Differences between the electronic environments of reduced and oxidized Escherichia coli DsbA inferred from heteronuclear magnetic resonance spectroscopy

Abstract

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, 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 'HN, I5N, I3Ca, "CO, 'Ha, and I3Cp 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 ( 'H-'3C-'5N) and double resonance ('H-'5N or 'H-I3C) experiments. Secondary structures were established using the CSI (Chemical Shift Index) method, NOE connectivity patterns, ? T H~H U 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 Pro15 1, 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 I3Ca 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.

Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy

“…The proton-detection based solid-state chemical shifts agree well with previous solution NMR assignments (Couprie et al 1998), yielding coefficients of determination R 2 = 0.978 for 1 HN, 0.995 for 15 N, 0.994 for C′, and 0.997 for Cα (Fig. S3).…”

“…Its chemical shifts have been determined in solution state (Couprie et al 1998) and in the solid state using 13 C-detected 3D and 4D experiments (Sperling et al 2010). Here proton-detected experiments were performed on this 21 kDa protein to demonstrate the capability of achieving residue-specific assignments for relatively large proteins of mixed secondary structure, by exploiting unique Cβ 13 C chemical shift information.…”

“…S3): 1 HN for G65 by over 1 ppm; 15 N of G65 and V150 by over 1.5 ppm; C′ of M64 and G65 by over 1.0 ppm; Cα of E38, G65 by over 1.5 ppm. Interestingly, these sites fall within the regions that demonstrated significant chemical shift perturbations between oxidized and reduced DsbA: regions around the active site (residues 26–43), around His60, Gln97, and Pro151 (Couprie et al 1998). In our previous study of non-deuterated nano-crystalline DsbA, large chemical shift variations for sites including E38, M64, G65, G66 were also observed between solid and solution states (Sperling et al 2010).…”

Assignment Strategies for Large Proteins by Magic-Angle Spinning NMR: The 21-kDa Disulfide-Bond-Forming Enzyme DsbA

“…The conformational changes in the backbone of DsbA upon oxidation/reduction are minimal and confined to the vicinity of the active site; the hinge motion of the thioredoxin and cap domains relative to each other do not correlate with redox state 18,19. Solution NMR has likewise indicated changes in chemical shift for residues in the vicinity of the active site, as expected for an altered chemical environment 19,20…”

Structural and Functional Characterization of ScsC, a Periplasmic Thioredoxin-Like Protein fromSalmonella entericaSerovar Typhimurium

“…NMR data processing was completed using NMRpipe. NMR backbone assignments have previously been completed for EcDsbA (10) and were used to confirm analysis. Two control spectra were recorded for each protein ( Supplementary Fig.…”