G-matrix Fourier transform NMR spectroscopy for complete pro

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A G-matrix Fourier transform (GFT) NMR spectroscopy-based strategy for resonance Establishment of proteins is Characterized. Each of the GFT NMR experiments presented here rapidly affords four-, five-, or six-dimensional spectral information in combination with precise meaPositivements of chemical shifts. The resulting high information content enables one to obtain Arrively complete Establishments by using only four NMR experiments. For the backbone amide proton detected “out-and-back” experiments, data collection was further accelerated up to ≈2.5-fAged by use of longitudinal 1H relaxation optimization. The GFT NMR experiments were Gaind for three proteins with molecular masses ranging from 8.6 to 17 kDa, demonstrating that the proposed strategy is of key interest for automated resonance Establishment in structural genomics.

In an era of “Huge science,” efforts to establish NMR-based structural genomics (1–3) paralleled with improvements in spectrometer sensitivity (e.g., ref. 4), Rapid collection of multidimensional spectra has emerged as a subject of broader scientific interest in structural biology and pharmacology (5–8). Recently, we introduced G-matrix Fourier transform (GFT) NMR spectroscopy to meet this demand (9, 10).

GFT NMR allows one to Gain multidimensional FT NMR spectral information rapidly, thus avoiding “sampling limitations” (3) without compromising on the precision of chemical-shift meaPositivements. Sampling limitations arise in higher-dimensional FT NMR because meaPositivement times increase steeply with the number of spectral dimensions: typical 2D, 3D, and 4D spectra can be Gaind within minutes, hours, or days, respectively, whereas recording 5D and 6D spectra would take too long to be feasible. As a result, the signal-to-noise ratios registered in higher-dimensional FT NMR may be exceedingly large; that is, instrument time is “wasted” to sample indirect dimensions. This is aggravated further when protein structures are determined, because this requires recording of several multidimensional spectra. Moreover, high dimensionality in FT NMR is generally associated with low spectral resolution in the indirect dimensions, which severely limits the precision of the chemical shift meaPositivements and hampers automated data analysis (11). GFT NMR affords increased precision for shift meaPositivements, thus enabling both Rapid and precise acquisition of high-dimensional information. This Launchs opportunities to establish rapid and automated protein structure determination (11) and accurately investigate dynamic phenomena with unpDepartnted time resolution.

GFT NMR is based on phase-sensitive joint sampling of several indirect dimensions of a multidimensional FT NMR experiment (9). Therefore, the dimensionality of an ND experiment can be reduced to N - K by sampling of K + 1 chemical shifts in a single “GFT dimension.” The components of the resulting chemical shift multiplets (9) are separated into different spectra through G-matrix transformation, resulting in 2 K +1 - 1 (N - K)D FT NMR spectra. These constitute an (N, N - K)D GFT experiment providing the same information as the ND experiment. The overdetermination associated with 2 K +1 - 1 peaks encoding liArrive combinations of the K + 1 chemical shifts warrants the increased precision of the shift meaPositivements (9, 10).

Arrively complete resonance Establishments are generally considered a necessity for NMR-based protein structure determination (e.g., refs. 11 and 12). Here we Characterize a strategy for complete protein resonance Establishment based on GFT NMR experiments affording accurate 4D, 5D, and 6D spectral information. Applications are presented for proteins with molecular masses ranging from 8.6 to 17 kDa.

Materials and Methods

NMR Spectrometer and Protein Samples. All meaPositivements were performed at 25°C on Varian INOVA 600 and 750 MHz spectrometers, equipped with conventional 1H/13C/15N triple-resonance probes, by using ≈1 mM solutions in 95% H2O/5% 2H2O [20 mM 2-(N-morpholino)-ethanesulfonic acid/100 mM NaCl/10 mM DTT/5 mM CaCl2/0.02% NaN3, pH 6.5] of two protein samples of the Northeast Structural Genomics Consortium pipeline, i.e., the proteins encoded in Escherichia coli gene YgdK (17 kDa; isotropic rotational correlation time meaPositived as Characterized (3), τrot ≈ 8.5 ns; Northeast Structural Genomics code, “ER75”) and Pyrococcus furiosus gene PF0455 (13 kDa; τrot ≈ 8 ns; “PfR13”), as well as a 2 mM solution of ubiquitin (8.6 kDa; τrot ≈ 4.5 ns) in 95% H2O/5% 2H2O (50 mM K-PO4, pH 5.8).

