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
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 . 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 ."
ABSTRACT: Phytochromes are dimeric biliprotein photoreceptors exhibiting characteristic red/far-red photocycles. Full-length cyanobacterial phytochrome Cph1 from Synechocystis 6803 is soluble initially but tends to aggregate in a concentration-dependent manner, hampering attempts to solve the structure using NMR and crystallization methods. Otherwise, the Cph1 sensory module (Cph1Δ2), photochemically indistinguishable from the native protein and used extensively in structural and other studies, can be purified to homogeneity in >10 mg amounts at mM concentrations quite easily. Bulk precipitation of full-length Cph1 by ammonium sulfate (AmS) was expected to allow us to produce samples for solid-state magic-angle spinning (MAS) NMR from dilute solutions before significant aggregation began. It was not clear, however, what effects the process of partial dehydration might have on the molecular structure. Here we test this by running solid-state MAS NMR experiments on AmS-precipitated Cph1Δ2 in its red-absorbing Pr state carrying uniformly (13)C/(15)N-labeled phycocyanobilin (PCB) chromophore. 2D (13)C-(13)C correlation experiments allowed a complete assignment of (13)C responses of the chromophore. Upon precipitation, (13)C chemical shifts for most of PCB carbons move upfield, in which we found major changes for C4 and C6 atoms associated with the A-ring positioning. Further, the broad spectral lines seen in the AmS (13)C spectrum reflect primarily the extensive inhomogeneous broadening presumably due to an increase in the distribution of conformational states in the protein, in which less free water is available to partake in the hydration shells. Our data suggest that the effect of dehydration process indeed leads to changes of electronic structure of the bilin chromophore and a decrease in its mobility within the binding pocket, but not restricted to the protein surface. The extent of the changes induced differs from the freezing process of the solution samples routinely used in previous MAS NMR and crystallographic studies. AmS precipitation might nevertheless provide useful protein structure/functional information for full-length Cph1 in cases where neither X-ray crystallography nor conventional NMR methods are available.
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- "A recent study of Akbey et al. depicts various experimental aspects of DONER and ways to achieve efficient coherence transfer in perdeuterated proteins with different proton/deuteron ratios. They observed long range correlations between carbon spins spatially separated by about $ 6 Å . "
ABSTRACT: A Floquet description of a phase alternated homonuclear recoupling scheme for perdeuterated systems is presented. As a result, we demonstrate improvements in the recoupling efficiency of the DOuble Nucleus Enhanced Recoupling [DONER; J. Am. Chem. Soc. 131 (2009) 17054] technique by utilizing Phase Alternated Recoupling Irradiation Schemes [PARIS; Chem. Phys. Lett. 469 (2009) 342]. The effect of proton and deuterium radio frequency irradiation during recoupling has been systematically studied and theoretical observations have been verified experimentally using a deuterated model compound, l-Alanine, at 10 and 20kHz magic angle spinning frequency. Experimental results are well in agreement with theoretical observations, thereby significantly increasing the recoupling efficiency of conventional DONER in perdeuterated systems.
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ABSTRACT: We have recently proposed sedimented solute NMR (SedNMR) as a solid-state method to access biomolecules without the need of crystallization or other sample manipulation. The drawback of SedNMR is that samples are intrinsically diluted and this is detrimental for the signal intensity. Ultracentrifugal devices can be used to increase the amount of sample inside the rotor, overcoming the intrinsic sensitivity limitation of the method. We designed two different devices and we here report the directions for using such devices and the relevant equations for determining the parameters for sedimentation.
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