MAGNETIC RESONANCE IN CHEMISTRY Magn. Reson. Chem. 2006; 44: S196–S205 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1818 Fast multidimensional NMR spectroscopy by spin-state selective off-resonance decoupling (SITAR) Rochus Keller, Christy Rani R. Grace and Roland Riek * Structural Biology Laboratory, The Salk Institute, La Jolla, CA, 92037, USA Received 30 November 2005; Revised 23 January 2006; Accepted 28 February 2006 Spin-state selective off-resonance decoupling (SITAR) is applied to the amide proton-to-nitrogen-to- alpha-carbon correlation (HNCA) triple-resonance experiment by measuring the 15 N chemical shift during the acquisition simultaneously with the 1 H chemical shift. The simultaneous detection of both 1 H and 15 N chemical shifts in SITAR reduces the dimensionality of the HNCA-type experiment from three dimensions to two dimensions with a 15 N chemical shift resolution of ∼0.4 ppm. This enables the recording of triple-resonance experiments in several minutes. SITAR is furthermore applied to the amide proton-to-nitrogen-to-alpha-carbon-and-beta-carbon correlation (HNCACB) triple-resonance experiment and the 15 N-resolved [ 1 H, 1 H]-nuclear Overhauser enhancement spectroscopy (NOESY) experiment with similar success. The accompanied peak crowding and chemical shift degeneracy of the amide protons in the SITAR two- dimensional (2D) spectra, which are inherent properties of pseudo-dimensional experiments, are resolved by local correlation of the two sub-spectra. With this procedure a 13 C– 1 H strip for each 15 N– 1 H moiety is generated resulting in a three-dimensional (3D) strip list known from the conventional 3D spectra. The quality of the strip list in terms of peak crowding and chemical shift degeneracy is comparable to their corresponding 3D counterparts. An analysis-software within the CARA package is presented, which generates, visualizes and manages the SITAR spectra, the corresponding strip lists and the assignment process. Copyright  2006 John Wiley & Sons, Ltd. Supplementary electronic material for this paper is available in Wiley InterScience at http://www.interscience.wiley.com/ jpages/0749-1581/suppmat/ KEYWORDS: fast multidimensional experiment; NMR; biological macromolecule; chemical shift-coded experiment; high-throughput resonance assignment; SITAR; spin state-selective off-resonance decoupling INTRODUCTION The recent development of the cryogenic probe increases the sensitivity of the nuclear magnetic resonance (NMR) spectrometers by a factor of ¾3 – 4 and concomitantly reduces the necessary measuring time by one order of magnitude. This achievement builds the basis for the so-called ‘non- sensitivity-limited’ data collection regime for well-behaving small and medium size proteins. In this regime, the minimum measurement time required is defined by the sampling rate of the multidimensional NMR experiment and not the sensitivity of the experiment. To circumvent the sampling problem, ‘fast multidimensional NMR’ methods have been recently proposed and applied to triple-resonance experiments, speeding up the measuring time by up to two orders of magnitude. These new schemes include G- matrix Fourier transform NMR spectroscopy (GFT-NMR), Hadamard spectroscopy and projection spectroscopy (for review see Ref. 1). 1 GFT-NMR spectroscopy 2 is a reduced- dimensionality approach, which is based on simultaneous L Correspondence to: Roland Riek, Structural Biology Laboratory, The Salk Institute, 10010 N Torrey Pines Road, La Jolla, CA, 92037, USA. E-mail: riek@salk.edu evolution of two or more different chemical shifts reducing the dimensionality of a triple-resonance experiment by one or more. Kupce and Freeman applied Hadamard spectroscopy to the triple-resonance experiments HNCO and amide proton-to-nitrogen-to-carbonyl correlation (HNCA) and obtained the assignment of a small peptide after measuring only for a few minutes. 3 They also applied the three-dimensional (3D) projection spectroscopy to HNCA and HNCO experiments and obtained the spectrum of a protein after a few minutes of measuring time. 1 Here, we are applying spin-state selective off-resonance decoupling (SITAR) 4 to obtain fast multidimensional NMR spectra. SITAR is based on the notion that chemical shift information can be extracted by off-resonance decoupling. 5 As we shall see, the use of SITAR for the detection of the 15 N chemical shift in HNCA-type triple-resonance experiments enables the measurement of a triple-resonance experiment in only several minutes. The chemical shift resolution is comparable to the conventional 3D counterpart. The crowding of the spectrum as well as the chemical shift degeneracy of the amide protons, which are inherent problems of pseudo-3D spectroscopy, are resolved by spin- state selection and an algorithm that uses local correlations Copyright  2006 John Wiley & Sons, Ltd.