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

Autor: Júnior JRP; Departament of Technology, Universidade Estadual de Maringá, Umuarama, PR, Brazil., Caruso ÍP; Department of Physics, Instituto de Biociências, Letras e Ciências Exatas - Universidade Estadual Paulista 'Júlio de Mesquita Filho', São José do Rio Preto, SP, Brazil.; National 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, Ilha do Fundão, Rio de Janeiro, RJ, Brazil., de Sá JM; Department of Physics, Instituto de Biociências, Letras e Ciências Exatas - Universidade Estadual Paulista 'Júlio de Mesquita Filho', São José do Rio Preto, SP, Brazil., Mezalira TS; Departament of Technology, Universidade Estadual de Maringá, Umuarama, PR, Brazil., de Souza Lima D; Departament of Technology, Universidade Estadual de Maringá, Umuarama, PR, Brazil., Pilau EJ; Departament of Chemistry, Universidade Estadual de Maringá, Maringá, PR, Brazil., Roper D; School of Life Sciences, The University of Warwick, Coventry, United Kingdom., Fernandez MA; Departament of Biotechnology, Genetics and Cell Biology, Universidade Estadual de Maringá, Maringá, PR, Brazil., Vicente Seixas FA; Departament of Technology, Universidade Estadual de Maringá, Umuarama, PR, Brazil.
Jazyk: angličtina
Zdroj: Protein and peptide letters [Protein Pept Lett] 2022; Vol. 29 (5), pp. 448-459.
DOI: 10.2174/0929866529666220405104446
Abstrakt: Background: 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 (T m ), calorimetry and Van't Hoff enthalpy changes of denaturation ( ΔH U cal and ΔH U 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 T m and ΔH U vH U 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.
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Databáze: MEDLINE
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