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1 University of Groningen Determination of the three-dimensional solution structure of the histidine-containing phosphocarrier protein HPr from Escherichia coli using multidimensional NMR spectroscopy Nuland, Nico A.J. van; Grötzinger, Joachim; Dijkstra, Klaas; Scheek, Ruud M.; Robillard, George T. Published in: Default journal DO: /j tb17492.x MPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 1992 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Nuland, N. A. J. V., Grötzinger, J., Dijkstra, K., Scheek, R. M., & Robillard, G. T. (1992). Determination of the three-dimensional solution structure of the histidine-containing phosphocarrier protein HPr from Escherichia coli using multidimensional NMR spectroscopy. Default journal. DO: /j tb17492.x Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy f you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 Eur. J. Biochcm. 210, (1992) 0 FEBS 1992 Determination of the three-dimensional solution structure of the histidine-containing phosphocarrier protein HPr from Escherichia coli using multidimensional NMR spectroscopy Nico A. J. van NULAND', Joachim GROTZNGER', Klaas DJKSTRA', Ruud M. SCHEEK' and George T. ROBLLARD' ' The Bioson Research nstitute, University of Groningen, The Netherlands nstitut fur Biochemie, Rheinisch-Westfalische Technische Hochschule Aachen, Federal Republic of Germany (Received July 7/August 24,1992) - EJB We recorded several types of heteronuclear three-dimensional (3D) NMR spectra on "N-enriched and 3C/l 'N-enriched histidine-containing phosphocarrier protein, HPr, to extend the backbone assignments [van Nuland, N. A. J., van Dijk, A. A., Dijkstra, K., van Hoesel, F. H. J., Scheek, R. M. & Robillard, G. T. (1992) Eur. J. Biochem. 203, to the side-chain 'H,5N and 3C resonances. From both 3D heteronuclear 'H-NOE 'H-3C and 'H-NOE 'H-"N multiple-quantum coherence (3D-NOESY-HMQC) and two-dimensional (2D) homonuclear NOE spectra, more than 1200 NOE were identified and used in a step-wise structure refinement process using distance geometry and restrained molecular dynamics involving a number of new features. A cluster of nine structures, each satisfying the set of NOE restraints, resulted from this procedure. The average root-mean-square positional difference for the Ca atoms is less than 0.12 nm. The secondary structure topology of the molecule is that of an open-face sandwich formed by four antiparallel fl strands packed against three a helices, resembling the recently published structure of Bacillus subtilis HPr, determined by X- ray crystallography [Herzberg, O., Reddy, P., Sutrina, S., Saier, M. H., Reizer, J. & Kapafia, G. (1992) Proc. Natl. Acad. Sci. USA 89, ). The histidine-containing phosphocarrier protein HPr plays a central role in the phosphoenolpyruvate-dependent carbohydrate uptake by Escherichia coli. t accepts a phosphoryl group from enzyme and donates it to enzyme 1 in a chain of phosphorylation/dephosphorylation reactions resulting in the phosphorylation and transport of carbohydrates at the expense of phosphoenolpyruvate. During phosphorylation, the phosphoryl group is attached to the N" position of the His15 imidazole ring (Weigel et al., 1982a,b; Waygood et al., 1985; van Dijk et al., 1990). The hydrogen bonds involving the histidine have been delineated by "N-NMR spectroscopy; they play an important role in the mechanism of phosphoryla- Correspondence to G. T. Robillard, The Bioson Research nstitute, University of Groningen, Nijenborgh 4, NL-9747 AG Gronicgen, The Netherlands Abbreviations. 2D, 3D, 4D, two-, three- and four-dimensional; HMQC, heteronuclear multiple-quantum coherence spectroscopy; NOESY, NOE spectroscopy; COSY, correlated spectroscopy; TOCSY, total correlation spectroscopy; HNCA, 3D triple-resonance 1HN-'5N-'3C~ correlation spectroscopy; "N-TOCSY-HMQC, 3D total correlation 'H-"N multiple-quantum coherence spectroscopy; "N-NOESY-HMQC, 3D 'H-NOE 'H"N multiple-quantum coherence spectroscopy; 13C-NOESY-HMQC, 3D 'H-NOE 'H-3C multiple quantum coherence spectroscopy; 13C-HCCH-COSY, 3D 'H- 13C-'3C-1H correlation spectroscopy via 'Jcc carbon couplings; 13C- HCCH-TOCSY, 3D 1H-'3C-'3C-'H total correlation spectroscopy with isotropic mixing of 3C magnetization; TPP, time-proportional phase incrementation; rf, radio frequency; rms, root mean square; DG, distance geometry; MD, molecular dynamics; DDD, distancebounds driven dynamics; HPr, histidine-containing phosphocarrier protein; P-HPr, phospho-hpr. tion of HPr (van Dijk et al., 1990). HPr can phosphorylate other HPr molecules in an autocatalytic exchange reaction; this has been used to determine the phosphoryl-group potential of mutant forms of phospho-hpr (P-HPr) relative to wildtype P-HPr (van Dijk et al., 1991). Complete assignments were obtained for the backbone 'H, "N and 3C resonances (van Nuland et al., 1992) using threedimensional (3D) heteronuclear total correlation 'H-' 'N multiple-quantum coherence spectroscopy ("N-TOCSY- HMQC; Marion et al., 1989a) and 3D heteronuclear 'H-NOE 'H-"N multiple-quantum coherence spectroscopy ("N- NOESY-HMQC; Fesik and Zuiderweg, 1988; Marion et al., 1989b) on "N-enriched HPr and an additional 3D tripleresonance 'HN-1sN-13Ca correlation (HNCA) spectrum (kura et al., 1990a; Kay et al., 1990a) of 13C/'5N-enriched protein. Many of the sequential 'H backbone assignments, derived from two-dimensional (2D) NMR studies (Klevit et al., 1986) were corrected (Hammen et al., 1991; van Nuland et al., 1992). Spectral overlap in the amide proton region, which had thwarted analysis of the 2D spectra, could be overcome completely by spreading these resonances in a third dimension according to the chemical shifts of the attached backbone "N nuclei. n this study we performed 3D 'H-3C- ' 3C-1H total correlation spectroscopy (' 3C-HCCH-TOCSY; Kay et al, 1990b; Bax et al., 1990b), 3D 1H-13C-'3C-'H correlation spectroscopy ('3C-HCCH-COSY; Bax et al., 1990a) and 3D 'H-NOE 'H-3C multiple-quantum coherence spectroscopy (13C-NOESY-HMQC; kura et al., 1990b; Zuidenveg et al., 1990) experiments on 13C/15N-enriched HPr to extend the assignments to the side chains. Using distance geometry (DG) and restrained molecular dynamics (MD),

