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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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Júnior, José Renato Pattaro, Caruso, Ícaro Putinhon, de Sá, Jéssica Maróstica, Mezalira, Taniara Suelen, de Souza Lima, Diego, Pilau, Eduardo Jorge, Roper, David I., Fernandez, Maria Aparecida and Vicente Seixas, Flavio Augusto (2022) Characterization of secondary structure and thermal stability by biophysical methods of the D-alanyl,D-alanine ligase B protein from Escherichia coli. Protein & Peptide Letters, 29 (5). pp. 448-459. doi:10.2174/0929866529666220405104446 ISSN 0929-8665.
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WRAP-Biophysical-characterization-recombinant-Escherichia-coli-D-alanyl-D-alanine-ligase-B-2022.pdf - Accepted Version - Requires a PDF viewer. Download (1219Kb) | Preview |
Official URL: https://doi.org/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. Objectives: We aimed the biophysical characterization of recombinant Escherichia coli DdlB (EcDdlB), regarding parameters of melting temperature (Tm), calorimetry and van’t Hoff enthalpy changes of denaturation ( and ), 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 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. Conclutions: 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.
Item Type: | Journal Article | ||||||||||||||||||
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Subjects: | Q Science > QC Physics Q Science > QD Chemistry Q Science > QR Microbiology T Technology > TP Chemical technology |
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Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||||||||||||||||
SWORD Depositor: | Library Publications Router | ||||||||||||||||||
Library of Congress Subject Headings (LCSH): | Peptidoglycans -- Synthesis, Bacterial cell walls, Ligases, Recombinant proteins, Circular dichroism, Calorimetry | ||||||||||||||||||
Journal or Publication Title: | Protein & Peptide Letters | ||||||||||||||||||
Publisher: | Bentham Science Publishers Ltd. | ||||||||||||||||||
ISSN: | 0929-8665 | ||||||||||||||||||
Official Date: | 19 May 2022 | ||||||||||||||||||
Dates: |
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Volume: | 29 | ||||||||||||||||||
Number: | 5 | ||||||||||||||||||
Page Range: | pp. 448-459 | ||||||||||||||||||
DOI: | 10.2174/0929866529666220405104446 | ||||||||||||||||||
Status: | Peer Reviewed | ||||||||||||||||||
Publication Status: | Published | ||||||||||||||||||
Reuse Statement (publisher, data, author rights): | “The published manuscript is available at EurekaSelect via http://www.eurekaselect.com/10.2174/0929866529666220405104446 | ||||||||||||||||||
Access rights to Published version: | Restricted or Subscription Access | ||||||||||||||||||
Date of first compliant deposit: | 22 July 2022 | ||||||||||||||||||
Date of first compliant Open Access: | 19 May 2023 | ||||||||||||||||||
RIOXX Funder/Project Grant: |
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