Practical aspects of high-sensitivity multidimensional 13C MAS NMR spectroscopy of perdeuterated proteins

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Practical aspects of high-sensitivity multidimensional 13C MAS NMR spectroscopy of perdeuterated proteins

April 2012
Publication year: 2012
Source:Journal of Magnetic Resonance, Volume 217



The double nucleus enhanced recoupling (DONER) experiment employs simultaneous irradiation of protons and deuterons to promote spin diffusion processes in a perdeuterated protein. This results in 4–5times higher sensitivity in 2D 13C–13C correlation experiments as compared to PDSD [1]. Here, a quantitative comparison of PDSD, 1H-DARR, 2H-DARR, and 1H+ 2H 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 the 2H-DARR experiment within a buildup range of ~18–45ms, whereas values between 24 and 69ms 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?–Calip regions of the 2D 13C–13C DONER spectrum applying proton CP (1H–13C). As a parameter determining the S/N in 13C–13C correlation experiments, proton CP efficiency is investigated using deuterated samples with proton/deuterium ratios at 20%, 40%, and 100% H2O. Sufficiently strong 13C 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 ~6Å by using DONER approach.
Graphical abstract

Highlights

? Double nucleus enhanced recoupling recovers spin-diffusion in deuterated proteins. ? DONER results in up to ~5times larger cross-peak intensities compared to PDSD. ? Long range distance restraints are observed up to ~6Å. ? High 13C resolution is only achieved with deuterium scalar-decoupling at MAS?20kHz. ? Coherent cross-relaxation broadens 13C lines of deuterated protein below 20kHz MAS.





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