The greatly improved sensitivity resulting from the use of TROSY during 15N evolution and amide proton acquisition enables the recording of HNCA spectra of large proteins with constant-time 13Cα evolution. In [13C]-ct-[15N,1H]-TROSY-HNCA experiments with a 2H/13C/15N-labeled 110kDa protein, 7,8-dihydroneopterin aldolase from Staphylococcus aureus, nearly all correlation peaks seen in the [15N,1H]-TROSY-HNCA spectrum were also detected. The improved resolution in the 13C dimension then enabled a significant number of sequential assignments that could not be obtained with [15N,1H]-TROSY-HNCA without [13C]-constant-time perio

Pervushin, Konstantin

Salzmann, Michael

Senn, Hans

Wider, Gerhard

Wüthrich, Kurt

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Journal of Biomolecular NMR, 14: 85–88, 1999.KLUWER/ESCOM© 1999 Kluwer Academic Publishers. Printed in the Netherlands.85[13C]-constant-time [15N,1H]-TROSY-HNCA for sequential assignments oflarge proteinsMichael Salzmanna,b, Konstantin Pervushina, Gerhard Widera, Hans Sennb & Kurt Wüthricha,∗aInstitut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093Zürich, SwitzerlandbF. Hoffmann-La Roche AG, Pharma Research, CH-4070 Basel, SwitzerlandReceived 1 March 1999; Accepted 17 March 1999Key words: HNCA with 13C constant-time evolution, protein NMR, resonance assignments, spectral resolution,triple resonance experiments, TROSYAbstractThe greatly improved sensitivity resulting from the use of TROSY during 15N evolution and amide protonacquisition enables the recording of HNCA spectra of large proteins with constant-time 13Ca evolution. In [13C]-ct-[15N,1H]-TROSY-HNCA experiments with a 2H/13C/15N-labeled 110 kDa protein, 7,8-dihydroneopterin aldolasefrom Staphylococcus aureus, nearly all correlation peaks seen in the [15N,1H]-TROSY-HNCA spectrum werealso detected. The improved resolution in the 13C dimension then enabled a significant number of sequentialassignments that could not be obtained with [15N,1H]-TROSY-HNCA without [13C]-constant-time period.Abbreviations: CSA, chemical shift anisotropy; ct, constant-time; DD, dipole–dipole; INEPT, insensitive nucleienhanced by polarization transfer; PFG, pulsed field gradient; rf, radio-frequency; ST2-PT, single transition-to-single transition polarization transfer; 3D, three-dimensional; TPPI, time-proportional phase incrementation;TROSY, transverse relaxation-optimized spectroscopy.IntroductionAssignment of chemical shifts to individual nucleiprovides the foundation for protein structure deter-mination by NMR (Wüthrich et al., 1982; Wüthrich,1986), and hence obtaining sequence-specific reso-nance assignments for larger proteins is a necessaryrequirement for further extension of the size limitfor NMR structure determination. Multidimensionaltriple-resonance NMR experiments with 2H/13C/15N-labeled proteins have so far been the most successfulapproach in this regard (LeMaster, 1994; Yamazakiet al., 1994a,b; Shan et al., 1996; Kay and Gardner,1997; Gardner et al., 1998), and with the introduc-tion of TROSY (Pervushin et al., 1997) into tripleresonance experiments (Salzmann et al., 1998, 1999)the potentialities of this approach have been furtherlargely extended. With the constructive use of the in-∗To whom correspondence should be addressed.terference between dipole–dipole (DD) coupling andchemical shift anisotropy (CSA) interactions in 15N–1HN moieties, up to three-fold sensitivity gains wereobtained using TROSY-type triple resonance experi-ments with a 23 kDa protein when compared to thecorresponding conventional implementations (Salz-mann et al., 1998, 1999; Yang and Kay, 1999). Inlarge proteins, full use of the improved sensitivity islimited by signal overlap in the 13C dimension, whichmay prevent unique correlation of backbone 15N–1HN