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

NMR Supported Structural Biology, Leibniz-Institut für Molekulare Pharmakologie, Robert-Roessle Str. 10, 13125 Berlin, Germany.
Journal of Magnetic Resonance (Impact Factor: 2.51). 03/2012; 217:77-85. DOI: 10.1016/j.jmr.2012.02.015
Source: PubMed


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 4-5 times higher sensitivity in 2D (13)C-(13)C correlation experiments as compared to PDSD [1]. Here, a quantitative comparison of PDSD, (1)H-DARR, (2)H-DARR, and (1)H+(2)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 the (2)H-DARR experiment within a buildup range of ∼18-45 ms, whereas values between 24 and 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(α)-CO and C(α)-C(alip) regions of the 2D (13)C-(13)C DONER spectrum applying proton CP ((1)H-(13)C). As a parameter determining the S/N in (13)C-(13)C correlation experiments, proton CP efficiency is investigated using deuterated samples with proton/deuterium ratios at 20%, 40%, and 100% H(2)O. Sufficiently strong (13)C 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.

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    • "ed in this sample , the maximum intensity reached is slightly smaller compared to the frozen solution ( Figure 3 ) . This could arise from relaxation effects and conformational transitions between different protein subconformations ( energy minima ) , both of which are hydration - dependent ( Zanotti et al . , 1999 ; Krushelnitsky et al . , 2009 ; Akbey et al . , 2012 ) . These data thus demonstrate that the process of partial dehydration causes changes of electronic structure of the bilin as well as its mobility within the pocket ."
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