High‐Resolution Solid‐State NMR Studies on Uniformly [¹³C,¹⁵N]‐Labeled Ubiquitin

Abstract

Understanding of the effects of intermolecular interactions, molecular dynamics, and sample preparation on high-resolution magic-angle spinning NMR data is currently limited. Using the example of a uniformly [13C,15N]-labeled sample of ubiquitin, we discuss solid-state NMR methods tailored to the construction of 3D molecular structure and study the influence of solid-phase protein preparation on solid-state NMR spectra. A comparative analysis of 13C', 13Calpha, and 13Cbeta resonance frequencies suggests that 13C chemical-shift variations are most likely to occur in protein regions that exhibit an enhanced degree of molecular mobility. Our results can be refined by additional solid-state NMR techniques and serve as a reference for ongoing efforts to characterize the structure and dynamics of (membrane) proteins, protein complexes, and other biomolecules by high-resolution solid-state NMR.

Characterization of the Conjugation Pattern in Large Polysaccharide–Protein Conjugates by NMR Spectroscopy

“…14 mg of material; U‐ 2 H, Lys‐ 1 H, 13 C, 15 N ca. 5 mg of material) were packed into Bruker 3.2 mm zirconia rotors, then rehydrated until the resolution of the 13 C 1D SSNMR spectrum stopped changing 30, 34, 35, 36, 42, 43, 44. Standard 13 C‐detected two‐dimensional spectra [ 13 C‐ 13 C DARR,45, 46 15 N‐ 13 C NCA,47 15 N‐ 13 C NCO48] were acquired according to reference 49.…”

“…However, the assignment of each H ζ ‐Ν ζ couple to the corresponding H‐N of the backbone is a difficult task because of the high overlap between H ζ ‐Ν ζ signals, and the lack of connections between the two H‐N couples across the side chain. For the solid‐state NMR investigation, ANSII (either free or its MenC conjugate) was freeze‐dried, packed into the magic‐angle spinning (MAS) rotors, then rehydrated to restore the resolution 30, 34, 35, 36. The spectra collected on the pelleted ANSII‐MenC conjugate (Figure 2) are largely superimposable to those of the native and PEGylated forms of the enzyme (see Figure S2 in the Supporting Information) making possible the assessment of the preservation of the protein fold after conjugation with the MenC polysaccharide 37, 38, 39, 40.…”

Probing Structural and Motional Features of the C-Terminal Part of the Human Centrin 2/P17-XPC Microcrystalline Complex by Solid-State NMR Spectroscopy

“…The spectral resolution of solid-state NMR spectra of proteins depends on the experimental conditions and procedures of sample precipitation or crystallization. 24−27 The relationship between the crystal quality or the crystalline form of the protein and the observed line widths of the 28 and recently related to local differences in dynamics and conformation. 29 As discussed above, different experimental conditions and procedures may be used for obtaining microcrystalline samples of the C-ter HsCen2/P17-XPC complex, in particular sea-urchin-like crystals (sample A) and plate-like crystals (sample B).…”

Investigating transport proteins by solid state NMR

“…Solid state NMR is increasingly applied to isotope labelled membrane proteins in order to derive data about protein structure and dynamics. The possibility of a complete de novo structure determination purely based on MAS NMR recoupling techniques was first demonstrated for small insoluble peptides (Jaroniec et al 2004;Rienstra et al 2002) and has been extended to soluble proteins studied in the solid state (Castellani et al 2002;Seidel et al 2005;Zech et al 2005) . However, similar studies have not yet been successfully applied to membrane proteins largely due to limitations in spectral resolution.…”