Energetics of MazG unfolding in correlation with its structural features.

TitleEnergetics of MazG unfolding in correlation with its structural features.
Publication TypeJournal Article
Year of Publication2009
AuthorsDrobnak, I., A. Korencic, R. Loris, I. Marianovsky, G. Glaser, A. Jamnik, G. Vesnaver, and J. Lah
JournalJ Mol Biol
Volume392
Issue1
Pagination63-74
Date Published2009 Sep 11
Type of Articleta
ISSN1089-8638
KeywordsCalorimetry, Differential Scanning, Circular Dichroism, Dimerization, Escherichia coli, Escherichia coli Proteins, Fluorometry, Hot Temperature, Models, Molecular, Protein Denaturation, Protein Folding, Protein Structure, Quaternary, Protein Structure, Tertiary, Pyrophosphatases
Abstract

MazG is a homodimeric alpha-helical protein that belongs to the superfamily of all-alpha NTP pyrophosphatases. Its function has been connected to the regulation of the toxin-antitoxin module mazEF, implicated in programmed growth arrest/cell death of Escherichia coli cells under conditions of amino acid starvation. The goal of the first detailed biophysical study of a member of the all-alpha NTP pyrophosphatase superfamily, presented here, is to improve molecular understanding of the unfolding of this type of proteins. Thermal unfolding of MazG monitored by differential scanning calorimetry, circular dichroism spectroscopy, and fluorimetry at neutral pH in the presence of a reducing agent (dithiothreitol) can be successfully described as a reversible four-state transition between a dimeric native state, two dimeric intermediate states, and a monomeric denatured state. The first intermediate state appears to have a structure similar to that of the native state while the final thermally denatured monomeric state is not fully unfolded and contains a significant fraction of residual alpha-helical structure. In the absence of dithiothreitol, disulfide cross-linking causes misfolding of MazG that appears to be responsible for the formation of multimeric aggregates. MazG is most stable at pH 7-8, while at pH <6, it exists in a molten-globule-like state. The thermodynamic parameters characterizing each step of MazG denaturation transition obtained by global fitting of the four-state model to differential scanning calorimetry, circular dichroism, and fluorimetry temperature profiles are in agreement with the observed structural characteristics of the MazG conformational states and their assumed functional role.

DOI10.1016/j.jmb.2009.05.086
Alternate JournalJ. Mol. Biol.
PubMed ID19523960