3 882 A W1 x 02 m3 x 01 x 13 C - waltz 13 c=o 1 decouple involving some new features, a low-resolution structure of E. coli HPr could be determined. An iterative procedure was then used to extract additional distance information from the NOE spectra, in order to refine the solution structure of HPr. MATERALS AND METHODS Sample preparation The production and purification of uniformly 15Nenriched and 13C/l 5N-enriched HPr have been described previously (van Nuland et al., 1992). The protein concentrations in the 15N-enriched and 13C/15N-enriched NMR samples were about 6 mm and 2 mm, respectively. NMR spectroscopy All NMR experiments were performed on a Varian VXR 500 MHz NMR spectrometer. The pulse sequences used for the 15N-TOCSY-HMQC (Marion et al., 1989a) and "N-NOESY-HMQC (Fesik and Zuiderweg, 1988; Marion et al., 1989b) experiments on "Nenriched HPr and for the triple-resonance HNCA experiment (kura et al., 1990a; Kay et al., 1990a) on 13C/'5N-enriched HPr have been described in detail previously (van Nuland et al., 1992). The pulse sequences used for 13C-HCCH-TOCSY (Kay et al., 1990b; Bax et al., 1990b), 13C-HCCH-COSY (Bax et al., 1990a) and 13C-NOESY-HMQC (kura et al., 1990b; Zuiderweg et al., 1990) are shown in Fig. 1. The strong

4 883 water resonance was suppressed by presaturation with a weak radio-frequency (rf) field. All spectra were recorded at 30 "C and ph 6.5. A series of 2D NOE spectra with mixing times of 40, 80, 120, 160 and 200 ms was recorded on "N-enriched protein dissolved in 'H20. Maximum tl and t2 values were 76.8 ms and ms, respectively, and spectral widths in the w1 and wz domains were both 6666 Hz. The 'H carrier was at the water resonance and, during the acquisition period, "Ndecoupling was achieved by a broad-band Waltz decoupling sequence using an rf field of yb1 = 1 khz. The residual water resonance was suppressed by presaturation for 1 s. The measurement of the exchange rates of the backbone amide protons was carried out by following the intensities of the 'H-"N correlation peaks in a series of HMQC experiments recorded at 20 C at ph 8. After freeze-drying, the "Nlabeled HPr, originally dissolved in 'H20, was dissolved in 'H20. The first spectrum was recorded within 25 min, starting 7 min after addition of 'HzO to the protein. Each subsequent spectrum was recorded in 18 min. The exchange lifetime z, for each amide proton was extracted from the spectra by fitting the increase in intensity of the 'H-"N cross-peaks to a single exponential. Typical experimental details: maximum tl and tz values were 64 ms and 154 ms, respectively, and spectral widths in the o1 ("N) and o2 ('H) domains were 2000 Hz and 6666 Hz. The 'H carrier was at the water resonance and, during the acquisition period, '5N-decoupling was achieved by a broad-band Waltz decoupling sequence using a rf field of yb1 = 1 khz. The water resonance was suppressed by presaturation for 1 s. Structure calculations: DG and MD. The 3D "N-NOESY-HMQC, 3D '3C-NOESY-HMQC and the 2D-NOESY build-up experiment in 'H20 yielded 928 unambiguous NOE restraints. NOE identified in the 3D NOE spectra were classified as strong or weak, corresponding to upper distance bounds of 0.35 nm and 0.5 nm, respectively. The 2D-NOE build-up series in 'H20 yielded more accurate distance estimates. We used the C3,5H - C4H NOE in phenylalanine rings (corresponding to 0.25 nm) for calibration. This resulted in reasonable distances ( nm) for the Ha- Ha pairs in the regular part of the antiparallel p sheet of HPr. Measured distances were converted to distance restraints, leaving at least 0.15 nm difference between the upper and lower distance bounds to allow for uncertainties in the calibration procedure, the cross-peak volume determination and for dynamic averaging of the NOE. n addition, the available information on the secondary structure was used to define 32 H-bonds, which were modeled by restraining the carbonyl oxygen and amide proton of the residues involved to between 0.22 nm and 0.17 nm (Table 2). Coordinates were calculated with these upper and lower distance bounds for all non-hydrogen atoms, as well as the amide and Ha, of HPr, as follows. After collecting all upper and lower distance bounds in a matrix, 16 four-dimensional (4D) coordinate frames were calculated using essentially Crippen's EMBED algorithm (Crippen and Havel, 1978). To impose the proper chiralities on all asymmetric carbons, and to ensure proper handedness of the a helices, a list of quadruples of atoms (chirals) was constructed for which the signed volume was constrained to its target value, calculated from a reference structure. This was performed for atoms connected to the asymmetric carbons, in the usual way (Havel et al., 1983), as well as for quadruples of N atoms on