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

Abstract

Summary We present strategies for chemical shift assignments of large proteins by magic-angle spinning (MAS) solid-state NMR (SSNMR), using the 21-kDa disulfide bond forming enzyme DsbA as a prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to ~17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy and 3D and 4D backbone correlation experiments yield highly confident assignments for more than 90% of the backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths under 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, TEDOR experiments enhanced sensitivity for Pro and Gly residues, 2-13C-glycerol labeling clarified Val, Ile and Leu assignments, IPAP correlation spectra enabled interpretation of otherwise crowded Glx/Asx sidechain regions, and 3D NCACX experiments on 2-13C-glycerol samples provided unique sets of aromatic (Phe, Tyr, Trp) correlations. Together with high sensitivity CANCOCA 4D and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution 2D 13C-13C spectrum.

BSH-CP based 3D solid-state NMR experiments for protein resonance assignment

“…The sequential resonance assignment is the basis of all further ssNMR spectroscopic studies (Pauli et al 2001;Schuetz et al 2010;Shi et al 2009;Sperling et al 2010). An important step was the development of efficient 15 N-13 C SPECIFIC-CP transfer which is widely used today (Baldus et al 1998).…”

Properties of the DREAM scheme and its optimization for application to proteins

“…The resonance assignment of proteins with more than 200 residues is now feasible using uniformly labeled samples, with the 33 kDa C-terminal domain of the yeast prion Ure2p (285 residues) presently being the largest system with extensive de novo assignments in the solid state . Other large systems with extensive resonance assignments include HET-s(1-227) (Schuetz et al 2010) and DsbA (223 residues) (Sperling et al 2010;Shi et al 2010). An important limitation is the need to acquire data showing a sufficiently high signal-to-noise ratio of the relevant cross peaks for each of the 3D spectra required for the assignment process (Schuetz et al 2010;Habenstein et al 2011).…”

Advanced solid-state NMR techniques for characterization of membrane protein structure and dynamics: Application to Anabaena Sensory Rhodopsin

“…This method creates proteins which are alternately labeled with 13 C patterns which do not contain adjacent 13 C labels in most amino acids [29]. This improves the resolution of individual resonances due to the suppression of many one-bond J-couplings, simplifies the overall spectra due to the reduction of the number of resonances, and specifically facilitates the assignment of the aromatic side chains [30,31]. These benefits were all observed in ASR, and experiments preformed on samples grown on alternately labeled glycerols allowed us to extend the assignments to a total of 206 of the 229 residues [32].…”