groups with the intraresidually and sequentiallyadjoining 13Ca, 13Cb or 13CO spins. Since the spin-selective state of 15N can be preserved during a [13C]-constant-time (ct) evolution period, the improved sen-sitivity due to the use of TROSY now opens an avenueto overcome this limitation by introducing ct 13C evo-lution periods, which eliminate homonuclear 13C–13Cscalar couplings and provide much narrower lines inthe 13C dimension (Vuister and Bax, 1992; Yamazaki,86Figure 1. Experimental scheme for the [13C]-ct-[15N,1H]-TROSY-HNCA experiment for deuterated proteins (for the modifications needed forprotonated proteins, see Salzmann et al., 1998). The radio-frequency (rf) pulses on 1H, 15N, 13CO, 13Ca and 2H were applied at 4.8, 119, 174,55 and 3.8 ppm, respectively. The narrow and wide black bars indicate non-selective 90◦ and 180◦ pulses, respectively. On the line marked1H, sine bell shapes indicate selective 90◦ pulses with a duration of 1 ms and a Gaussian shape truncated at 5%, which are applied on thewater resonance. The line marked PFG indicates durations and amplitudes of Gaussian shape pulsed magnetic field gradients applied along thez-axis: G1: 800 ms, 15 G/cm; G2: 800 ms, 9 G/cm; G3: 800 ms, 22 G/cm. The delays are t = 2.7 ms, TN = 18.2 ms and TC = 26.2 ms.The phase cycle is 91 = {y, −y, x, −x}, 92 = {−y}, 93 = {-y}, #1 = {4x, 4(−x)} and #rec = {y, −y, −x, x, −y, y, x, −x}. All otherrf-pulses are applied either with phase x or as indicated above the pulses. In the 15N(t1) dimension a phase-sensitive spectrum is obtained byrecording a second FID for each increment of t1, with 91 = {y, −y, −x, x}; 92 = {y}; 93 = {y}, and the data is processed as described byKay et al. (1992). Quadrature detection in the 13Ca(t2) dimension is achieved by the States-TPPI method (Marion et al., 1989) applied to thephase #1. The water magnetization stays aligned along the +z-axis throughout the experiment by the use of water flip-back pulses (Grzesiekand Bax, 1993) at times a, b and f . Residual transverse water magnetization is suppressed by the WATERGATE sequence (Piotto et al., 1992)immediately before data acquisition. For 2H-decoupling, WALTZ-16 (Shaka et al., 1983) was used with a field strength of 2.5 kHz. 13COdecoupling was performed using off-resonance SEDUCE-1 (McCoy and Mueller, 1992) with a field strength of 0.83 kHz, which allows theapplication of rf-pulses on the 13Ca channel with a field strength of 20.83 kHz.1994a). This communication describes an implemen-tation of [13C]-ct-[15N,1H]-TROSY-HNCA and its ap-plication with a 2H/13C/15N-labeled 110 kDa protein,the 7,8-dihydroneopterin aldolase from Staphylococ-cus aureus (Hennig et al., 1998).A 3D HNCA experiment yields two 13Ca corre-lation peaks for each 15N(i)–1HN(i) amide moiety,the intraresidual peak with 13Ca(i) and the sequentialpeak with 13Ca(i-1), so that for larger proteins oneends up with a crowding of cross peaks along the13C frequency axis (Montelione and Wagner, 1989;Ikura et al., 1990; Grzesiek and Bax, 1992). In theimplementation of the HNCA or [15N,1H]-TROSY-HNCA experiments one works with limited resolutionin the 13C dimension to prevent that the homonuclearscalar couplings between 13Ca and 13Cb are resolved(Wider, 1998). The introduction of a [13C]-ct evolu-tion period of duration TC = 1/1J(13Ca,13Cb) enablesa three-fold increase in resolution for the 13Ca dimen-sion without the appearance of 13Ca–13Cb couplings,and the carbonyl carbons can in any case readily bedecoupled from 13Ca by selective composite pulsedecoupling (McCoy and Mueller, 1992). The use ofa [13C]-ct evolution period, however, increases thetime period during which transverse 13Ca magneti-zation is active by approximately 20 ms. In view ofthe concomitant loss of sensitivity, we present here aninitial application