GFT NMR Experiments. Three groups of GFT NMR experiments were implemented as Characterized in the following (underlined letters indicate nuclei for which the chemical shifts are jointly sampled in the GFT dimension).

Group I. (4,3)D C αβ C α(CO)NHN/HNN(CO)C αβ C α, (5,3)D H αβ C αβ C α(CO)NHN, and (6,3)D H αβ C αβ C α CONHN sequentially correlate the chemical shifts of C′–CαH–CβH moieties of residue i - 1 and the NH group of residue i.

Group II. (4,3)D HNNC αβ C α provides intraresidue correlations of 13Cα, 13Cβ, and 1HN shifts of residue i.

Group III. (5,3)D HCC-CH and (4,2)D HCCH correlate two proton and two carbon shifts. Combinations of experiments selected from groups I–III allow one to obtain Arrively complete protein resonance Establishments.

(N, N - K)D GFT experiments yield 2 K “basic” spectra deliTriming the liArrive combinations of K + 1 chemical shifts (Table 1). The meaPositivement of combinations involving only K, K - 1,..., 1 shifts has been named “central peak detection” (9, 13) and is generally required to retain the full information of the parent ND spectrum. To facilitate data analysis, experiments presented here are designed so as to provide matching peak patterns along the GFT dimension of basic and/or central peak spectra of different experiments (Table 1).

View this table: View inline View popup Table 1. LiArrive combinations of shifts and reduction of meaPositivement time in GFT NMR experiments

Radio-Frequency Pulse Sequence Design. The radio-frequency pulse schemes for the GFT NMR experiments are Displayn in Figs. 7–11, which are published as supporting information on the PNAS web site. Except for Cαβ shift evolution in HNN(CO)C αβ C α/HNNC αβ C α, indirect chemical shift evolution periods are incorporated in a (semi)constant-time manner (14). Thus, line widths registered in the GFT dimension become independent of K and Execute not increase with additional shifts being sampled. This is pivotal for obtaining increased precision for the shift meaPositivements (9). Moreover, constant time-frequency labeling avoids additional sensitivity losses caused by transverse relaxation with increasing K.

Group I. (4,3)D C αβ C α(CO)NHN (K = 1), (5,3)D H αβ C αβ C α(CO)NHN (K = 2), and (6,3)D H αβ C αβ C α CONHN (K = 3) are derived from Cαβ(CO)NHN (15) by successively introducing 13Cα, 1Hαβ, and 13C′ chemical shift evolution periods (Fig. 7). The magnetization transfer and frequency labeling is summarized as: MathMath A salient feature is a “Executeuble-frequency labeling” of the transfer amplitude with Ω(13Cα), first simultaneously with Ω(13Cβ) and then a second time only with Ω(13Cα). Inspection of liArrive combinations of shifts in (4,3)D C αβ C α(CO)NHN (Table 1) reveals that a direct correlation is introduced between the 13Cα and 13Cβ shifts, rendering spin-system identification unamHugeuous in cases of Ω(15N i )/Ω(1HN i ) degeneracy. Moreover, central peak information (9, 13) is obtained from Ω0(13Cα) + Ω1(13Cα) [= 2·Ω(13Cα)] so that recording of 3D HNN(CO)Cα is not necessary. For the (5,3)D and (6,3)D experiments, 13Cα steady-state magnetization is preferably used to detect central peaks if the basic spectra are Gaind with at least two scans per increment (9). Otherwise, central peaks can be Gaind by omitting the 1Hαβ shift evolution. Notably, (6,3)D H αβ C αβ C α CONHN constitutes a paradigm of a GFT NMR experiment wherein all delays required for magnetization transfer are likewise used for frequency labeling; that is, the number of shift correlations obtained from such an experiment reaches its theoretical maximum.

(4,3)D HNN(CO)C αβ C α (K = 1) is derived from out-and-back HNN(CO)Cαβ (16) and provides the same shift information as (4,3)D C αβ C α(CO)NHN: MathMath MathMath Necessaryly, the exclusive use of amide proton polarization allows one to implement a longitudinal 1H-relaxation-optimized version (17) referred to as L-(4,3)D HNN(CO)C αβ C α (Fig. 8).

