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. 1997 Nov 11;94(23):12366-71.
doi: 10.1073/pnas.94.23.12366.

Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution

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Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution

K Pervushin et al. Proc Natl Acad Sci U S A. .

Abstract

Fast transverse relaxation of 1H, 15N, and 13C by dipole-dipole coupling (DD) and chemical shift anisotropy (CSA) modulated by rotational molecular motions has a dominant impact on the size limit for biomacromolecular structures that can be studied by NMR spectroscopy in solution. Transverse relaxation-optimized spectroscopy (TROSY) is an approach for suppression of transverse relaxation in multidimensional NMR experiments, which is based on constructive use of interference between DD coupling and CSA. For example, a TROSY-type two-dimensional 1H,15N-correlation experiment with a uniformly 15N-labeled protein in a DNA complex of molecular mass 17 kDa at a 1H frequency of 750 MHz showed that 15N relaxation during 15N chemical shift evolution and 1HN relaxation during signal acquisition both are significantly reduced by mutual compensation of the DD and CSA interactions. The reduction of the linewidths when compared with a conventional two-dimensional 1H,15N-correlation experiment was 60% and 40%, respectively, and the residual linewidths were 5 Hz for 15N and 15 Hz for 1HN at 4 degrees C. Because the ratio of the DD and CSA relaxation rates is nearly independent of the molecular size, a similar percentagewise reduction of the overall transverse relaxation rates is expected for larger proteins. For a 15N-labeled protein of 150 kDa at 750 MHz and 20 degrees C one predicts residual linewidths of 10 Hz for 15N and 45 Hz for 1HN, and for the corresponding uniformly 15N,2H-labeled protein the residual linewidths are predicted to be smaller than 5 Hz and 15 Hz, respectively. The TROSY principle should benefit a variety of multidimensional solution NMR experiments, especially with future use of yet somewhat higher polarizing magnetic fields than are presently available, and thus largely eliminate one of the key factors that limit work with larger molecules.

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Figures

Figure 1
Figure 1
Experimental scheme for TROSY-type two-dimensional 1H,15N correlation spectroscopy. In the rows marked 1H and 15N, narrow and wide bars stand for nonselective 90° and 180° rf-pulses, respectively. Water suppression is achieved by watergate (34), using the two off-resonance rf-pulses indicated by curved shapes. The 1H and 15N carrier frequencies are placed at 9 and 127 ppm, respectively. The delay τ1 corresponds to 1/(41J(1H,15N)) = 2.7 ms. Phases used are ψ1 = {y,−y,−x,x,y,−y,−x,x}; ψ2 = {4(x),4(-x)}; φ1 = {4(y),4(-y)}; φ2 (receiver) = {x,−x,−y,y,x,−x,y,−y}; x on all other pulses. The row marked PFG (pulsed field gradient) indicates the applied magnetic field gradients along the z-axis: G1, amplitude = 30 G/cm, duration = 0.4 ms; G2, −60 G/cm, 1 ms; G3, 50 G/cm, 0.4 ms; G4, 48 G/cm, 0.6 ms. Two free induction decays are recorded per t1 delay, with ψ1 incremented by 90° in between and stored as the real and imaginary parts of the interferogram in t1. The Fourier transformation results in a two-dimensional 1H,15N correlation spectrum that contains only the component of the four-line 15N–1H multiplet that has the slowest T2 relaxation rates for both nuclei. With this scheme, DD/CSA relaxation interference, which has been known for many years (35, 36), can be used to extend the limits of protein NMR.
Figure 2
Figure 2
Contour plots of 15N,1H correlation spectra showing the indole 15N–1H spin system of Trp-48 recorded in a 2 mM solution of uniformly 15N-labeled ftz homeodomain complexed with an unlabeled 14-bp DNA duplex in 95% H2O/5% 2H2O at 4°C, pH = 6.0, measured at the 1H frequency of 750 MHz. (a) Conventional broad-band decoupled [15N,1H]COSY spectrum (22, 23). The evolution caused by the 1J(1H,15N) scalar coupling was refocused in the ω1 and ω2 dimensions by a 180° proton pulse in the middle of the 15N evolution time t1, and by waltz composite pulse decoupling of 15N during data acquisition, respectively. (b) Conventional [15N,1H]COSY spectrum recorded without decoupling during t1 and t2. (c) TROSY-type 15N,1H correlation spectrum recorded with the pulse scheme of Fig. 1. Chemical shifts relative to DSS in ppm and shifts in Hz relative to the center of the multiplet are indicated in both dimensions. The arrows identify the locations of the cross-sections shown in Fig. 3.
Figure 3
Figure 3
Cross-sections through the spectra of Fig. 2 (solid lines). To facilitate a comparison of the linewidths in the different spectra the cross-sections were normalized to the same maximal signal amplitude. (a1), (a2), etc. refer to the arrows in Fig. 2. Simulated line shapes (dashed lines in a and b) were calculated using 1J(1H,15N) = −105 Hz, a rotational correlation time of τc = 20 ns, and chemical shift anisotropies of ΔσH = −16 ppm and ΔσN = −160 ppm. A long-range scalar coupling 2J(1Hδ1,15Nɛ1) = −5 Hz was included in the simulation of the 15N lineshapes (24), but possible effects of the small scalar couplings 3J(1Hδ1,1Hɛ1) and 3J(1Hζ2,15Nɛ1) were neglected. For 1HN the relaxation due to DD coupling with other protons in the nondeuterated complex was approximated by three protons placed at a distance of 0.24 nm from 1HN.
Figure 4
Figure 4
1H and 15N lineshapes predicted for the broad and narrow multiplet components of 1HN and 15N of the 15N–1H moiety in a [15N,1H]COSY experiment of the type of Fig. 3 b1 and b2 for large proteins in H2O solution at 20°C and a 1H frequency of 750 MHz. (a1 and a2) Spherical protein of size 150 kDa. For the calculation a rotational correlation time of 60 ns, ΔσH = −16 ppm and ΔσN = −160 ppm were used, and all nonlabile protons were replaced with deuterons. Relaxation due to DD coupling with other labile protons was modeled by placing two protons at a distance of 0.29 nm from 1HN. The full linewidths at half height are indicated. (b1 and b2) Spherical protein of size 800 kDa. The calculation used τc = 320 ns and otherwise the same parameters as in a.

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