with the intrinsically highly sensi-tive [15N,1H]-TROSY-HNCA experiment (Salzmannet al., 1998), using uniformly deuterated proteins.Figure 1 shows an experimental scheme for [13C]-ct-[15N,1H]-TROSY-HNCA, where TROSY is imple-mented as recently described (Salzmann et al., 1998,1999). Both the 1H and 15N steady-state magnetiza-tions are used at the outset of the experiment (Per-vushin et al., 1998a,b). Between the time points aand d, the magnetization is transferred from 1HN via15N to 13Ca, using two successive INEPT steps. Dur-ing the [13C]-ct evolution period, 13Ca evolves dueto the 13Ca chemical shift only, since 13CO, 15N and87Figure 2. Comparison of corresponding [&2(13C),&3(1H)] strips from HNCA spectra obtained with and without 13C constant time evolution:(a) [13C]-ct-[15N,1H]-TROSY-HNCA recorded with the scheme of Figure 1; (b) [15N,1H]-TROSY-HNCA (Salzmann et al., 1998). The stripswere taken at the 15N chemical shifts indicated at the bottom, and are centered about the corresponding 1HN chemical shifts. At the topthe sequence-specific assignments are indicated by the one-letter amino acid symbol and the sequence number. In (a) the three sequentialconnectivities are indicated by horizontal lines. (a′) and (b′) show cross sections along the &2(13C) dimension through the centers of the stripsin (a) and (b), respectively, where the chemical shift range from 52 to 62 ppm is plotted. The signal-to-noise ratios for the main peak in eachcross section are indicated at the top. Both spectra were recorded at 20 ◦C with the same 0.5 mM sample of uniformly 2H/13C/15N-labeled7,8-dihydroneopterin aldolase from S. aureus (Hennig et al., 1998) in a mixed solvent of 95% H2O/5% D2O at pH 6.5, using a BrukerDRX-750 spectrometer equipped with four rf channels for generating the 1H, 2H, 13C and 15N rf-pulses, a pulsed field gradient unit and atriple resonance probehead with an actively shielded z-gradient coil. When recording the spectrum (b), the previous implementation of the[15N,1H]-TROSY-HNCA experiment (Salzmann et al., 1998) was modified so that 13CO decoupling by Gaussian off-resonance pulses wasreplaced by 0.83 kHz off-resonance SEDUCE-1 decoupling (McCoy and Mueller, 1992) and the rf-pulses on the 13Ca channel were appliedwith a field strength of 20.83 kHz, exactly as described for [13C]-ct-[15N,1H]-TROSY-HNCA in Figure 1. The following parameter settingswere used for (a): data size 42(t1) × 128(t2) × 1024(t3) complex points; t1max(15N) = 17.4, t2max(13Ca) = 25.6, t3max(1H) = 75.6 ms;spectral widths in &1(15N), &2(13Ca) and &3(1H) 2400, 5000 and 13550 Hz, respectively; 16 scans per increment were acquired, resulting in4 days of measuring time. The data set was zero-filled to 128(t1) × 256(t2) × 2048(t3) complex points. In (b) the same parameters were usedexcept for the following: data size in t2 was 40 points, t2max (13Ca) = 8 ms, 48 scans per increment were acquired in 4 days of measuringtime, and zero-filling in t2 was to 128 complex points. Prior to Fourier transformation both data matrices were multiplied with a 75◦-shiftedsine bell window in the indirect dimensions and a 60◦-shifted sine bell window in the acquisition dimension (DeMarco and Wüthrich, 1976).Data processing was performed using the program PROSA (Güntert et al., 1992), and for the data analysis the program XEASY (Bartels et al.,1995) was used.2H are decoupled during t2, and TC is chosen suchthat TC = 1/1J(13Ca,13Cb) (Vuister and Bax, 1992).The magnetization is transferred back to 15N at timepoint e. Between e and f the 15N nuclei evolve withtheir chemical shifts during a ct period, and from timepoint f onward the ST2-PT element transfers the 15Nsingle-transition to the 1H single transition selectedin TROSY (Pervushin et al., 1998a). The water mag-netization is maintained