Intraresidual HNCA-TROSY

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°...

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... 

Fig. 6. The efficiency of the coherence transfer, for the first increment, as a function of delay 2Ta for the HNCA-TROSY. The transfer amplitudes for the intraresidual (a) and sequential (b) cross peaks were calculated with the following parameters,...

The HNCA-TROSY experiment can be readily extended to correlate the 13C spin of the preceding residue with intraresidual 11 IN(/), 15N(i), and 13C° (/ ) frequencies. This kind of four-dimensional HNCO CA-TROSY experiment (Fig. 7) was recently introduced by Konrat et al.79 The coherence flows through the following pathway... 

Fig. 19. Pulse scheme of the MP-HNCA-TROSY experiment. Delay durations A = 1/(4/hn) 2T a = 27 ms 2Ta= 18-27 ms 2TN = 1/(2JNC-) <5 = gradient + field recovery delay 0 < k < Ta/t2,inax- Phase cycling scheme for the in-phase spectrum is 0i = y 02 = x, — x + States-TPPI 03 = x 0rec = x, — x 0 = y. For the antiphase spectrum, f is incremented by 90°. The intraresidual and sequential connectivities are distinguished from each other by recording the antiphase and in-phase data sets in an interleaved manner and subsequently adding and subtracting two data sets to yield two subspectra.

Fig. 21. Schematic illustration of MP-HNCA-TROSY antiphase (a) and in-phase (b) spectra with long acquisition time in q. The corresponding subspectra are shown after addition of the antiphase and in-phase data sets (c) and after subtraction of the antiphase and in-phase data sets (d). Due to very small Vcc > the intraresidual cross peaks are almost entirely cancelled out from the antiphase spectrum (a). In the subspectra, the intraresidual cross peaks are shown as doublets, separated by 53 Hz splitting in Fi-dimension, whereas sequential cross peaks are shown as singlets, and they exhibit 53 Hz offset for the upheld and downfield components between the subspectra.

Although, the MP-HNCA-TROSY experiment alone can yield sequential assignment, it can be also used concomitantly with the HNCA-TROSY experiment. This strategy is explained later, but let us first focus on the coherence transfer efficiency of the MP-HNCA-TROSY experiment. The transfer functions for the antiphase experiment (the efficiency for the in-phase experiment is practically the same) are calculated according to Eqs. (10) and (11) for the intraresidual... 

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