Backbone resonance assignments of 13C,15N-labeled proteins are usually based on triple-resonance experiments. Many of these experiments include the amide moiety and can easily be optimized for large molecules using TROSY. ttese experiments contain extended time periods with transverse amide proton or amide nitrogen magnetization (Fig. 5.3b) and yield a dramatic increase in signal intensities when using TROSY. tte basis for most of the multidimensional TROSY experiments (e.g., Fig. 5.3b) were standard triple-resonance experiments (Ikura et al. 1990; Bax and Grzesiek 1993; Yamazaki et al. 1994; Sattler et al. 1999) that were used with 2H,13C,15N-labeled proteins, typically up to molecular masses of about 30 kDa. For work with larger molecules, the 2D [15N,1H]-TROSY described earlier (Fig. 5.3a) is introduced into these pulse sequences (Salzmann et al. 1998, 1999a, b; Konrat et al. 1999; Loria et al. 1999; Yang and Kay 1999b). Especially the 15N constant-time evolution periods in the TROSY version of these experiments yield large sensitivity gains (Fig. 5.3b), which in turn can be used, for example, to improve spectral resolution in the 13C dimension by using constant-time evolution times (Salzmann et al. 1999a). Further sensitivity improvements could be obtained by a variety of sensitivity enhancement schemes (Meissner and Sorensen 1999; Ranee et al. 1999; Yang and Kay 1999a).
With TROSY techniques, resonance assignments have be obtained for much larger proteins than ever thought possible with conventional, non-TROSY NMR
techniques. Ms was first demonstrated with a homo-octameric 110-kDa protein (Salzmann et al. 2000), where 20-50 fold sensitivity gains were observed. For example, Fig. 5.4 shows the dramatic improvements in spectral quality that can be obtained with TROSY in a molecular complex of about 60 kDa. tte HNCA triple-resonance spectrum shown in Fig. 5.4, panels C and D forms the basis for complete assignments of the backbone atoms in a polypeptide chain since it correlates the XH and 15N nuclei of an amide moiety with the intraresidue and the preceding a-carbon nuclei. Whereas the TROSY version shows clear cross peaks, the conventional spectrum cannot at all yield the desired correlations. In general, the application of TROSY to triple-resonance experiments results in sensitivity gains of more than 1 order of magnitude with proteins in the molecular mass range from 25 to 150 kDa (Konrat et al. 1999; Salzmann et al. 1998,1999b, 2000; Yang and Kay 1999b).
To date, sequence-specific backbone resonance assignments based on TROSY triple-resonance experiments have been described for numerous proteins. Important examples are the chemical shift assignments of the 723-residue monomeric protein malate synthase, which is the largest single polypeptide chain for which chemical shift assignments have been obtained (Tugarinov et al. 2002) (Fig. 5.6), and the NMR studies of the 91-kDa 11-meric TRAP protein (McElroy et al. 2002) and the 67-kDa dimeric form of the tumor suppression protein p53 (Mulder et al. 2000).
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