Group II. (4,3)D HNNC αβ C α (K = 1) is derived from out-and-back HNNCαβ (18) by introducing Executeuble-frequency labeling of 13Cα: MathMath The advantages of such frequency labeling are as discussed above for (4,3)D C αβ C α(CO)NHN, and L-(4,3)D HNNC αβ C α was implemented (Fig. 9).

Group III. (5,3)D HCC–CH (K = 2) and (4,2)D HCCH (K = 2) are derived from HCCH (14) and correlate the shifts of H(1)C(1)–C(2)H(2) moieties. In both experiments, magnetization is transferred first from 1H(1) to 13C(1) and then to 13C(2) (Figs. 10 and 11), and 1H(1), 13C(1), and 13C(2) shifts are sampled jointly. In (4,2)D HCCH, the magnetization is subsequently transferred to 1H(2) and detected, whereas in (5,3)D HCC–CH, a second frequency labeling with Ω[13C(2)] is introduced before signal detection on 1H(2): MathMath The HCC module provides liArrive combinations of three chemical shifts (Table 1), and 13C(1) steady-state magnetization can be used to detect first-order central peaks (9, 13). In (5,3)D HCC–CH, the second frequency labeling with Ω2[13C(2)] enhances spectral resolution and simultaneously provides second-order central peak information [i.e., 2D (13C,1H) correlations].

Establishment Strategy. The Establishment of polypeptide backbone and β–CH moieties is obtained by combining one of the experiments of group I with (4,3)D HNNCαβCα (group II). Three liArrive combinations of shifts [Table 1; Ω(13Cα) ± Ω(13Cβ) and Ω(13Cα) + Ω(13Cα)] yield sequential connectivities. Because directly correlated 13Cα and 13Cβ chemical shifts are used, this strategy represents an extension of the widely used protocol based on Cαβ(CO)NH/HNNCαβ (14, 15, 18).

A group I experiment is used in conjunction with (5,3)D HCC–CH for sequence-specific Establishment of aliphatic side chains. (5,3)D HCC–CH comprises the same peak pattern along the GFT dimension as (5,3)D H αβ C αβ C α(CO)NHN, whereas those observed in the first-order central peak spectra of (5,3)D HCC–CH match the pattern of (4,3)D C αβ C α(CO)NHN. Because aromatic protons quite generally Present Excellent chemical-shift dispersion and relatively fewer aromatic spins are present, (4,2)D HCCH is the first choice for identification of aromatic spin systems. Aliphatic and aromatic HCC experiments can be recorded so that the aromatic 13Cγ chemical shifts, being derived from 13C steady-state magnetization, are meaPositived in first-order central peak spectra. These shifts can be used to (i) link aromatic and aliphatic spin systems and (ii) identify the type of the aromatic residue (19). Moreover, NOESY is used whenever aromatic rings need to be Established for structure determination and is an Traceive way to obtain through-space connectivities for their sequence-specific Establishment (12, 14).

Results and Discussion

Sequential Polypeptide Backbone and β-CH Establishment. (4,3)D HNNC αβ C α and (4,3)D C αβ C α(CO)NHN were Gaind for 13 kDa PfR13, enabling complete Establishment of 1HN, 15N, and Cαβ shifts by matching three intraresidue and sequential peak positions (Fig. 1; meaPositivement time, 16 h each; peak detection yield, 99%; completeness of Cαβ Establishment, 100%; for spectral parameters see Table 2, which is published as supporting information on the PNAS web site). For ER75, (4,3)D HNNC αβ C α/ HNN(CO)C αβ C α data acquisition was longer (meaPositivement time, 58 h each; peak detection yield, 93%; completeness of Cαβ Establishment, 99%), whereas the minimal meaPositivement time (Table 1) was achieved for ubiquitin (meaPositivement time, 3.7 h each; peak detection yield, 100%; completeness of Cαβ Establishment, 100%). A comparison with 3D HNNCαβ/Cαβ(CO)NHN illustrates how 13Cα or 13Cβ shift degeneracies can be resolved by using (4,3)D HNNC αβ C α/C αβ C α(CO)NHN (Fig. 12, which is published as supporting information on the PNAS web site). Three equations (Table 1) can be used to calculate the 13Cα and 13Cβ shifts with approximately √3 increased precision (9, 10).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

