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NMR spectroscopy is a well-established tool in structural biology. NMR constitutes an alternative technique to X-ray crystallography to obtain structural and dynamic information at atomic resolution and to study intermolecular interactions of biological macromolecules at near-physiological solution conditions (Wuthrich 1986; Ferentz and Wagner 2000; Wider 2000). Considerable efforts are being devoted to extend applications of solution NMR to larger molecular systems, because a large number of biologically important macromolecules and macromolecular complexes have molecular masses beyond the practical range amenable to conventional NMR spectroscopy in solution. Increasing this size limit allows, for instance, structure determinations of proteins that cannot be crystallized, including integral membrane proteins, investigations of intermolecular interactions involving large molecules and macromolecular assemblies, and the structure determination of larger oligonucleotides and their complexes with proteins.

During the past 2 decades, solution NMR studies of biological macromolecules have been limited to relatively small structures, ttis is directly reflected by the size distribution of the NMR structure entries in the Protein Data Bank, where most of the NMR structures are in the range 2-25 kDa, with an average around 10 kDa (Guntert 1998). In the 1980s, when the first 3D protein structures were elucidated by NMR (Braun et al. 1983; Williamson et al. 1985; Wuthrich 1986; Wagner et al. 1987), one of the main bottlenecks when investigating "larger" proteins was the daunting analysis of their crowded homonuclear proton-proton 2D NMR spectra. In the early 1990s, biochemical labeling of proteins with stable 15N and 13C isotopes (LeMaster 1994; Kainosho 1997; Gardner and Kay 1998) became a widespread solution for this problem, particularly for the structure determination of biological macromolecules with molecular masses above approximately 10 kDa. Isotope labeling in combination with heteronuclear multidimensional NMR spectroscopy, particularly the development of triple-resonance and 13C/15N-resolved 3D and 4D NMR experiments (Ikura et al. 1990; Bax and Grzesiek 1993; Sattler et al. 1999), was a significant step ahead enabling an increase of the molecular sizes amenable to NMR studies to about 20-25 kDa.

For proteins or protein complexes with molecular masses above 25-30 kDa the spectral quality rapidly deteriorates A major limitation when working with these larger macromolecules arises from the high relaxation rates of the NMR signal, causing severe line broadening, which translates into poor spectral resolution and low

Springer Series in Biophysics J.L.R. Arrondo and A. Alonso Advanced Techniques in Biophysics © Springer-Verlag Berlin Heidelberg 2006

signal-to-noise ratios. Substantial improvements of the quality of NMR spectra of biological macromolecules with molecular masses above approximately 25 kDa can be obtained with deuteration, a technique that has been applied in biological NMR for more than 30 years. Combined with 15N- and 13C-labeling, 2H-labeling experienced an impressive revival about 10 years ago and has become an essential tool for determining large structures in solution (for reviews, see LeMaster 1990, 1994; Gardner and Kay 1998; Goto and Kay 2000).

Deuteration extended the applicability of conventional NMR spectroscopy in solution to molecular sizes of up to 50 kDa. For even larger molecular structures, the spectral quality decreases again rapidly owing to relaxation, despite optimized partial deuteration. Full deuteration of biological macromolecules is not practical because the most valuable information comes from the hydrogen atoms. With the introduction of transverse relaxation-optimized spectroscopy (TROSY) (Pervushin et al. 1997; Wider and Wüthrich 1999; Pervushin 2000, 2003; Riek et al. 2000; Riek 2003; Wüthrich and Wider 2003), relaxation could be reduced to such an extent that satisfactory linewidths and sensitivity can be achieved in NMR experiments with very large molecules. TROSY works best with deuterated proteins and is especially suited for application to protonated amide groups. In concert with improved instrumentation, TROSY has greatly extended the size limit for macromolecules that can be studied by solution NMR. ttis technical progres has opened a wide range of new applications for solution NMR (Wüthrich 1998; Venters et al. 2002; Fernández and Wider 2003; Tugarinov et al. 2004) and, for instance, has made possible studies of molecular systems with molecular masses upto lxlO6 Da (Fiaux et al. 2002; Riek et al. 2002),

In the following, the theoretical background of TROSY, important practical considerations regarding the implementation of TROSY, including isotope labeling of macromolecules, and numerous recent applications of TROSY for structural and functional studies oflarge biological macromolecules will be discussed.

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