Practical Aspects of High-Sensitivity MultidimensionalC MAS NMR Spectroscopy of Perdeuterated Proteins

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Practical Aspects of High-Sensitivity MultidimensionalC MAS NMR Spectroscopy of Perdeuterated Proteins


Publication year: 2012
Source: Journal of Magnetic Resonance, Available online 1 March 2012

Ümit*Akbey, Barth-Jan*van Rossum, Hartmut*Oschkinat

Thedouble nucleus enhanced recoupling(DONER) experiment employs simultaneous irradiation of protons and deuterons to promote spin diffusion processes in a perdeuterated protein. This results in 4-5 times higher sensitivity in 2DC-C correlation experiments as compared to PDSD.Here, a quantitative comparison of PDSD,H-DARR,H-DARR, andH+H DONER has been performed to analyze the influence of spin diffusion on polarization transfer processes. Cross peak buildup curves were analyzed to obtain guidelines for choosing the best experimental parameters. The largest cross peak intensities were observed for the DONER experiments. The fastest build-up rate was observed in theH-DARR experiment within a buildup range of ~18-45 ms, whereas values between 24-69 ms are observed for the DONER experiment. Furthermore, the effects of direct excitation and cross polarization (CP) are compared. A comparison between DONER and RFDR experiments reveal ~50% more intense cross peaks in the C?-CO and C?-Calipregions of the 2DC-C DONER spectrum applying proton CP (H-to-C). As a parameter determining the S/N inC-C correlation experiments, proton CP efficiency is investigated using deuterated samples with proton/deuterium ratios at 20, 40, and 100% H2O. Sufficiently strongC CPMAS signal intensity is observed for such proteins even with very low proton concentration. The effect of proton and/or deuterium decoupling is analyzed at various MAS spinning frequencies. Deuterium decoupling was found most crucial for obtaining high resolution. Long range correlations are readily observed representing distances up to ~6 Å by using DONER approach.

Graphical abstract



Highlights

? DOuble Nucleus Enhanced Recoupling recovers spin-diffusion in deuterated proteins. ? DONER results in up to ~5 times larger cross-peak intensities compared to PDSD. ? Long range distance restraints are observed up to ~6 Å. ? HighC resolution is only achieved with deuterium scalar-decoupling at MAS ?20kHz. ? Coherent cross-relaxation broadensC lines of deuterated protein below 20 kHz MAS.



Source: Journal of Magnetic Resonance