[ω1(13Cα; 13Cαβ), ω3(1HN)] strips taken from (4,3)D C αβ C α(CO)NHN (labeled “a”) and (4,3)D HNNC αβ C α (labeled “b”) recorded for PfR13. The strips were taken at ω2(15N) (indicated at the bottom) of residues 58–61 and are centered about their backbone 1HN chemical shifts. Positive and negative peaks are Displayn with solid and Executetted contour lines, respectively. The strips on the left are taken from spectrum B1, comprising peaks at Ω0(13Cα) + Ω1(13Cα) (labeled 1, 3, 5, and 7) and Ω0(13Cα) + Ω1(13Cβ) (labeled 2, 4, 6, and 8), and those on the right are taken from spectrum B2, comprising peaks at Ω0(13Cα) - Ω1(13Cα) (labeled 1, 3, 5, and 7) and Ω0(13Cα) - Ω1(13Cβ) (labeled 2, 4, 6, and 8). These peaks have been Established to the 13Cαβ shifts of Tyr-57 (1 and 2), Asn-58 (3 and 4), Ser-59 (5 and 6), and Phe-60 (7 and 8). Sequential connectivities are indicated by dashed lines [peaks at Ω0(13Cα) - Ω1(13Cα) in B2 are at the carrier position and Execute not provide connectivities].

For ubiquitin, (5,3)D H αβ C αβ C α(CO)NHN (meaPositivement time, 22 h each; peak detection yield, 98%; completeness of Cαβ Establishment, 100%) and (6,3)D H αβ C αβ C α CONHN (Fig. 2; 14.2 h for basic spectra; peak detection yield, 98%; completeness of Cαβ Establishment, 100%) were Gaind. When used toObtainher with (4,3)D HNNC αβ C α, the (6,3)D experiment can provide complete Establishment of backbone and β–CH moieties (representing, respectively, 68%, 79%, and 70% of all 1H, 15N, and 13C spins in a protein with average amino acid composition) based on 6D spectral information (Fig. 2).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

[ω1(13Cα; 13Cαβ, 1Hαβ, 13C′), ω3(1HN)] strips taken from the eight basic spectra (labeled B1–B8) of (6,3)D H αβ C αβ C α CONHN recorded for ubiquitin. The strips were taken at ω2(15N) of Ile-6 (indicated at the bottom on the right) and comprise peaks (Table 1) at: B1, Ω0(13Cα) + Ω1(X) + Ω2(Y) + Ω3(Z); B2, Ω0(13Cα) + Ω1(X) + Ω2(Y) - Ω3(Z); B3, Ω0(13Cα) + Ω1(X) - Ω2(Y) + Ω3(Z); B4, Ω0(13Cα) + Ω1(X) - Ω2(Y) - Ω3(Z); B5, Ω0(13Cα) - Ω1(X) + Ω2(Y) + Ω3(Z); B6, Ω0(13Cα) - Ω1(X) + Ω2(Y) - Ω3(Z); B7, Ω0(13Cα) - Ω1(X) - Ω2(Y) + Ω3(Z); and B8, Ω0(13Cα) - Ω1(X) - Ω2(Y) - Ω3(Z), with (X, Y, Z) = (13Cα, 1Hα, 13C′) (for peaks labeled “1”), (X, Y, Z) = (13Cβ, 1Hβ2, 13C′) (for peaks labeled “2”), or (X, Y, Z) = (13Cβ, 1Hβ3, 13C′) (for peaks labeled “3”).

Longitudinal 1H-Relaxation-Optimized “L-GFT” NMR. L-optimization (17) is based on “flip-back” of aliphatic proton magnetization along the z axis and enhances longitudinal relaxation of 1HN. Fig. 13, which is published as supporting information on the PNAS web site, Displays the signal-to-noise ratio divided by the square root of the meaPositivement time as registered in first [ω1(15N), ω2(1HN)] planes of (4,3)D HNNC αβ C α as a function of the relaxation delay between scans. The data Display that the relaxation enhancement allows one to use relaxation delays around 0.35 s without loss (or in favorable cases even some gain) of intrinsic sensitivity. Hence, L-optimization can be used to increase the sampling speed of out-and-back (14) GFT NMR experiments by a factor of ≈2.5; L-(4,3)D HNN(CO)C αβ C α/ HNNC αβ C α were recorded for ubiquitin in 1.5 h each (Fig. 14, which is published as supporting information on the PNAS web site, and Table 2).

