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

José Renato   Pattaro   Júnior; Ícaro   Putinhon   Caruso; Jéssica   Maróstica   de Sá; Taniara   Suelen   Mezalira; Diego      de Souza Lima; Eduardo   Jorge   Pilau; David      Roper; Maria   Aparecida   Fernandez; Flavio Augusto      Vicente  Seixas

doi:10.2174/0929866529666220405104446

Abstract

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.

Keywords: D-alanyl, D-alanine ligase, recombinant protein, circular dichroism, differential scanning calorimetry, bacterial cell wall, Escherichia coli, heterologous expression.

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DOI https://dx.doi.org/10.2174/0929866529666220405104446	Print ISSN 0929-8665
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Protein & Peptide Letters

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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