N Jamin, F. Toma/ Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001)83-114 04 a 0.3 0.2 0.1 00bb855655650558505 sbs bsbsbsbs bsbs bsbsbsbs bsbs bs b 00 10 20 3.0 Fig. 5. Backbone(b) and side-chain(s)relaxation parameters of the Tlp(upper graph) and [H-NI NOE (lower graph) at 600 MHz for the ee(black bars)and the DNA-bound (hatched bars) lac repressor headpiece. The backbone and side-chain parameters are indicated with"b d"s",respectively. For Asn, 'side-chain'refers to the N GIn and Arg, this refers to N.( Fig. 4 from Ref. [401). Reprinted with the permission of R. Kaptein and of the American Chemical Society (o 1997) approach, Wuthrich and coworkers observed a signif- benefit from the implementation of the TROSY icant reduction in the linewidth for N and H in a 2D principle H,N correlation experiment performed with a uniformly N-labeled protein complex with a DNA 2 7. Long-range distance constr fragment at 750 MHz and 4C (TC 20+/-2 ns This TROSY principle has been implemented in the Bax and coworkers have proposed the use of the conventional triple resonance experiments HNCA, magnetic field dependence of the dipolar H-N and HNCO, HN(CO)CA, HN(CA)CO, HNCACB and H-C couplings [37] and of the N shift [38]to HN(CO)CACB. A 2-3-fold enhancement in the measure the orientation of Nh, Ch or Cc bond signal-to-noise ratio has been observed when applied vectors relative to the magnetic susceptibility tensor to H/C/N-labeled proteins and significant gains of Thus, these measurements will provide long-range sensitivity were measured or predicted for protonated constraints between distinct regions of the complex proteins. The highest sensitivity gains are obtained for Molecules with an anisotropic magnetic susceptibility the regular secondary structure elements in the protein will align along the static magnetic field to a degree core. Studies of protein-DNA complexes should which is proportional to the product of the anisotropyapproach, WuÈthrich and coworkers observed a signif￾icant reduction in the linewidth for 15N and 1 H in a 2D 1 H,15N correlation experiment performed with a uniformly 15N-labeled protein complex with a DNA fragment at 750 MHz and 48C …tc ˆ 20 1 = 2 2 ns†: This TROSY principle has been implemented in the conventional triple resonance experiments HNCA, HNCO, HN(CO)CA, HN(CA)CO, HNCACB and HN(CO)CACB. A 2±3-fold enhancement in the signal-to-noise ratio has been observed when applied to 2 H/13C/15N-labeled proteins and signi®cant gains of sensitivity were measured or predicted for protonated proteins. The highest sensitivity gains are obtained for the regular secondary structure elements in the protein core. Studies of protein±DNA complexes should bene®t from the implementation of the TROSY principle. 2.7. Long-range distance constraints Bax and coworkers have proposed the use of the magnetic ®eld dependence of the dipolar 1 H±15N and 1 H±13C couplings [37] and of the 15N shift [38] to measure the orientation of NH, CH or CC bond vectors relative to the magnetic susceptibility tensor. Thus, these measurements will provide long-range constraints between distinct regions of the complex. Molecules with an anisotropic magnetic susceptibility will align along the static magnetic ®eld to a degree which is proportional to the product of the anisotropy 90 N. Jamin, F. Toma / Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001) 83±114 Fig. 5. Backbone (b) and side-chain (s) relaxation parameters of the T1r (upper graph) and [1 H±15N] NOE (lower graph) at 600 MHz for the free (black bars) and the DNA-bound (hatched bars) lac repressor headpiece. The backbone and side-chain parameters are indicated with ªbº and ªsº, respectively. For Asn, `side-chain' refers to the Nd ; Gln and Arg, this refers to Ne . (Fig. 4 from Ref. [40]). Reprinted with the permission of R. Kaptein and of the American Chemical Society (q 1997)