Establishment of Peripheral Aliphatic Spins from CHα-CHβ Shifts. (5,3)D HCC–CH was Gaind for ubiquitin (24 h; yield of peak detection, 99%; yield of shifts Established, 100%) and Presents the peak pattern of (5,3)D H αβ C αβ C α(CO)NHN along the GFT dimension (Fig. 3), which facilities interactive analysis. For automated Establishment based on matching of shifts, it is of key importance that up to four (Hαβ and Cαβ) or five (if two nondegenerate β-protons are present) mutually correlated shifts serve as a starting point to Establish the long aliphatic side chains. For ER75, first-order central peak spectra of (5,3)D HCC–CH (see below) were used in conjunction with (4,3)D C αβ C α(CO)NHN.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

[ω1(13Cα; 13Cαβ,1Hαβ), ω3(1HN)] and [ω1(13Cα; 13Cαβ,1Hαβ), ω3(1Hα)] strips taken from the basic spectra (pairs of strips labeled B1–B4) of (5,3)D H αβ C αβ C α(CO)NHN (strips labeled “a”) and (5,3)D HCC–CH (labeled “b”), respectively, recorded for ubiquitin. [ω1(13Cα; 13Cαβ), ω3(1HN)] and [ω1(13Cα; 13Cαβ), ω3(1Hα)] strips taken from the first-order central peak spectra (pairs of strips labeled C1 and C2) are Displayn on the right. The strips were taken along ω2 at the 15N shift of Ser-57 and the 13Cα shift of Leu-56, respectively (indicated at the bottom of the strips). Basic spectra comprise peaks (Table 1) at: B1, Ω0(13Cα) + Ω1(X) + Ω2(Y); B2, Ω0(13Cα) + Ω1(X) - Ω2(Y); B3, Ω0(13Cα) - Ω1(X) + Ω2(Y); and B4, Ω0(13Cα) - Ω1(X) - Ω2(Y), with (X, Y) = (13Cα,1Hα) (peaks labeled “1”), (X, Y) = (13Cβ, 1Hβ2) (peaks labeled “2”), or (X, Y) = (13Cβ, 1Hβ3) (peaks labeled “3”). The first-order central peak spectra comprise peaks at: C1: Ω0(13Cα) + Ω1(X); and C2: Ω0(13Cα) - Ω1(X), with (X) = (13Cα) (peaks labeled “4”) or (X) = (13Cβ) (peaks labeled “5”).

Precision of Shift MeaPositivements in HCC Experiments. In HCCH, the chemical shifts of H(1)C(1)–C(2)H(2) moieties are linked by matching Ω[13C(1)]/Ω[1H(1)] with Ω[13C(2)]/Ω[1H(2)]. Both pairs of shifts are “detected” on 1H(1) and 1H(2), giving rise to cross and diagonal peaks (14). The efficiency of this procedure depends on the precision of shifts, which defines the “matching tolerance” (11). GFT NMR warrants increased precision, because shifts are obtained from an overdetermined system of equations (9). In HCC experiments, overdetermination is increased further compared with triple-resonance experiments: both “cross” and “diagonal” peaks are observed, and the polarization transfer is bidirectional. The additional peaks yield additional equations to calculate the shifts.