consecutive residues in the three a helices of HPr. An error function was constructed, consisting of a distance part and a chiral part (Havel et al., 1983). While for the distance part the full 4D coordinates were used, for the chiral part only 3D coordinates were used, corresponding to the three largest radii of inertia of the 4D structures. This function was minimized in three stages: 100 steps of conjugate-gradient optimization were followed by a simplified MD calculation [distance-bounds driven dynamics (DDD); Kaptein et al., 1988; Scheek et al ; first 3000 time steps at an elevated temperature (1000 K), followed by an annealing phase of another 1000 steps, during which the kinetic energy was slowly taken away by temperature coupling to an external bath of 1 K (Berendsen et al., 1984). The next step was to project the 4D coordinate frames into 3D space, which was achieved by using the EMBED algorithm again to calculate the coordinates corresponding to the three largest radii of inertia. The same error function, now completely defined in 3D, was minimized further by a DDD calculation similar to that described above. Of the 16 starting structures, 13 reached acceptable minima and showed essentially the same folding of the backbone. These 13 structures were used for further refinement. The set of NOE-derived distance restraints was extended by reexamining all NOE that had not yet been unambiguously ascribed to one pair of protons. n many cases the correct assignment followed immediately from the knowledge about the 3D structure acquired at this stage. n addition, a number of non-noe (de Vlieg et al., 1986) was added to the list of NOE-derived distance restraints. This was performed for pairs of protons that, in at least one of the low-resolution structures, were close enough to show NOE, but for which definitely no NOE was observed. Care was taken to avoid cases where NOE could have been missed due to other reasons than too great a distance between the protons involved. Thus, the absence of NOE was used to define a lower bound of only 0.35 nm on the corresponding proton-proton distance for amide protons, and a and p protons, and only in cases where both protons were also involved in other strong NOE. Moreover, the absence of NOE was only used if overlap or water presaturation could not have hindered its detection. Using this extended set of restraints, another DDD calculation was started with the 13 coordinate frames. All protons were added at this stage. A slightly modified protocol was used to avoid the use of pseudo atoms (Wiithrich, 1986). Pairs of protons (or proton groups) that could not be discriminated spectrally (such as the C2,6 or C3,5 protons of rapidly flipping Phe rings, or overlapping methylene protons or methyl groups) were treated as follows. Since only the sum of NOE involving such protons could be measured, the resulting NOE was halved and interpreted as a pair of identical restraints, each referred to one of the equivalent protons. From the structure, the corresponding distances were calculated and averaged as (rp6)- before they were compared with the restraints. Restraining forces were calculated, if necessary, and distributed equally among the pair of protons, thus avoiding any distortion of the position of the protons relative to each other. Using this procedure pseudo atoms and corresponding pseudo corrections are no longer necessary to treat such cases of equivalent protons. For non-equivalent methylene protons and methyl groups, we arbitrarily labeled the low-field and high-field resonances 1 and 2, respectively. Obviously, this is allowed only when protons (or groups of equivalent protons) 1 and 2 are involved in exactly identical restraints, in which case no stereospecific assignment can be made on the basis of NOE alone. When this was not the case, the pair was included in a

5 884 Table 1. Side-chain 'H, 3C and 15N chemical shifts for E. coli HPr at ph 6.5 and 3OoC, 50 mm potassium phosphate. Residue Chemical shift ca CY Cfi other PPm 1 Met 2 Phe 3 Gln 4 Gln 5 Glu 6 Val 7 Thr 8 le 9 Thr 10 Ala 11 Pro 12 Asn 14 Leu 15 His 16 Thr 17 Arg 18 Pro 19 Ala 20 Ala 21 Gln 22 Phe 23 Val 24 Lys 25 Glu 26 Ala 27 Lys 29 Phe 30 Thr 31 Ser 32 Glu 33 le 34 Thr 35 Val 36 Thr 37 Ser 38 Asn 40 Lys 41 Ser 42 Ala 43 Ser 44 Ala 45 Lys 46 Ser 47 Leu 48 Phe 49 Lys 50 Leu 51 Gln 52 Thr 53 Leu 55 Leu 56 Thr 57 Gln 59 Thr 60 Val 61 Val 62 Thr 63 le 64 Ser 65 Ala 66 Glu 2.28, 2.10 (32.6) 3.16, 2.62 (42.7) 1.88, 1.73 (33.0) 2.41, 1.86 (34.1) 1.84 (32.0) 2.13 (35.2) 3.88 (69.8) 2.10 (36.9) 4.42 (69.5) 1.35 (18.5) 2.36, 1.92 (31.8) 2.85, 2.72 (38.2) 1.99, 1.03 (40.9) 3.23, 3.49 (30.2) 4.23 (68.4) 2.00 (27.4) 2.21 ', 1.74" (31.O) 1.22 (17.4) 3.58 (17.4) 2.26 (28.0) 3.58, 3.01 (39.5) 2.09 (31.8) 2.04, 2.04' (32.0) 2.00 (28.8) 1.23 (18.4) 1.95', 1.95 (32.6) 3.35, 2.78 (40.2) 4.22 (68.9) 3.53, 3.20 (65.5) 2.09', 2.09 (30.1) 1.40 (41.6) 3.89 (71.4) 1.85 (33.4) 3.78 (71.4) 3.67', 3.67 (65.5) 3.10, 2.84 (39.3) 1.86, 1.76' (33.5) 3.64, 3.52 (66.5) 1.20 (23.1) 4.14, 3.73 (64.0) 1.30 (19.8) 2.25', 1.80 (32.9) 3.85 (63.7) 1.82 (42.0) 3.20, 2.99 (38.6) 1.99 (31.8) 1.84, 1.70 (41.9) 2.22, 2.14 (28.6) 4.35 (69.5) 1.87 (42.2) 1.69, 1.47" 4.36" (69.5) 2.17', 1.99 (27.1) 4.06 (69.2) 1.96 (31.9) 2.03 (34.3) 3.85 (69.8) 1.87 (38.2) 3.70 (65.9) 1.34 (25.1) 2.04, 1.94 (33.4) 2.48(31.0) 2.07, 1.97 (32.4) 2.41, 1.86 (34.5) 2.15 (36.8) 1.07 (21.4), 0.89 (20.4) 0.93 (21.4) 1.65', 1.17' 0.87 (17.3) 1.18 (21.4) 2.16" (27.4) 1.27 (27.5) 1.29 (21.7) 1.82, 1.65 (27.1) 1.78" 2.52, 2.47 (34.8) 1.15 (22.7), 0.92 (21.1) 1.56, 1.39 (24.9) 2.57, 2.34 (36.8) 1.49 (24.5) 1.30 (21.6) 2.3Sb, 2.30b( 35.8) 1.61 ', 0.82 (26.8) 0.74" (16.1) 1.10 (21.6) 1.00 (21.5), 0.71 (21.5) 1.06 (20.9) 1.52, 1.41 (24.5) 1.51, 1.46 (24.5) 1.51 (26.5) 1.59 (25.2) 1.62 (26.3) 2.61, 2.22 (34.9) 1.30 (21.7) 1.34' 1.36" 1.19 (21.5) 2.17, 2.17" (33.8) 1.25 (22.0) 1.01 (21.0), 0.79 (21.0) 0.79 (22.7), 0.79 (20.2) 1.01 (21.4) 1.71", 1.10 (28.0) 0.93 (3 7.5) 2.19 (37.1) 7.26' 0.74 (13.5) (C3,5) 7.35", (C4) 7.45" (NH,) 7.44, 6.69 (113.7(15N)) (NH,) 7.46, 6.88 (112.9(15N)) 3.91, 4.02 (50.8) (NH,) 7.48, 6.92 (115.8('5N)) 0.69 (23.9), 0.51 (25.0) 7.27" (C2) 7.9Ob." 3.32 (42.9) 3.17", 3.03'(48.9) 7.14' 1.71 (29.4) 1.79 (29.2) 7.29' 0.53' 1.76b, 1.76 (28.9) (NH,) 7.89, 6.88 [114.2(15N)] (C3,5) 7.27,', (C4) 6.93' (C6) 2.99 (41.7) (C6) 3.05 (42.0) (C3,5) 6.89", (C4) 6.60" (OyH) 5.74 (NH,) 7.54, 7.02 [115.9(15N)] (C6) 3.06 (42.1) 1.81 (28.9) (C6) 2.98 (42.1) 0.92 (25.8) 7.23' (C3,5) 7.37', (C4) 7.32" 1.84, 1.71 " (28.5) (C6) 3.10 (42.1) 0.79 (24.6) (NH,) 7.30, 6.80 [ 12.9(15N)] 0.80' (25.8), 0.68 (25.8) 0.78, 0.68 (25.8) 0.93b (13.7) (NH,) 7.28, 6.96 [113.8('5N)]

6 885 Table 1 (continued). Residue Chemical shift CY C6 other 68 Glu 69 Asp 70 Glu 71 Gln 72 Lys 73 Ala 74 Val 75 Glu 76 His 77 Leu 78 Val 79 Lys 80 Leu 81 Met 82 Ala 83 Glu 84 Leu 85 Glu 2.01 (29.7) 3.33, 2.61 (39.6) 1.84, 2.33b (27.5) 1.52, 1.42 (26.8) 1.80, 1.80b (32.1) 0.63 (18.4) 2.19 (31.8) 1.93b, 2.08 (29.2) 3.29b, 3.14 (32.5) 1.84, 1.57 (40.6) 2.15 (33.3) 1.95, 1.86b (31.8) 1.86, 1.69'(42.5) 2.32b, 2.10 (32.7) 1.57 (18.8) 2.19, 2.06 (30.4) 1.82b, 1.47 (42.7) 2.12, 2.00 (30.2) 2.31 (35.9) 2.74, 2.12b (34.3) 1.52, 1.05 (32.1) 1.55, 1.47 (24.9) 1.80b (29.2) 1.15 (24.2), 0.95 (22.5) 2.54, 2.24 (37.2) 6.70b3" (NH,) 7.47, 6.57 (113.2("N)) (C6) 3.04 (42.0) 1.92b (26.5) 0.61 (26.4), 0.26 (20.6) 1.01 (19.3), 0.95 (21.1) 1.52, 1.35 (24.7) 1.68b(29.2) (C6) 2.98 (42.1) 1.55'(26.5) 0.92 (25.6), 0.92 (24.5) 2.56 (33.7) 2.46, 2.30 (36.1) 1.47b (27.1) 0.91 (25.8) 2.28 (36.4) (C2) 7.98'r" a 'H chemical shifts are expressed relative to trimethylsilylpropanesulfonic acid, 15N shifts relative to liquid NH3 (Live et al., 1984), and 13C shifts relative to hypothetical internal trimethylsilylpropionic acid (Bax and Subramanian, 1986). 5N and shifts between brackets. Stereospecifically assigned protons are printed in italics. n discrepancy with values reported by Hammen et al. (1991). 3C chemical shift not observed. list of prochirals, and omitted from the list of chirals (compare the concept of floating chiralities; Weber et al., 1988). During the DDD run, at every 10 steps, the atoms in the list of prochirals were swapped (i. e. their coordinates and velocities were interchanged; cf. Williamson and Madison, 1990), and after each swap of a pair of protons, the error function was calculated again. The swap was accepted unconditionally when it caused the error function to decrease; when the error function increased, a Monte-Carlo criterion was used to decide whether the swap was still acceptable. When two or more pairs in the list of prochirals are involved in NOE among themselves (e. g. methylene pairs of the same residue), they cannot be treated one by one in an uncorrelated manner. Such pairs were flagged in the list, signalling the DDD program that these must be swapped simultaneously, as well as one by one. The procedure ensures, not only that all possibly relevant conformers are sampled, but also, in favorable cases it may result in stereospecific assignments for some of the prochiral pairs of protons (or methyl groups). Apart from this feature (flipping chiralities), the DDD run was performed in a way similar to the previous one: first 1000 steps at 1000 K, followed by an annealing phase, again of 2000 steps. The list of NOE-derived restraints was extended once more (Table 3) and the last DDD calculation repeated to yield a final cluster of nine structures, with acceptably small violations of the restraints remaining (see below). RESULTS Side-chain 'H, "N and 13C assignments The polypeptide backbone 'HN, 15N, 'Ha and 3Ca chemical shifts of E. coli HPr in 50 mm potassium phosphate, ph 6.5, at 30 C, obtained from heteronuclear 3D NMR ex- periments, were previously reported (van Nuland et al., 1992). The backbone 'HN, 15N and 'Ha chemical shifts were also reported recently by Hammen et al. (1991), who corrected a significant fraction of their backbone assignments after reexamination of their original work (Klevit et al., 1986; Klevit and Drobny, 1986; Klevit and Waygood, 1986), still leaving three discrepancies in the assignment of backbone nuclei : the 5N chemical shifts of Glu32 and Glu85 were interchanged by Hammen et al. (1991), and the assignment of the 15N resonance of Thr56 differs in the two data sets. Other discrepancies between the resonance assignments of Hammen et al. (1991) and ours are marked in Table 1. The present study extends the backbone spin systems to the side chains, using the previously reported NMR experiments, complemented by the '3C-HCCH-COSY (Bax et al., 1990a) and '3C-HCCH-TOCSY (Kay et al., 1990b; Bax et al., 1990b) techniques. With all the 'Ha assignments in hand, together with the 13Ca assignments obtained from the HNCA experiment, it is now possible to use the HCCH-COSY and HCCH- TOCSY experiments to delineate the amino acid spin systems. n the HCCH experiments, 'H magnetization is transferred to its directly bonded 13C nucleus via the ' JCH coupling. Next, this 3C magnetization is transferred to neighboring ' 3C nuclei via 'JcC couplings, and finally transferred back to the 'H via the 'JCH coupling. The HCCH-COSY and HCCH-TOCSY experiments differ only in the mechanism used for the 13C- 13C magnetization transfer. nstead of a 90" 13C mixing pulse in the COSY experiment, an isotropic mixing scheme, DPS- 3 (Shaka et al., 1988), is used to transfer the 13C magnetization in the TOCSY (Fig. 1). The rate at which magnetization is transferred among the J-coupled carbons depends on the effective J-coupling present during this mixing period, which is substantially smaller than the normal 'J coupling for coupled 13C nuclei with widely different chemical shifts (Bax et al.,

