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Production, biophysical characterization and initial crystallization studies of the N‐ and C‐terminal domains of DsbD, an essential enzyme in Neisseria meningitidis
Author(s) -
Smith Roxanne P.,
Whitten Andrew E.,
Paxman Jason J.,
Kahler Charlene M.,
Scanlon Martin J.,
Heras Begoña
Publication year - 2018
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x17017800
Subject(s) - periplasmic space , transmembrane protein , biology , cytoplasm , obligate , thioredoxin , biochemistry , enzyme , escherichia coli , botany , receptor , gene
The membrane protein DsbD is a reductase that acts as an electron hub, translocating reducing equivalents from cytoplasmic thioredoxin to a number of periplasmic substrates involved in oxidative protein folding, cytochrome c maturation and oxidative stress defence. DsbD is a multi‐domain protein consisting of a transmembrane domain (t‐DsbD) flanked by two periplasmic domains (n‐DsbD and c‐DsbD). Previous studies have shown that DsbD is required for the survival of the obligate human pathogen Neisseria meningitidis . To help understand the structural and functional aspects of N. meningitidis DsbD, the two periplasmic domains which are required for electron transfer are being studied. Here, the expression, purification and biophysical properties of n‐ Nm DsbD and c‐ Nm DsbD are described. The crystallization and crystallographic analysis of n‐ Nm DsbD and c‐ Nm DsbD are also described in both redox states, which differ only in the presence or absence of a disulfide bond but which crystallized in completely different conditions. Crystals of n‐ Nm DsbD Ox , n‐ Nm DsbD Red , c‐ Nm DsbD Ox and c‐ Nm DsbD Red diffracted to 2.3, 1.6, 2.3 and 1.7 Å resolution and belonged to space groups P 2 1 3, P 321, P 4 1 and P 12 1 1, respectively.