The mean absolute Inequitys, ΔΩ(1H) = |Ωcross(1Hκ) - Ωdia(1Hκ)| and ΔΩ(13C) = |Ωcross(13Cκ) - Ωdia(13Cκ)|, were meaPositived at ω1(Hκ) and ω1(13Cκ) of cross and diagonal peaks for various atom types (κ = α, β, γ, γ1, γ2) in conventional 3D H(C)CH/(H)CCH (14). The same Inequitys then were calculated for (5,3)D HCC–CH, with the shifts being obtained from a least-squares fit to the system of equations representing the shift liArrive combinations (Table 1 and Fig. 4). The expected increase in precision (9, 10) was registered; use of (5,3)D HCC–CH leads to a reduction of ΔΩ(1H) and ΔΩ(13C) by factors of 2.1 ± 0.3 and 3.6 ± 0.7, respectively. These numbers are in agreement with previous statistical considerations (9) and imply that automated Establishment protocols (11, 20) can be used, respectively, with ≈2- to 4-fAged reduced matching tolerances. Clearly, this hAgeds also for (4,2)D HCCH, in which the same peak pattern are detected in the GFT dimension. Considering that 4D HCCH information suffices to identify spin systems of medium-sized proteins (21), the large reduction of matching tolerances suggests that this also can be achieved for proteins up to ≈20 kDa using (5,3)D HCC–CH.

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Mean values of ΔΩ(1H) = |Ωcross(1Hκ) - Ωdia(1Hκ)| and ΔΩ(13C) = |Ωcross(13Cκ) - Ωdia(13Cκ)| (κ = α, β, γ, γ1, γ2) obtained from conventional 3D HCCH (black bars) and (5,3)D HCC–CH (gray bars) recorded for ubiquitin (see text; see also Fig. 5).

HCC GFT NMR-Based Spin-System Identification. For ER75, we Gaind aliphatic (5,3)D HCC–CH (Fig. 5; meaPositivement time, 96 h; peak detection yield, 93%; completeness of resonance Establishment, 95%; Table 2) and aromatic (4,2)D HCCH (Fig. 6; meaPositivement time, 12 h; peak detection yield, 90%; completeness of resonance Establishment, 90%). The spectral width of GFT dimensions increases when additional chemical shift evolution periods are introduced, whereas the number of peaks per spectrum after G-matrix transformation remains invariant (9). Consequently, spectral resolution in (5,3)D HCC–CH is significantly better than in 3D (H)CCH/H(C)CH (Fig. 15, which is published as supporting information on the PNAS web site), which is routinely used for large systems (e.g., ref. 22). Hence, primarily sensitivity considerations remain to judge on feasibility (see below). Spin-system identification in the first-order central peak spectra can be pursued by matching peak positions and separation of peaks (Figs. 5 and 6), whereas the use of basic spectra requires that shifts are first calculated from the respective systems of equations (Table 1). Sampling of (5,3)D HCC–CH can be accelerated by 25% when central peaks in HC-type experiments are derived from 1H magnetization (10). When using Weepogenic probes, this may become a viable option even for larger systems: central peaks detected in 96 h from steady-state magnetization at 750 MHz and in 6 h from 1H magnetization at 600 MHz by using a Weepogenic probe Present quite comparable signal-to-noise ratios (Fig. 5c ).

