine ligase-
Palavras-chave: dc.subjectD-alanyl-
Palavras-chave: dc.subjectdifferential scanning calorimetry-
Palavras-chave: dc.subjectEscherichia coli-
Palavras-chave: dc.subjectheterologous expression-
Palavras-chave: dc.subjectrecombinant protein-
Título: dc.titleCharacterization of Secondary Structure and Thermal Stability by Biophysical Methods of the D-alanyl,D-alanine Ligase B Protein from Escherichia coli-
Tipo de arquivo: dc.typelivro digital-
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