Flip-back pulse

The effect could be considerable for solvent-exposed parts of the backbone and could render the NOE values inaccurate. These systematic errors could be minimized by using water flip-back pulses in order to avoid saturation of H20 magnetization [11]. The NOE data are generally more susceptible to errors than Ri and R2 because (i) the NOE experiments start with the equilibrium 15N magnetization that is 10 fold lower than that of (XH) in the Hi and R2 experiments, hence relatively low sensitivity, and (ii) the NOE values are derived from only two sets of measurements, whereas R1 and R2 data are obtained from fitting multiple sets of data the latter is expected to result in a more efficient averaging of experimental errors. 

The 15N,1H shift correlation maps are most conveniently recorded with a sensitivity-enhanced HSQC sequence with incorporated water flip-back pulses for reduced saturation transfer and pulsed-field gradients for coherence selection. The pulse sequence of the experiment is shown in Fig. 14.4 A. 

Another commonly used technique is the water flip-back pulse, a shaped pulse designed to selectively rotate only the water magnetization by 90°, putting it back on the +z axis after a hard (nonselective) pulse has rotated all of the sample magnetization into the x-y plane. Water can be viewed as a wild and powerful bucking bronco—it must be tamed and never allowed to get out of its pen. The best place for water is on the +z axis where it will not do any harm. This is the rationale behind the flip-back pulse every time water is moved from the +z axis, use a selective pulse to put it back there. 

The sequential mainchain assignment of perdeuterated proteins is achieved by collecting and analyzing 3D HNCACB, 3D HN(CO)CACB, and 4D HN(CACO)NH data (1). These sequences include "H decoupling when C is transverse and work best if H2O flip-back pulses and pulsed field gradients are employed. Complete aliphatic deuteration increases both resolution and sensitivity in these experiments by eliminating partially deuterated CHnDm moieties, which have different C chemical shifts due to the "H isotope shift. 

Fig. 26. IWo-dimensional NOESY experiment (conditions as forRg. 25), without (A) and with (B) RD amplification used as a water flip-back pulse. The spectrum in (B) has noticeably better water suppression due to the complete alignment of the water magnetization along the +z axis at the end of the detection peri. (From Abergel et al. with permission.)...

Spectra with the form of the two terms on the right-hand side of Eq. (18) can be obtained by two extra modulation periods of length t in the exchange pulse sequence, one before and one after rm. The sine.sine and cosine.cosine terms of Eq. (18) are selected by suitable choices of the phases of the flip back pulses, labelled a and b in Fig. 38. The spectrum of the form of Eq. (18) is produced by the pulse sequence in Fig. 38(a), for non-spinning samples, while the spectrum of the form 1/2 S is produced using the sequence of Fig. 38(b), which matches that in Fig. 38(a) in terms of pulses and delays and so should produce a matched intensity spectrum, so that when the relevant spectra from the pulse sequence in Fig. 38(a) are subtracted from it, the desired pure-exchange spectrum is obtained. The A periods in both sequences are simply Hahn echoes, implemented to achieve non-distorted powder patterns in both spectral dimensions.2... 

Fig. 3. HNCA (a) and two implementations of HNCA-TROSY (b-c) experiments for recording intraresidual HN(/), 15N(/), 13C"(i) and sequential 1 HN(7), l5N(/), 13Ca(i — 1) correlations in 13C/15N/2H labelled proteins. Narrow and wide bars correspond to 90° and 180° flip angles, respectively, applied with phase x unless otherwise indicated. Half-ellipse denotes water selective 90° pulse to obtain water-flip-back.88,89 All 90°...

Fig. 9.1 Basic HNN COSY pulse sequence. Narrow and wide pulses correspond to flip angles of 90° and 180°, respectively, whereas low-power (water flip-back) 90° l-l pulses are illustrated as smaller narrow pulses. Delays 8 = 2.25 ms 7=15 ms (can be shorter or longer) (a = 2.5 ms fb = 0.25 ms fc= 2.25 ms fd = 0.5 ms. Unless indicated, the phase of all pulses are applied along...

Fig. 14.4 Pulse sequences used for the experiments described in this chapter. A [ N HJ-HSQC with water flip back and PFGs. The shaped pulse on the proton channel is a sine-shaped, 1.5 ms soft pulse all other pulses are hard pulses. Gradients are applied as square or sine-shaped pulses. The sign of the last gradient is reversed for anti-echo selection together with the sign of phase 6. B CPMG sequence. C bpPFGLED sequence. The delay T denotes the diffusion delay. Typically, r is set to 1 ms, T to 50-100 ms and Te to 1.2 ms.

HOHAHA experiment with minimal water saturation was developed by Schleucher et al. (1995a) and Dhalluin et al. (1996) proposed a water-flip-back TOCSY where water-selective pulses are used to flip a maximal fraction of the water magnetization back along the +z axis at the start of the acquisition time. TOCSY experiments with excellent water suppression based on excitation sculpting (Stott et al., 1995 Hwang et al., 1995) were reported by Callihan et al. (1996). 

Figure 2 Gradient-echo-based water suppression pulse sequences, (a) WATERGATE (b) water-flip-back (c) excitation sculpting (d and e) examples of the S pulse train that is sandwiched between the gradient echo (d) water-selective inversion...

Fig. 20. A fast flip-back WATERGATE HSQC (FHSQC) that avoids water saturation and radiation damping. The WATERGATE subunit uses a 3-9-19 pulse. Details of the phase cycling, delays and gradient strengths can be found elsewhere. ...

Finally, a 90° pulse flips the cosine term back to the x -yf plane ... 

Take a moment to look at review problem 2.7 and Figures A.2 and A.3 in Appendix 1. In this problem the 0.20-ms pulse resulted in an inversion of M (a flip angle of 180° resulting from a 180t. pulse). Then M decayed exponentially back to Mo as described by the equation... 

---