Chemical shift assignment of the transmembrane helices of DsbB, a 20‐kDa integral membrane enzyme, by 3D magic‐angle spinning NMR spectroscopy

Abstract

The Escherichia coli inner membrane enzyme DsbB catalyzes disulfide bond formation in periplasmic proteins, by transferring electrons to ubiquinone from DsbA, which in turn directly oxidizes cysteines in substrate proteins. We have previously shown that DsbB can be prepared in a state that gives highly resolved magic-angle spinning (MAS) NMR spectra. Here we report sequential 13 C and 15 N chemical shift assignments for the majority of the residues in the transmembrane helices, achieved by threedimensional (3D) correlation experiments on a uniformly 13 C, 15 N-labeled sample at 750-MHz 1 H frequency. We also present a four-dimensional (4D) correlation spectrum, which confirms assignments in some highly congested regions of the 3D spectra. Overall, our results show the potential to assign larger membrane proteins using 3D and 4D correlation experiments and form the basis of further structural and dynamical studies of DsbB by MAS NMR.

Insights into the production and characterization of electrospun fibers from regenerated silk fibroin

“…Solid-state NMR (SSNMR) can be applied under a variety of conditions not accessible to X-ray crystallography and solution NMR [60], and several protein structures have been successfully determined in the solid state by magic-angle-spinning (MAS) NMR [61][62][63].…”

“…However, a single broad peak is observed in this region of the spectrum of our regenerated SF after freeze-drying (L). This coalescence of signals typically occurs in inter-changeable and/or disordered conformations when NMR is performed in solution, and it can be associated with static disorder in solid-state NMR [62]. Therefore, fibroin molecules in lyophilized powder (L) would adopt random coil configurations or a mixture of coiled-structures, rather than the ordered poly-p-turn attributed to Silk I [64].…”

Mutant Protein A30P α-Synuclein Adopts Wild-type Fibril Structure, Despite Slower Fibrillation Kinetics

“…Once these cross-polarization transfers have occurred, the resulting polarization on the 13 CЈ nucleus is transferred through space to the side chain 13 C nuclei using the DARR mixing scheme. Using these three multidimensional experiments, we conducted chemical shift assignments of sequential residues using well established techniques for making de novo site-specific chemical shift assignments (22,(43)(44)(45)(46)(47)(48)(49). Fig.…”

Fuzzy complexes of myelin basic protein: NMR spectroscopic investigations of a polymorphic organizational linker of the central nervous systemThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

“…Peripheral membrane-associated MBP represents a very different system from that of the microcrystalline (Igumenova et al 2004), amyloid fibril (Siemer et al 2006), or transmembrane protein (Frericks et al 2006;Li et al 2008;Shi et al 2009) samples for which ssNMR spectroscopic residue-specific assignments have been achieved. Due to the fast molecular motions inherent in MBP, which averaged the dipolar interactions, traditional ssNMR techniques were not applicable and necessitated the development of a complementary suite of NMR experiments to obtain sequence-specific assignments (Zhong et al 2007).…”