along the +z-axis during theentire experiment, using selective pulses on the waterresonance at the time points a, b and f (Grzesiek andBax, 1993). The coherence flow in [13C]-ct-[15N,1H]-TROSY-HNCA can thus be described byHi → Ni → Cai,i−1(t2)→ Ni(t1)→ Hi(t3) (1)where H, N and Ca represent the 1HN, 15N and 13Camagnetizations, t1 and t2 are the 15N and 13C evo-lution times, and t3 is the 1H acquisition time. Theindices i and i − 1 indicate that coherence is trans-ferred from 15N(i)–1HN(i) to both the sequentially andthe intraresidually adjoining 13Cas. The periods dur-ing which TROSY is active are underlined: on 15Nit is active during the 15N–13Ca INEPT and the re-verse 13Ca–15N INEPT, including the ct 15N evolutionperiod t1, and on 1HN it acts during the data acqui-sition period t3. During the [13C]-ct evolution periodthe 15N spin-selective state is preserved. In principle,88the pulse sequence of Figure 1 could be adapted foruse with protonated proteins by replacing deuteriumdecoupling with selective decoupling of the a-protons(Salzmann et al., 1998), but fast 13Ca relaxation willlimit such applications.Figure 2, a and b, shows corresponding[&2(13C),&3(1H)] strips from 3D [13C]-ct-[15N,1H]-TROSY-HNCA and 3D [15N,1H]-TROSY-HNCAspectra of a uniformly 2H/13C/15N-labeled proteinrecorded with identical conditions. The protein studiedis the 7,8-dihydroneopterin aldolase from Staphylo-coccus aureus, which has a molecular weight of 110kDa and consists of eight identical subunits with 121amino acid residues. The protein concentration was0.5 mM, i.e., 4 mM in subunits. From measure-ments of T1(15N)/T2(15N) we obtained an estimatefor the lower limit on the effective rotational cor-relation time at 20 ◦C of tc ≥ 45 ns (Kay et al.,1989). The strips were taken at the 15N chemical shiftsof the residues 68, 69, 119, 120, 90 and 91, andthe horizontal lines in Figure 2a indicate the sequen-tial connectivities for the three pairs of neighboringresidues. The improved spectral resolution in [13C]-ct-[15N,1H]-TROSY-HNCA enabled the unambiguousidentification of the sequential 1HN–13Ca connectiv-ities in the crowded 13Ca frequency range around56 ppm, whereas with [15N,1H]-TROSY-HNCA noreliable sequential assignment was possible because ofspectral overlap (Figure 2b).The [13C]-ct period in [15N,1H]-TROSY-HNCAis of special interest for large proteins with crowdedspectra (Gardner et al., 1998). In spite of the decreaseof signal-to-noise (see Figure 2, a′ and b′), a workablesensitivity is maintained. For example, in the presentlystudied aldolase one expects 117 intraresidual and 117sequential HNCA cross peaks. The numbers of peaksseen by [15N,1H]-TROSY-HNCA were 117 and 114,and by [13C]-ct-[15N,1H]-TROSY-HNCA they were110 and 103, i.e., 94% of all expected intraresidualand 88% of all expected sequential peaks were seenusing 3D [13C]-ct-[15N,1H]-TROSY-HNCA.AcknowledgementsFinancial support was obtained from the Schweiz-erischer Nationalfonds (project 31.49047.96). Wethank Bernard Gsell for help in preparing the NMRsample of Staphylococcus aureus 7,8-dihydroneopterinaldolase.ReferencesBartels, C., Xia, T., Billeter, M., Güntert, P. and Wüthrich, K. (1995)J. Biomol. NMR, 6, 1–10.DeMarco, A. and Wüthrich, K. (1976) J. Magn. Reson., 24, 201–204.Gardner, K.H., Zhang, X., Gehring, K. and Kay, L.E. (1998) J. Am.Chem. Soc., 120, 11738–11748.Grzesiek, S. and Bax, A. (1992) J. Magn. Reson., 96, 432–440.Grzesiek, S. and Bax, A. (1993) J. Am. Chem. 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[13c]-Constant-Time [15n,1h]-Trosy-Hnca for Sequential Assignments of Large Proteins

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[13c]-Constant-Time [15n,1h]-Trosy-Hnca for Sequential Assignments of Large Proteins

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