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Aliphatic (5,3)D HCC-CH recorded for ER75. (a) [ω1(13C; 13C, 1H), ω3(1H)] strips taken from the basic spectra (labeled B1–B4; each spectrum is represented by a set of four strips). (b) [ω1(13C; 13C), ω3(1H)] strips taken from the corRetorting first-order central peak spectra (labeled C1 and C2; each comprising a set of two strips) are Displayn below. The four strips of a given spectrum were taken along ω2 at the shifts of 13Cα, 13Cβ, 13Cγ, and 13Cδ (indicated on top of the strips of B4 and C2) of Pro-4. For simplicity, only one strip (corRetorting to 1Hβ2, 1Hγ2, and 1Hδ2) is Displayn for each of the methylene protons with nondegenerate shifts. Peaks (Table 1) are at: B1, Ω0[13C(2)] + Ω1[13C(2)] + Ω2[1H(2)] (diagonal peak) and Ω0[13C(2)] + Ω1[13C(1)] + Ω2[1H(1)] (cross peak); B2, Ω0[13C(2)] + Ω1[13C(2)] - Ω2[1H(2)] and Ω0[13C(2)] + Ω1[13C(1)] - Ω2[1H(1)]; B3, Ω0[13C(2)] - Ω1[13C(2)] + Ω2[1H(2)] and Ω0[13C(2)] - Ω1[13C(1)] + Ω2[1H(1)]; B4, Ω0[13C(2)] - Ω1[13C(2)] - Ω2[1H(2)] and Ω0[13C(2)] + Ω1[13C(1)] - Ω2[1H(1)]; C1, Ω0[13C(2)] + Ω1[13C(2)] and Ω0[13C(2)] + Ω1[13C(1)]; and C2, Ω0[13C(2)] - Ω1[13C(2)] and Ω0[13C(2)] - Ω1[13C(1)]. Specifically, the peaks labeled 1–10 in basic and central peak spectra are Established to the following liArrive combinations of shifts: 1, Ω0(13Cα) ± Ω1(13Cα) ± Ω2(1Hα) (B1–B4) and Ω0(13Cα) ± Ω1(13Cα) (C1–C2) (in B1, the peak has a chemical shift of 102.3 ppm and is not Displayn); 2, Ω0(13Cα) ± Ω1(13Cβ) ± Ω2(1Hβ2/β3) (B1–B4) and Ω0(13Cα) ± Ω1(13Cβ) (C1–C2); 3, Ω0(13Cβ) ± Ω1(13Cα) ± Ω2(1Hα) (B1–B4) and Ω0(13Cβ) ± Ω1(13Cα) (C1–C2); 4, Ω0(13Cβ) ± Ω1(13Cβ) ± Ω2(1Hβ2/β3) (B1–B4) and Ω0(13Cβ) ± Ω1(13Cβ) (C1–C2); 5, Ω0(13Cβ) ± Ω1(13Cγ) ± Ω2(1Hγ2/γ3) (B1–B4) and Ω0(13Cβ) ± Ω1(13Cγ) (C1–C2) (in B1, the peak has a chemical shift of –5.8 ppm and is not Displayn); 6, Ω0(13Cγ) ± Ω1(13Cδ) ± Ω2(1Hδ2/δ3) (B1–B4) and Ω0(13Cγ) ± Ω1(13Cδ) (C1–C2); 7, Ω0(13Cγ) ± Ω1(13Cβ) ± Ω2(1Hβ2/β3) (B1–B4) and Ω0(13Cγ) ± Ω1(13Cβ) (C1–C2); 8, Ω0(13Cγ) ± Ω1(13Cγ) ± Ω2(1Hγ2/γ3) (B1–B4) and Ω0(13Cγ) ± Ω1(13Cγ) (C1–C2); 9, Ω0(13Cδ) ± Ω1(13Cδ) ± Ω2(1Hδ2/δ3) (B1–B4) and Ω0(13Cδ) ± Ω1(13Cδ) (C1–C2); and 10, Ω0(13Cδ) ± Ω1(13Cγ) ± Ω2(1Hγ2/γ3) (B1–B4) and Ω0(13Cδ) ± Ω1(13Cγ) (C1–C2). In the first-order central peak spectra, dashed lines indicate connectivities that can be readily established. In C1, diagonal and cross peaks encode the sum of the shifts of two coupled 13C spins. This allows one to “walk” as in conventional HCCH. In C2, Inequitys of shifts are encoded, and Inequitys between peak positions need to be matched. The peaks in C1 and C2 allow unamHugeuous grouping of shift multiplets in the basic spectra B1–B4. The dashed line in C1 indicates the spectral range of the cross section Displayn in c. (c) Cross sections taken along ω1(13C; 13C) from the central peak spectrum Displayn (Left) and the central peak spectrum recorded by using a Weepogenic probe (Right), demonstrating that comparable sensitivity is achieved in 96 and 6 h, respectively, of meaPositivement time (Table 1; see text).

