Magic angle spinning NMR spectroscopy of thioredoxin reassemblies

Abstract

Differentially isotopically enriched 1-73((13)C,(15)N)/74-108((15)N) and 1-73((15)N)/74-108((13)C,(15)N) Escherichia coli thioredoxin reassemblies prepared by fragment complementation were investigated by high-resolution magic angle spinning solid-state NMR spectroscopy. Nearly complete resonance assignments, secondary and tertiary structure analysis are reported for 1-73((13)C,(15)N)/74-108((15)N) reassembled thioredoxin. Temperature dependence of the dipolar-assisted rotational resonance (DARR) spectra reveals the residues undergoing intermediate timescale motions at temperatures below - 15 degrees C. Analysis of the DARR intensity buildups as a function of mixing time in these reassemblies indicates that at long mixing times medium- and long-range cross-peaks do not experience dipolar truncation, suggesting that isotopic dilution is not required for gaining nontrivial distance restraints for structure calculations.

Sensitivity gains, linearity, and spectral reproducibility in nonuniformly sampled multidimensional MAS NMR spectra of high dynamic range

“…The 1–73-(U- 13 C, 15 N)/74–108-(U- 15 N) reassembled thioredoxin sample was prepared by controlled precipitation as described previously (Marulanda et al 2004, 2005; Yang et al 2007, 2008, 2009); 11 mg of 1–73(U- 13 C, 15 N)/74–108(U- 15 N) reassembled thioredoxin were packed into a 3.2 mm Varian MAS rotor and sealed with a spacer and spinner.…”

Solid-State NMR Spectroscopy of Protein Complexes

“…All spectra are recorded at 14.1 T with the MAS frequency of 10 kHz. Reproduced from Magnetic Resonance in Chemistry 2007, 45: S73–84 (22) with permission from John Wiley and Sons.…”

“…Solid-state NMR spectroscopy has emerged as one of the very few techniques that can yield atomic level structural information for these types of systems. Recently, several studies have been reported on solid-state NMR applications for analysis of protein assemblies, such as bacteriophage viruses (6), oligomeric membrane peptides and proteins (7–10), amyloid fibrils (11–17), HIV-1 capsid protein assemblies (18), microtubule-associated protein assemblies (19), as well as assemblies of soluble proteins (20–22). The major strength of solid-state NMR spectroscopy is that there is no intrinsic limitation on molecular size or solubility, and long-range order is not required.…”

“…The major strength of solid-state NMR spectroscopy is that there is no intrinsic limitation on molecular size or solubility, and long-range order is not required. In large systems where the resonance lines are narrow but spectral congestion presents a challenge, sparse (23), differential (20), and selective isotopic labeling(24, 25) enables simplification of SSNMR spectra and hence detailed atomic-resolution information can be attained (21, 22, 26, 27). Furthermore, with solid-state NMR spectroscopy residue-specific dynamics can be probed for protein complexes on multiple timescales ranging from picoseconds to many seconds (28), which fosters a deeper understanding of their biological function.…”

“…In this chapter, we present experimental protocols for sample preparation techniques, resonance assignments by MAS NMR spectroscopy, structure analysis and dynamics studies of protein complexes based on our work on three classes of protein complexes: thioredoxin reassembly, HIV-1 capsid protein assembly and microtubule/CAP-Gly assembly (18, 19, 22, 27, 28). Figure 6 illustrates representative morphologies of HIV-1 CA assemblies, microtubules (MT) and CAP-Gly/MT assemblies before and after magic angle spinning.…”