Application Note 22
[2,3-13C]-Labeled Aromatic Residues as a Means to Improving Signal Intensities and Kick-Starting the Assignment of Membrane Proteins by Solid-State MAS-NMR
Matthias Hiller, Victoria A. Higman, Stefan Jehle, Barth-Jan van Rossum, Werner Kühlbrandt, and Hartmut Oschkinat
Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, Germany
and Max-Planck-Institut für Biophysik, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany
Over the past few years solid-state MAS-NMR has rapidly been developing into a structure determination technique for biological macromolecules.1-6 Its advantages include the ability to study membrane proteins in their native lipid environment,7-10 as well as making possible the study of nonsoluble or noncrystallisable protein states, such as amyloid fibrils.11-15 However, a prerequisite for structure determination is a high level of resonance assignment. There are numerous examples of small- and medium-sized proteins for which this has been possible,4,16-20 but for large membrane proteins, such as the 281-residue outer membrane protein G (OmpG)21,22 resonance assignment still remains a challenge. Signal overlap is an obvious problem, but fast longitudinal and transverse relaxation rates also contribute toward lower signal/noise ratios. Furthermore, membrane proteins can reduce the Q-factor of the coil, and the experimentalist is then left with the difficult task of balancing increased decoupling powers against possible sample heating which could lead to sample degradation. These problems result in lower signal intensities in proton-driven spin diffusion (PDSD) spectra; however, they can be addressed using several different strategies, such as by improving coil design,23-25 by developing spectral editing pulse sequences,26,27 or by using novel labeling strategies.28,29 It is this last approach, using a novel labeling strategy, which is presented here in this application note.
It has been shown that samples produced using [1,3-13C]- and [2-13C]-labeled glycerol in the bacterial growth medium give rise to spectra with improved cross-relaxation properties and which prove very helpful at the restraint generation stage.1 But assignment using solely these samples is not possible, since the C’, Cα and Cβ atoms are never simultaneously labeled. An alternative approach would be to use [1,2,3-13C]-, [1,2-13C]- and/or [2,3-13C]-labeled amino-acids. This would retain the favorable cross-relaxation properties of the glycerol samples by keeping the number of consecutively bonded labeled carbon atoms low, but would provide a more useful set of labels for assignment. The two former labeling patterns can easily be generated with uniformly labeled short amino acids such as alanine, serine, cysteine, or glycine. Additional incorporation of other amino acids with [2,3-13C] labeling would increase the number of detectable amino acids but reduce the overlap in NCO-type spectra,30 thus providing scope for many unambiguous sequential assignments from these spectra. When considering which amino acid types to select for [2,3-13C] labeling, it is worth noting that Cβ signals from aromatic amino acids are often very weak or not visible at all. [2,3-13C] labeling aromatic amino acids would reduce J-couplings at the Cα and Cβ positions, as well as ensure that the aromatic ring (which undergoes fast relaxation) does not draw away magnetization and effectively act as a magnetization “sink.” The [2,3-13C] labeling would then not only aid assignment, but also improve the spectral quality and identification of aromatic spin systems.
This application note describes the use of a novel labeling strategy to increase the spectral quality and level of resonance assignment in MAS-NMR spectra of the biological macromolecule OmpG. Based on the protein’s sequence, a labeling scheme of fully [15N,13C]-labeled Ala and Gly and [2,3-13C,15N]-labeled Tyr and Phe was selected. The sample (hereafter referred to as OmpG-GAFY) was prepared using established protocols and incorporating [2,3-13C,15N]-Tyr (CNLM-7610), [2,3-13C, 15N]-Phe (CNLM-7611), [U-13C3,15N]-Ala (CNLM-534) and [U-13C2,15N]-Gly (CNLM-1673).
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