7 886 B a 0 OB A fl O Q Fig. 2. Selected contour plots of slices taken from the two HCCH spectra of '5N/13C-enriched HPr in 'HzO at 3OoC, in 50 mm potassium phosphate, ph6.5. Each slice shows a part of the 'H (wl) frequency domain and a narrow band around the 13C (0.12) frequency of the corresponding 13C- 'H spin pair. The 13C (w2) chemical shifts of the identified 13C - 'H spin pairs are shown in the figure. (A) Slices taken from the HCCH-TOCSY spectrum (mixing time, 20 ms) at the 'H (03) frequencies of the CnH (4.98 pprn), CPH (2.03 pprn), Cy'H3 and Cy2H3 (0.79 ppm) protons of Va161. Note the resolved 13C chemical shifts for the two CyH3 groups while the 'H chemical shifts are degenerated. (B) Slices taken from the HCCH-COSY spectrum at the 'H (w3) frequencies of the CclH protons of Asnl2 (4.91 pprn), Ser41 (5.83 ppm), Ala65 (5.68 ppm) and Asp69 (4.93 ppm), in descending order. Cross-peaks arising from CPH protons of the different types of spin systems can easily be identified in each slice. 1990b; Clore et al., 1990). We recorded two HCCH-TOCSY spectra with two different mixing times, 20 ms and 35 ms, respectively. Fig. 2 illustrates how both types of HCCH experiments were used. Fig. 2A shows four 2D slices taken from the 3D HCCH-TOCSY spectrum (mixing time of 20 ms) at the 'H (03) frequencies corresponding, in descending order, to the CaH, CPH and CyH protons of Va161. Each slice covers a part of the 'H spectral width (01) and a narrow band around the 3C (02) frequency of the 3C-lH spin pair. The typical connectivity pattern for the valine spin system is clear. Although the 'H frequencies of the two CyH3 groups are the same, they can be discriminated by their different 3C chemical shifts. The relayed connectivities from the different CxH (x = a,p,y) groups to the others, yield the complete assignments of the valine spin systems. The HCCH-COSY experiment is particularly useful for identifying the Gly, Ala, Thr and Val spin systems, as well as amino acids of the AMX type (e.g. Ser, Asn, Asp, His and Phe) in HPr. This is illustrated in Fig. 2B, which shows four 2D slices taken from the 3D HCCH-COSY spectrum at the 'H (03) frequency corresponding to the CclH protons of Asnl2, Ser41, Ala65 and Asp69. Again, each slice covers a part of the 'H spectral width (01) and a narrow band around the 3C (02) frequency of the identified 13Ca-1Ha spin pair. Cross-peaks arising from the CPH protons can easily be identified and can be checked by finding the same connectivity pattern, starting from the CjH protons at the corresponding 3C chemical shift of the 3CP- 'HP spin pair, which differs substantial from the 3C chemical shift of the Ca for these residues. Most of the spin systems could be assigned by using both HCCH experiments, resulting in an almost complete set of 'H and 3C chemical shifts for the side chain atoms, which are summarized in Table 1. n the "N-HMQC spectrum (van Nuland et al., 1992), eight pairs of cross peaks were observed at ppm (lh) and ppm (5N) arising from the side-chain NH2 groups of Asn (12 and 38) and Gln (3, 4, 21, 51, 57 and 71). The 'H and 5N chemical shifts of these NH2 groups could be assigned on the basis of NOE between the amide protons and the terminal CH2 group (CPH, for Asn and C,H2 for Gln) observed in the 100 ms "N-NOESY-HMQC spectrum. n the same spectrum a strong cross-peak could be observed between the amide proton of Ser31 and a proton with an unusual chemical shift of 5.74ppm. t is assumed that this frequency belongs to the side-chain hydroxy proton of Ser31. n small linear peptides dissolved in dimethyl sulfoxide the chemical shift of the hydroxy proton of serines is 5.92 ppm (Wiithrich, 1976), which is close to the value reported here. The existence of such a slowly exchanging proton suggests that it is strongly hydrogen bonded and not water accessible. Hammen et al. (1991) identified another hydroxy proton belonging to Thr59 (5.31 ppm) in addition to the OyH of Ser31, which we did not observe in any of our spectra. The assignments of the aromatic protons of the two histidines (15 and 76) and four phenylalanines (2, 22, 29 and 48) were based on the NOE spectra in 'H20, where the NOE interaction could be observed between the aromatic protons and the neighboring aliphatic protons that had been assigned independently. Four typical NOE patterns of three resonances, identifying C2,6, C3,5 and C4 protons in the aromatic ring, were observed for the four phenylalanines in HPr. Strong NOE to the CaH and CPHz protons are typical for C2,6 protons, while weaker NOE identify C3,5 protons. No NOE could be observed for the C4 proton of the phenylalanine ring to the aliphatic protons. n the case of the two histidines, the C5 proton of the aromatic ring show NOE interaction to the CPH2, while such an