High-resolution four-dimensional 1H-13C NOE spectroscopy using methyl-TROSY, sparse data acquisition, and multidimensional decomposition.
An approach for recording four-dimensional (4D) methyl (1)H-(13)C-(13)C-(1)H NOESY spectra with high resolution and sensitivity is presented and applied to Malate Synthase G (723 residues, 82 kDa). Sensitivity and resolution have been optimized using a highly deuterated, methyl-protonated sample in concert with methyl-TROSY, sparse data sampling in the three indirect dimensions, and 4D spectral reconstruction using multidimensional decomposition (MDD). A sparse data acquisition protocol is introduced that ensures that sufficiently long indirect acquisition times can be employed to exploit the decreased relaxation rates associated with methyl-TROSY, without increasing the duration of the 4D experiment beyond acceptable measurement times. In this manner, only a fraction ( approximately 30%) of the experimental data that would normally be needed to achieve a spectrum of high resolution is acquired. The reconstructed 4D spectrum is of similar resolution and sensitivity to three-dimensional (3D) (13)C-edited NOE spectra, is straightforward to analyze, and resolves ambiguities that emerge when 3D data sets only are considered.
Tugarinov
Vitali
V
Protein Engineering Network Centers of Excellence and Departments of Medical Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Kay
Lewis E
LE
Ibraghimov
Ilghiz
I
Orekhov
Vladislav Yu
VY
D002247:Carbon Isotopes; D008292:Malate Synthase / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011522:Protons
D002247:Carbon Isotopes; D011522:Protons; D008292:Malate Synthase