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Differences between the electronic environments of reduced and oxidized Escherichia coli DsbA inferred from heteronuclear magnetic resonance spectroscopy
Author(s) -
Couprie Joel,
Remerowski M. Lyndsay,
Bailleul Arnaud,
Courçon Marie,
Gilles Nicolas,
Quéméneur Eric,
Jamin Nadege
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560071003
Subject(s) - dsba , chemistry , chemical shift , active site , periplasmic space , heteronuclear molecule , nuclear magnetic resonance spectroscopy , crystallography , stereochemistry , escherichia coli , organic chemistry , biochemistry , gene , enzyme
DsbA is the strongest protein disulfide oxidant yet known and is involved in catalyzing protein folding in the bacterial periplasm. Its strong oxidizing power has been attributed to the lowered pK a of its reactive active site cysteine and to the difference in thermodynamic stability between the oxidized and the reduced form. However, no structural data are available for the reduced state. Therefore, an NMR study of DsbA in its two redox states was undertaken. We report here the backbone 1 H N , 15 N, 13 C α , 13 CO, 1 H α , and 13 C ß NMR assignments for both oxidized and reduced Escherichia coli DsbA (189 residues). Ninety‐nine percent of the frequencies were assigned using a combination of triple ( 1 H‐ 13 C‐ 15 N) and double resonance ( 1 H‐ 15 N or 1 H‐ 13 C) experiments. Secondary structures were established using the CSI (Chemical Shift Index) method, NOE connectivity patterns, 3 J H N H α and amide proton exchange data. Comparison of chemical shifts for both forms reveals four regions of the protein, which undergo some changes in the electronic environment. These regions are around the active site (residues 26 to 43), around His60 and Pro151, and also around Gln97. Both the number and the amplitude of observed chemical shift variations are more substantial in DsbA than in E. coli thioredoxin. Large 13 Cα chemical shift variations for residues of the active site and residues Phe28, Tyr34, Phe36, Ile42, Ser43, and Lys98 suggest that the backbone conformation of these residues is affected upon reduction.