interaction is absent for the C2 proton. Since the aromatic carbons resonate at about 130 ppm (kura et al., 1991), which is too far from the 3C carrier in the HCCH-COSY (43.8 ppm) and in the HCCH-TOCSY (48.0 ppm) experiments, no information on the 3C chemical shifts of aromatic carbons is obtained from the HCCH experiments. Fig. 3 summarizes the 3C chemical shifts observed for each type of amino acid in HPr. The distribution of these shifts is in good agreement with that reported for interleukin- D (Clore et al., 1990) and calmodulin (kura et al., 1991). Secondary structure analysis Fig. 4 summarizes experimental results that are known to correlate well with the local secondary structure, including the short-range and medium-range NOE, slow exchange of amide protons with water and the deviations of the 'Ha, 3Ca and 3CP chemical shifts from their random-coil values. These results will now be presented. Characteristic patterns of NOE connectivities can be useful to identify elements of secondary structure in proteins (Wiithrich, 1986). Strong dnn(i,i + 1) connectivities as well as medium dan(i,i + 3) and dblg(iri + 3) connectivities are indicative of an a-helical conformation. Antiparallel P-sheet struc-

8 887 ture is associated with strong darn(i,i + 1) and also with longrange cross-strand dolol(i,j), doln(ij + 1) and dnn(i,j + 1) connectivities between two polypeptide segments comprising the sheet. n this study, the 100-ms 3D "N-NOESY-HMQC and 200-ms 3D 13C-NOESY-HMQC spectra were used to identify such patterns. The NOE that identify the four-strand antiparallel P sheet of HPr were already published in an earlier paper (Fig. 8 in van Nuland et al., 1992). The measurement of the exchange rates of the backbone amide protons was carried out by dissolving freeze-dried HPr, with all amide protons replaced by deuterons, in 'HzO and following the intensities of the 'H-15N correlation peaks in a series of HMQC experiments recorded at 20 C at ph 8. Rap- 3C chemical shift (pprn) Fig. 3. Summary of the 3C chemical shift distribution observed for the different carbon atoms and different residue types in E. coli HPr. idly exchanging amide protons with an exchange lifetime z,, smaller than 0.3 h, could be detected in the first spectrum. The amide protons of residue 12, 38, 39, 47, 54 and 68, although still not visible in the HMQC spectrum recorded 3 days after the protein was dissolved in 'HzO, probably undergo exchange under our conditions too fast to be detectable at all. n a previous report, these protons were visible in the HMQC spectrum at ph 6.5 and 20 C (van Nuland et al., 1992). These protons, except for Leu47, are all positioned in loops or turns, which tend to be exposed near the molecular surface where the exchange of these amide protons is generally rapid (Wiithrich, 1986). The amide-proton-exchange lifetimes, zex, are divided into three groups in Fig. 4: fast, z, < 0.3 h, medium, 0.3 h < z, < 1 h, and slow, 1 h < z,, < 2 h. Recently, correlations were found between protein backbone conformation and the chemical shifts of the 'Ha protons (Wishart et al., 1992), and the 13Ca and 13CP atoms (Spera and Bax, 1991; Wishart et al., 1991). The deviations of the 'Ha, 13C~ and 13CP chemical shifts from their random-coil values (secondary shift A) show typical patterns for different types of secondary structure. An upfield shift for both the 'HE and 13CP chemical shifts was observed for a-helical residues, while a downfield shift of 3.1 ppm (Spera and Bax, 1991) or 2.3 ppm (Wishart et al., 1991) is typical for the 3Ca atom in an a helix. 13Ca nuclei in P-sheet residues typically show an upfield shift of 1.5 ppm (Spera and Bax, 1991) or 1.3 ppm (Wishart et al., 1991). Fig. 4 demonstrates stretches of negative and positive secondary shifts for the different atom types along the amino acid sequence, identifying the different secondary structure elements in HPr. Fig. 5 shows the distribution of secondary shifts in a helix and P sheet in HPr. Only residues for which all NMR data predict either a helix or P sheet are included. The average Ca secondary shift is 2.55 f 1.23 ppm for 29 residues in a helix and the average CP secondary shift is f 0.69 ppm. For 25 residues in fl Fig. 4. Summary of the short-range and medium-range NOE between residue i and residue i + l/i + 3 involving HN, CaH and CPH protons together with the slowly exchanging amide protons. n addition, the secondary shifts of the 'Ha (Wishart et al., 1992), 13Ca and 13CP (Spera and Bax, 1991 ; Wishart et al., 1991) atoms of each amino acid residue, are plotted as a function of the residue number in the amino acid sequence. The NOE intensities are indicated by black bars and are classified into three groups according to the number of contour levels in the NOE spectra. Blank bars indicate NOE involving protons with the same frequencies for Hai and HU~,~ in the case of dan(i,i+ 1) connectivities, for HNi and N;+ 1 in the case of d"(i,i + 1) connectivities, and for Hpi and Hpi+ 1 in the case of d~~(i,i + 1) connectivities. Dotted lines indicate NOE involving overlapping Hai and or Hai+3 protons in the case of dmn(i,i+ 3) connectivities, or overlapping HP,+3 and HPi or HPi-l, in the case of dep(i,i+ 3) connectivities. For the connectivities involving prolines the C5 protons are used in place of the HN protons. Amide protons that exchange fast at ph 8, z, < 0.3 h, are not marked. Open circles indicate HN protons with 0.3 h < z,, < 1 h, and an asterisk is used for protons with 1 h < T,, < 2 h.

