TROSY

2 TROSY. The number of publications in this area was substantially reduced relative to the previous year. A new TROSY element was proposed by Riek et al. The method utilises DD/ C CSA cross-correlation 

Other publications dealt with optimisations of the previously proposed sequences. Pervushin et al. proposed a new element N, H/ C, H-TROSY 

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The cross-correlation effects between the DD and CSA interactions also influence the transverse relaxation and lead to the phenomenon known as differential line broadening in a doublet [40], cf Figure Bl.13.8. There is a recent experiment, designed for protein studies, that I wish to mention at tire end of this section. It has been proposed by Pervushin etal [4T], is called TROSY (transverse relaxation optimized spectroscopy) and... 

Luy, B., Marino, J. P. JE-TROSY combined /- and TROSY-spectroscopy for the measurement of one-bond couplings in macromolecules. J. Magn. Reson. 2003, 163, 92-98. 

A HNCOi.,CA-TROSY 0 HNCOCA-TROSY HNCACO-TROSY... 

Exploitation of the TROSY effect is rather straightforward. In contrast to 15N-HSQC (Heteronuclear Single Quantum Coherence) or standard triple-resonance experiments based on 15N-HSQC, no radio frequency pulses or composite pulse decoupling should be applied on amide protons when HN spin is not in the transverse plane. Likewise the 15N decoupling should be... 

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. 4. The HNCO-TROSY experiment for recording solely interresidual 1HN, 15N, 13C correlations in 13C/15N/2H labelled proteins. All 90° (180°) pulses for the 13C and 13C spins are applied with a strength of 2/ /l5 (p/ /3), where 2 is the frequency difference between the centres of the 13C and 13Ca regions. All 13Ca pulses are applied off-resonance with phase modulation by Q. A = 1/(4/hn) Tn = l/(4/NC ) S = gradient + field recovery delay 0 < k < TN/z2,max- Phase cycling i = y 4>2 = x, — x + States-TPPI 03 = x 0rec = x, — x.

The magnetization has now been successfully transferred from the HN spin to the intraresidual and sequential 13C spins or alternatively to the interresidue 13C spin either using HNCA-TROSY or HNCO-TROSY schemes, respectively. It is inevitable that the HNCO-TROSY spectrum cannot be used for the sequential assignment alone because it does not bridge two sequential N shifts through common carbonyl carbon frequency. The... 

In the alternative approach, the HN(i), 15N( j, 13C (i/i— 1) correlations in the HNCA-TROSY spectrum can be supplemented with the data from the HN(CO)CA-TROSY experiment72 73 yielding solely 11 IN(/), 15N( ), 13C (i- 1) correlations. To this end, the HNCO-TROSY experiment is extended with the 13C —> 13C INEPT step, which utilizes rather large (ca. 51-55 Hz) one-bond scalar coupling between the 13C and 13C spins in order to transfer magnetization from the 13C (< — 1) nucleus further to the 13C ( — 1) spin. 

In the last section, we will focus on the suite of novel triple-resonance TROSY experiments designed for accomplishing the sequence-specific assignment of backbone nuclei in high molecular weight proteins. 

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