Sequential 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,...

Thus, the magnetization is transferred from the amide proton to the attached nitrogen and then simultaneously to the intra- and interresidual 13C spins and sequential 13C spin. The 13C chemical shift is labelled during /, and 13C frequency during t2. The desired coherence is transferred back to the amide proton in the identical but reverse coherence transfer pathway. The 15N chemical shift is frequency labelled during t3, and implemented into the 13C 15N back-INEPT step. The sensitivity of the HNCOmCA-TROSY experiment is excellent and nearly similar to HNCA-TROSY except for the inherent sensitivity loss by a factor of /2, arising from additional quadrature detection needed for 13C frequency discrimination in the fourth dimension. The excellent sensitivity is due to a very efficient coherence transfer pathway,... 

Fig. 15. Coherence transfer efficiencies as a function of delay 2TC for the residues in a-helix and ffisheet in the sequential HNCA-TROSY scheme. Equation (8) is plotted using the following parameters T2 un-trosy = 50 ms, T2 o = 25 ms, 2Ta = 25 ms,...

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... 

Heteronuclear triple resonance 3D sequential HNCA-TROSY... 

The first part, before the t period, of the experiment is identical to the HN(CO)CA-TROSY scheme. This step chooses solely the sequential pathway in an HN(CO)CA-TROSY manner. The chemical shift of the 13C nucleus is recorded during the t evolution period. The back-transfer route is, however, quite different. We transfer the desired coherence from 13C directly back to the 15N nucleus and remove the second 13C -> 13C INEPT step found in HN(CO)CA-TROSY and replace it with the HNCA like back-transfer step. The antiphase 2./N<> coupling then refocuses simultaneously with VNc during the 13C 15N back-INEPT step. Thus, the HN(CO)CANH-TROSY... 

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