Our Latest Developments in NMR Methodology
Sensitivity Enhancement in Solid-State NMR Experiments
We present a new method for enhancing the sensitivity in static solid-state NMR experiments for a gain in signal-to-noise ratio of up to 40%. This sensitivity enhancement is different from the corresponding solution NMR sensitivity enhancement schemes and is achieved by combining single- and multiple-quantum dipolar coherences. While this new approach is demonstrated for the polarization inversion spin exchange at magic angle (PISEMA) experiment, it can be generalized to the other separated local field experiments for solid-state NMR spectroscopy. This method will have a direct impact on solid-state NMR spectroscopy of liquid crystals as well as of membrane proteins aligned in lipid membranes.
(top) Comparison of two-dimensional spectra of N-acetylleucine from the (left) conventional PISEMA and (right) SE-PISEMA experiments. (bottom) Comparison of one-dimensional slices from the two-dimensional experiments taken at 187 ppm showing increase in signal to noise.
Gopinath, T., Veglia, G., Sensitivity Enhancement in Solid-State NMR Experiments via Single- and Multiple-Quantum Dipolar Coherences, J. Am. Chem. Soc., 2009, 131, 16, 5754-56
One-sample approach for protein complexes
We present a procedure for isolating subspectra corresponding to individual protein or peptide components in a ternary mixture or complex. Each of the three-component species is labeled differently: species A uniformly with 15N, species B uniformly with 15N and 13C, and species C uniformly with 15N but selectively with 13C' or 13Calpha. By using the dual carbon label selective HSQC (DCLS-HSQC) pulse sequence and exploiting differences in 1J 15N-13C coupling patterns to filter selected 15N resonances from detection during a constant time period, a subspectrum from each species can be generated from three spectra acquired from a single sample. Many important biological pathways involve dynamic interactions among members of multicomponent protein assemblies, and this approach offers a powerful way to monitor such processes.
(A) The DCLS-HSQC pulse sequence. (B) Representative spectra obtained at 37 °C on a Varian VNMRS 800 MHz spectrometer equipped with a cryogenic probe: i. full ternary mixture consisting of 1 mm [U–2H, U–15N]MBP, 0.8 mM [U–13C, U–15N]-ubiquitin, and 0.8 mM [13C′]-Ala4, [15N]-Ser5 Kemptide; ii. MBP subspectrum; iii. ubiquitin subspectrum; iv. Kemptide subspectrum.
Masterson, L.R., Tonelli, M., Markley, J.L., Veglia, G., Simultaneous Detection and Deconvolusion of Congested NMR Spectra Containing Three Isotopically Labeled Species, J. Am. Chem. Soc., 2008, 130, 25, 7818-19
Assymetric Methyl Group Labeling Approach
We present an asymmetric isotopic labeling strategy for probing unambiguously membrane protein binding interfaces in homo-oligomers. This technique is highly sensitive, rapid, and requires only one 13C methyl-edited nuclear Overhauser effect spectroscopy (NOESY) spectrum. As a proof of principle, we apply this technique to the homo-pentameric membrane protein phospholamban, defining the leucine-isoleucine zipper interface between its monomers with atomistic resolution.
(A) Isotopic labeling scheme as shown on the pinwheel model of phospholamban. The red monomer of PLN is labeled [U-2H, 12C, 14N, 13CH3-Ileδ1], while the blue monomer is labeled [U-2H, 12C, 14N, 13CH3-Valγ1,2, 13CH3-Leuδ1,2]. Owing to the precursor chosen, only one of the methyl groups in the Val and Leu sample is 13CH3. Constant-time HSQC experiment on the 1/1 mixed sample showing Ileδ1 (red, B) and Valγ1,2/Leuδ1,2 (blue, C) methyl resonances.
Traaseth, N.J., Verardi, R., Veglia, G., Asymmetric Methyl Group Labeling as a Probe of Membrane Protein Homo-oligomers by NMR Spectroscopy, J. Am. Chem. Soc., 2008, 130, 8, 2400-01.