9 888 A B <-helix E B-sheet 8 0 Fig. 5. Histograms of secondary shift A distribution in a helix (shaded bars) and fl sheet (white bars) of E. coli HPr for the Ca (A) and the Cfl (B) atoms. 13C chemical shifts are relative to hypothetical internal trimethylsilylpropionic acid (Bax and Subramanian, 1986). Randomcoil values used for calculating the secondary shifts are taken from Spera and Bax (1991). 37/ -6 Fig. 7. llustration of the iterative refinement procedure. Vertical bars in A and C represent protons which are within 0.5 nm distance of the amide proton of Gln57 in at least one of the nine structures after the first DG calculations (A) and after the refinement procedure (C). B shows contour plots of the slice taken from the 3D 15N-NOESY- HMQC spectrum at 8.84 pprn (133) that corresponds to the observed backbone amidc proton of Gln-57. The slice covers the amide 'H region (wl) and a narrow band around the "N (132, 124.4ppm) frequency of the "N-HN spin pair. The proton resonance which corresponds to the frequency of the amide proton of Gln57 is indicated by a vertical bar in this slice. Table 2. H-bond restraints used in the three steps of the structure determination and refinement. Donor Acceptor Donor Acceptor H 0 H $0 40 5b 6b 70 80, residue number Fig. 6. rms Ca positional differences along the amino acid sequence after the first step of calculations (white bars) and after the refinement procedure (black bars). sheet, the average Ca and Cp secondary shifts are f 0.85 and ppm, respectively. These average secondary shifts differ only slightly from the values found by Spera and Bax (1991) and Wishart et al. (1991). Qualitative interpretation of all the NMR data, including NOE connectivities, amide-proton-exchange rates and secondary shifts of the 'Ha, 13Ca and 13Cp atoms, lead to the identification of three helices, residues 19-28, and 71-84, and to a fourstrand antiparallel p sheet in E. colihpr (Klevit and Waygood, 1986; van Nuland et al., 1992). Structure calculations and refinement Fig. 6 shows the root-mean-square (rms) Ca positional differences along the amino acid sequence for the set of nine structures after the first step of the structure calculations. The largest fluctuations after these first calculations appear in the regions of residues 9-17 and Therefore, the first iteration of the refinement procedure focussed on these regions. Fig. 7 illustrates how this procedure was carried out. n Fig. 7B, contour plots are shown of a slice taken from the 3D "N-NOESY-HMQC spectrum at 8.84 ppm (wj), corresponding to the amide proton of Gln57. The slice covers the amide 'H spectral region (wl) and a narrow band around the "N (02, ppm) frequency of the 15N-HN spin pair. The proton resonance which corresponds to the frequency of the amide proton of Gln57 is also indicated. Fig. 7A shows the predicted NOE involving the amide proton of Gln57, based on the set of structures that resulted from the first DG calculations. Protons within 0.5 nm of the amide proton of Gln57, Phe2 Ala65 Gln4 le63 Val6 Val61 Lys24 Glu25 Ala26 Lys27 Glu32 Glu66 Thr34 Ser64 Val35 Ala42 Ah65 Phe2 lc63 Gln4 Val61 Val6 Ah20 Gln21 Phe22 Val23 Glu66 Glu32 Ser64 Thr34 Ah42 Val35 Thr36 Thr62 Ser37 Lys40 Gln5 1 Thr52 Glu75 His76 Leu77 Val78 Lys79 Leu80 Met81 Ah82 Glu83 Leu84 Thr62 Thr36 Lys40 Ser37 Leu47 Phe48 Gln71 Lys72 Ah73 Val74 Glu75 His76 Leu77 Val78 Lys79 Leu80 in at least one of these structures, are indicated at their known chemical shift positions. We then went back to the NOESY data, to examine whether any of these predicted contacts could be unambiguously assigned. This type of analysis was used to increase the number of positively identified NOE, and to generate a list of non-noe (see Material and Methods). Table 3 summarizes the number of restraints used in the subsequent stages of the structure determination. Fig. 7C shows the predicted NOE involving the amide proton of Gln57 based on the refined structures. The three predicted NOE that were not observed experimentally correspond to the amide protons of Thr59, Leu55 and Leul4. n none of these cases is the distance to the amide proton of Gln57 less than 0.35 nm in any of the structures. With the increase in the number of NOE rcstraints, the structures converged, as can be concluded from the decrease in the average rms positional differences (Table 3). This is also illustrated in Fig. 6, where the rms Ccr positional differences along the amino acid sequence after the first step of calculations and the last refinement step are shown for the same set of nine structures.

