Solid-State NMR Study of the Charge-Transfer Complex between Ubiquinone-8 and Disulfide Bond Generating Membrane Protein DsbB

Abstract

Ubiquinone (Coenzyme Q) plays an important role in the mitochondrial respiratory chain and also acts as an antioxidant in its reduced form, protecting cellular membranes from peroxidation. De novo disulfide bond generation in the E. coli periplasm involves a transient complex consisting of DsbA, DsbB and ubiquinone (UQ). It is hypothesized that a charge-transfer complex intermediate is formed between the UQ ring and the DsbB-C44 thiolate during the reoxidation of DsbA, which gives a distinctive ∼500 nm absorbance band. No enzymological precedent exists for an UQ-protein thiolate charge transfer complex, and definitive evidence of this unique reaction pathway for DsbB has not been fully demonstrated. In order to study the UQ-8-DsbB complex in the presence of native lipids, we have prepared isotopically labeled samples of precipitated DsbB (WT and C41S) with endogenous UQ-8 and lipids, and we have applied advanced multidimensional solid-state NMR methods. Double-quantum filter and dipolar dephasing experiments facilitated assignments of UQ isoprenoid chain resonances not previously observed and headgroup sites important for the characterization of the UQ redox states: methyls (∼ 20 ppm), methoxys (∼ 60 ppm), olefin carbons (120∼140 ppm) and carbonyls (150∼160 ppm). Upon increasing the DsbB(C41S) pH from 5.5 to 8.0, we observed a 10.8 ppm upfield shift for the UQ C1 and C4 carbonyls indicating an increase of electron density on the carbonyls. This observation is consistent with the deprotonation of the DsbB-C44 thiolate at pH 8.0 and provides direct evidence of the charge transfer complex formation. A similar trend was noted for the UQ chemical shifts of the DsbA(C33S)-DsbB(WT) heterodimer, confirming that the charge-transfer complex is unperturbed by the DsbB(C41S) mutant used to mimic the intermediate state of the disulfide bond generating reaction pathway.

NMR structures of membrane proteins in phospholipid bilayers

“…An important characterization of the temperature effects on the spectral quality of membrane proteins in lipid bilayers, especially in the presence of endogenous lipids, has also been made using a DsbB sample (De Angelis & Opella, 2007; Tang et al, 2011b). Notably, this study aimed to capture the highest-resolution spectra of a membrane protein, which often occurs when the lipids are in the cooler gel phase.…”

High-resolution membrane protein structure by joint calculations with solid-state NMR and X-ray experimental data

“…Many magic-angle-spinning (MAS) SSNMR techniques have been developed to elucidate both structure and dynamics of membrane proteins (McDermott 2009). SSNMR does not rely upon crystallized samples; membrane protein samples, prepared by removing excess detergent, retain native lipids and hydrophobic cofactors (Tang et al 2011), enabling high-resolution despite a lack of long range order. For soluble proteins, joint calculations of X-ray and solution NMR data have already produced structures of commensurate quality to those structures produced from either method alone (Chen et al 2000; Koharudin et al 2009; Liu et al 2009; Matei et al 2008; Schwieters et al 2007; Shaanan et al 1992).…”

“…By combining the strengths of X-ray crystallography for framing the protein fold and SSNMR for the ability to define the local structure at atomic-resolution, this protocol of joint structure determination can address the challenges in solving structures of membrane proteins to atomic resolution. SSNMR can also provide additional information on the cofactors in the active site of membrane proteins (Tang et al 2011). Taking advantage of versatile sample conditions in membrane proteins, SSNMR can potentially be used to examine various mutants representing the different stages in the electron transfer pathway of disulfide bond formation.…”

A rapid and robust method for selective isotope labeling of proteins

“…As we have previously shown, the overall spectral intensity improves at lower temperature, but the relative peak intensities among different spin systems does not change; this effect is due to a combination of enhanced Boltzmann polarization, improved probe performance, and slight increases in the CP enhancement as the temperature is lowered from −20 C to −50 C[22]. More recently, we improved instrumentation for collecting data closer to the phase transition and determined that membrane protein spectral resolution is superior at −20 C [24]. The relative intensity among spin systems does not change over this temperature range.…”