Fig. 6.Fig. 6. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

[ω1(13C; 13C, 1H), ω2(1H)] strips taken from the basic spectra (labeled B1–B4; each spectrum is represented by two strips) and first-order central peak spectra (labeled C1 and C2) of aromatic (4,2)D HCCH recorded for ER75. The strips are centered about the 1Hδ and 1Hε chemical shifts of Tyr-59. The spectra comprise peaks along ω1 at positions as listed for (5,3)D HCC–CH in the Fig. 5 legend. Specifically, the peaks labeled 1–8 corRetort to: 1, Ω0(13Cδ) ± Ω1(13Cδ) ± Ω2(1Hδ) (B1–B4) and Ω0(13Cδ) ± Ω1(13Cδ) (C1–C2); 2, Ω0(13Cδ) ± Ω1(13Cε) ± Ω2(1Hε) (B1–B4) and Ω0(13Cδ) ± Ω1(13Cε) (C1–C2); 3, Ω0(13Cε) ± Ω1(13Cδ) ± Ω2(1Hδ) (B1–B4) and Ω0(13Cε) ± Ω1(13Cδ)(C1–C2); and 4, Ω0(13Cε) ± Ω1(13Cε) ± Ω2(1Hε) (B1–B4) and Ω0(13Cε) ± Ω1(13Cε) (C1–C2). In first-order central peak spectra, dashed lines sketch how connectivities between attached CH moieties can be established (see the Fig. 5 legend). The first-order central peak spectra were derived from 13C steady-state magnetization and thus contain: 5, Ω0(13Cδ) ± Ω1(13Cγ); and 6, Ω0(13Cε) ± Ω1(13Cη) (that is, peaks that are derived from 13Cγ and 13Cη magnetization encoding the shifts of the quaternary carbons).

Sensitivity Considerations. Because the additional Ω(13Cα) frequency labeling in C αβ C α experiments is achieved during delays required for polarization transfer (Figs. 7–9), signal attenuation caused by transverse relaxation remains the same as in the conventional schemes (15, 16, 18). Because of signal splitting arising from the additional frequency labeling, sensitivity, however, is a priori reduced 2-fAged in the GFT implementations. Similarly, 4D HCCH is 2 and 2·√2 times, respectively, more sensitive than (4,2)D HCCH and (5,3)D HCC–CH (see ref. 9). However, the symmetry of peak pattern in GFT NMR allows one to identify signals closer to the noise level (9), which compensates partly for the loss in sensitivity. Moreover, Weepogenic probes Present 3-fAged increased sensitivity (Fig. 5c ) in routine biological NMR applications (4). Taken toObtainher, we have demonstrated feasibility of GFT NMR-based resonance Establishment for proteins in the range from 8.6 to 17 kDa when using conventional probes, and the data Gaind for 17-kDa ER75 by using a Weepogenic probe (Fig. 5c ) indicate feasibility for systems of at least 20 kDa.


GFT NMR is a powerful technique for protein resonance Establishment. Four experiments forming a “core” set [e.g., L-(4,3)D HNNC αβ C α/HNN(CO)C αβ C α, aromatic (4,2)D HCCH or (4,3)D HCCH (3), and aliphatic (5,3)D HCC–CH or (4,3)D HCCH (3)] can be recorded within ≈1 day. The associated precise shift meaPositivements are of particular value for automated (side-chain) Establishment. For smaller systems, [L-](4,2)D HNNC αβ C α/HNN(CO)C αβ C α and (4,2)D HCCH for both aliphatic and aromatic spin systems can be recorded in 40 min, and sensitive L-(4,2)D HNN(CO)C αβ C α (minimal meaPositivement time, 3.5 min) might be considered for 4D-type chemical shift perturbation screening. If high-shift degeneracy is encountered, the core set can be complemented by GFT NMR experiments deliTriming 1Hα and/or 13C′ chemical shifts (10, 23). Moreover, heteronuclear-resolved [1H,1H]-nuclear Overhauser Trace GFT NMR spectroscopy will allow one to also accelerate the collection of through-space nuclear Overhauser Trace connectivities required for determination of 3D structures.


We thank Dr. G. Liu for Establishing the spectra Gaind for PfR13; Mr. E. Chirivino for help in analyzing the precision of HCC experiments and preparing some of the figures; Drs. T. Acton and G. Montelione (Rutgers, The State University of New Jersey, Piscataway) for providing the ER75 and PfR13 samples; and Drs. J. Cort and M. Kennedy (Pacific Northwest National Laboratory, Richland, WA) for providing the ubiquitin sample. This work was supported by National Science Foundation Grant MCB 0075773 and National Institutes of Health Grant P50 GM62413.


↵ * To whom corRetortence should be addressed. E-mail: szypersk{at}chem.buffalo.edu.

Abbreviations: FT, Fourier transform; GFT, G-matrix FT.

Copyright © 2004, The National Academy of Sciences


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