13Cα decoupling during direct observation of carbonyl resonances in solution NMR of isotopically enriched proteins

Abstract

Direct detection of 13C can be advantageous when studying uniformly enriched proteins, in particular for paramagnetic proteins or when hydrogen exchange with solvent is fast. A scheme recently introduced for long-observation-window band-selective homonuclear decoupling in solid state NMR, LOW-BASHD (Struppe et al., J. Magn. Reson. 236, 89–94, 2013) is shown to be effective for 13Cα decoupling during direct 13C′ observation in solution NMR experiments too. For this purpose, adjustment of the decoupling pulse parameters and delays is demonstrated to be important for increasing spectral resolution, to reduce three-spin effects, and to decrease the intensity of decoupling side-bands. LOW-BASHD then yields 13C′ line widths comparable to those obtained with the popular IPAP method, while enhancing sensitivity by ca 35%. As a practical application of LOW-BASHD decoupling, requiring quantitative intensity measurement over a wide dynamic range, the impact of lipid binding on the 13C′-detected NCO spectrum of the intrinsically disordered protein α-synuclein is compared with that on the 1H-detected 1H-15N HSQC spectrum. Results confirm that synuclein’s “dark state” behavior is not caused by paramagnetic relaxation or rapid hydrogen exchange.

Sensitivity‐Enhanced 13C‐NMR Spectroscopy for Monitoring Multisite Phosphorylation at Physiological Temperature and pH

“…[6][7][8] The non-phosphopeaks (either from the phosphosites themselves or from neighboring residues) provide the most reliable information: reference intensities at 0 % phosphorylation are known and permit a straightforward normalization. However, at physiological pH and temperature, disordered proteins generate overlapping and weak 1 H- 15 N signals, as mentioned earlier. The counterpart phosphopeaks display generally more favorable intensities because phosphate groups on the sidechain slow down water-amide proton exchange, but exploiting them in a quantitative manner is often problematic: it requires normalization by peak intensities at 100 % phosphorylation, a stoichiometry that is often impossible to reach using in vitro phosphorylation by purified kinases, especially when these are purchased commercially-commonly at high cost.…”

“…To reach a more favorable NMR sensitivity, we decided to build on the ( 1 Hflip) 13 Ca- 13 CO correlation experiment, which is the most sensitive 2D 13 C-detected pulse sequence. [12] This type of experiment is not negatively affected by water-amide proton exchange, in contrast to the 13 CO- 15 N experiments (Supporting Information, Figure S3). 13 Ca- 13 CO experiments are usually thought to generate unresolved spectra showing abundant crosspeak overlaps for unfolded peptides.…”

“…These popular methods are sensitive, but provide a comprehensive characterization only with difficulties in the common cases of multiple neighboring or degenerate phosphosites. High-resolution NMR spectroscopy has emerged as a complementary approach to decipher complex phosphorylation schemes: using the classical detection of 1 H- 15 N amide resonances, residue-specific information is obtained in 2D 1 H- 15 N correlation spectra, even on multiple phosphorylation sites that were not identified by MS/WB. [6][7][8] In frequent cases where 2D NMR peaks of IDRs overlap, 3D BT-HNCO combined with non-uniform sampling (NUS) improves the resolution by further spreading the signals.…”

“…[12][13][14] Still, notably because of the lower 13 C gyromagnetic ratio, it suffers from lower signal-to-noise than conventional 1 H-NMR spectroscopy. The 13 CO- 15 N correlation experiments are used to study IDRs because they offer the best crosspeak dispersion, but necessitate high protein quantities to provide quality spectra (approximately 300 mL at 500 mm), which is not suitable for phosphorylation studies: a whole batch (commonly about 10 mg) of commercially available kinase is usually necessary to phosphorylate approximately 100-1000 nmol of high-preference phosphosites. To reach a more favorable NMR sensitivity, we decided to build on the ( 1 Hflip) 13 Ca- 13 CO correlation experiment, which is the most sensitive 2D 13 C-detected pulse sequence.…”

“…Then, we replaced the final IPAP scheme with a homonuclear decoupling scheme LOW-BASHD, which is applied during the acquisition (Supporting Information, Figure S4). [15] We also implemented a 120 ms triply compensated pulse G5 [16] to invert both carbonyl and alkyl 13 C nuclei in the final INEPT block. This yielded on average a 40 % increase in the signal-to-noise ratio without any side-effects on peak line-width (Figure 2 a,b), as demonstrated by Bax and colleagues.…”