10 Table 3. Number of restraints used in the three steps of the structure determination and refinement, and average rms positional differences for the Ca and all atoms (between brackets) after each step for the set of nine structures. Step i,i+ 1 i,i + 2 i,i+ 3 i,i + 4 Rest Non-NOE ltlls nm (0.37) (0.29) (0.21) residue number Fig. 8. Summary of the NOE involving backbone and side-chain protons used in the last step of the structure determination. They were obtained from the "N-NOESY-HMQC, 13C-NOESY-HMQC and the 2D NOE spectra of HPr. violation (nm) Fig. 9. Distribution of the upper-bound (white bars) and lower-bound (black bars) violations averaged over the set of nine structures. Lowerbound violations are only reported for pairs of atoms for which NOE were observed. There are 76 upper-bound violations less than 0.01 nm. The total procedure described above resulted in a set of nine structures, each satisfying the set of NOE restraints used as input for the DG calculations. Fig. 8 summarizes the NOE involving backbone and side-chain protons used for obtaining the final set of nine structures. All upper- and lower-bound violations are smaller than 0.06 nm and 0.04 nm, respectively. Fig. 9 shows the distribution of the upper bound and lower bound violations averaged over the set of nine structures. More than 99% of the NOE are satisfied within 0.02 nm. n all structures, Thr7 and le8 are involved in the largest upperbound violations. Convergence of the structures during refinement was poor for residues near those involved in the violated NOE restraints, as can be seen in Fig. 6. DSCUSSON This paper describes the side-chain 'H, 3C and 15N resonance assignments and structure determination of E. coli HPr derived from an analysis of the HCCH-COSY and HCCH- TOCSY experiments as well as the NOESY experiments on isotope-enriched protein. The power of the HCCH experiments stems from the use of the well-resolved one-bond 'H- 3C (about 140 Hz) and 13C-'3C (about 35 Hz) J couplings to transfer magnetization, versus the poorly resolved 'H-'H (about 12 Hz) J couplings used in conventional 2D COSY techniques. A significant improvement of the spectral dispersion is also achieved by spreading the resonances in a third dimension. These 3D heteronuclear experiments resulted in an almost complete set of resonance assignments, including the identification of the Ser31 OyH proton which was also observed by Hammenet al. (1991). n addition, the 13C chemical shift contains valuable information regarding protein conformation. n particular, 13Ca and 13Cj?, but also the 'Ha chemical shifts appear to be useful probes for secondary structure analysis (Fig. 4) in addition to the well-known 'H-'H NOE and J-coupling patterns. This figure also shows a salient upfield shift of 2.5 ppm relative to its random coil value for the Ha proton of Ala73, which is probably caused by a ringcurrent effect due to Phe29. Preliminary DG calculations with the initial list of unambiguous NOE often yielded structures, parts of which could be regarded as local mirror images of the correct structure. Obviously this was caused by the limited number of NOE that had been unambiguously assigned at this stage. We solved this problem by introducing a new type of chiral restraint, which ensures the right-handedness of identified a helices. Minimization of the resulting error function by simulated annealing turned out to be most effective in a 4D space, where possible conflicts between the distance and chiral terms in the error function are avoided (Havel, 1991). The resulting cluster of structures, albeit of low resolution, was subsequently used to solve many of the remaining ambiguities in the assignments of the NOE and to improve the structure in an iterative fashion. The use of flipping chiralities in the DDD program resulted in tentative stereospecific assignments for 14 pairs of protons [see Table 1, and, in addition, for both Ha protons of Gly28 (3.91 ppm and 3.40 ppm, respectively) and of Gly67 (3.95 ppm and 4.85 ppm, respectively)]. For all these pairs, the positioning of the low-field and high-field protons was identical in the nine structures after the final annealing phase, resulting in chiral volumes with the same sign in all structures. The iterative refinement procedure using DG calculations and restrained MD resulted in a set of nine structures with an average rms Ca positional difference of 0.12 nm. Fig. 10B shows the final set of nine structures as a stereoscopic representation of the a carbons in the molecule. The numbering of the residues is given for one of these structures in Fig. 10A. Although the remaining violations of the NOE restraints are

11 890 A B Fig. 10. Fold of HPr from E. coli after the final refinement. Stereoscopic representation showing a-carbon positions in the molecule. (A) One out of the set of nine structures to show the amino acid positions in B. Every fifth amino acid is labeled. (B) All nine structures superimposed. acceptably small, they seem to cluster in the less-well-defined part of the molecule. Whether this is due to an increased mobility in this part of the molecule is being investigated by "N T1, T2 and "N-'H NOE measurements, and by further refinement using time-averaged restrained MD (Torda et al., 1990). E. coli HPr forms a classical open-faced p sandwich (Richardson, 1981), made of a curved four-strand antiparallel p sheet and three helices packed against one face of the sheet. The antiparallel position of the two amphipathic helices, 1 and 3, is energetically favorable due to the alignment of the helix dipoles (Hol et al., 1981). Their hydrophobic sides are facing the hydrophobic side of the four-strand antiparallel p sheet. The topology is consistent with the recently published structure of Bacillus subtilis HPr (Herzberg et al., 1992). t is also consistent with the earlier published topology of the NMR structure of E. coli HPr (Klevit and Waygood, 1986). t differs significantly from the topology of the E. coli HPr crystal structure (El-kabbani et al., 1987) determined at acidic ph, which does not form an open-face p sandwich. The small helix at resitkes was identified in the earlier NMR work by Klevit and Waygood (1986). However, in their more recent publication, with corrected assignments, they did not report enough helical restraints to be certain of the existence of this second helix (Hammen et al., 1993). The use of "N and 13C enrichment in the present study leads to a more detailed interpretation of the NOE spectra. The additional information obtained from the 13C chemical shifts of the Ca and Cp carbon atoms and the 'Ha chemical shifts support the existence of this helix. Herzberg et al. (1992) reported this helix in the X-ray structure of B. subtilis HPr (with a slightly different orientation), but Wittekind et al. (1990) did not report it in the NMR study on the same HPr. Some features of the NMR structure are relevant for the active-site configuration. The active-site Hisl 5 is located on the surface of the protein at the N-terminus of the first helix. The NOE observed between the C2 proton of the imidazole ring and the C5 protons of Pro18 confirm the proximity of these side chains as also reported in the B. subtilis HPr X-ray structure (Herzberg et al., 1992). However, in contrast to the crystallographic work, the NMR structure is not of sufficient resolution to define the relative orientation of the two rings. The dipole of the N-terminal helix stabilizes the negatively charged phosphorylated histidine of P-HPr (Hol et al., 1978, 1981). No NOE were observed between the side chains of Hisl 5 and Argl7, in contrast to earlier NMR work by Klevit and Waygood (1986). n the B. subtilis HPr X-ray crystal structure, Herzberg et al. (1992) found the guanidino group complexed to a sulfate ion which also makes contacts to the active-site histidine as well as two other HPr molecules in the crystal. The authors suggest an altered side-chain conformational state of Argl7 in the unphosphorylated state to avoid the unfavorable electrostatic interaction with the dipole of the helix N-terminus. Our NOE data are not in conflict with this statement. The possible role of the C-terminal glutamate suggested by Klevit and Waygood (1986), based on the observation of NOE between the active site and the C-terminus and site-directed mutagenesis studies on HPr (Anderson et al., 1991), is not supported by our NMR work: no NOE were observed between the active site and the residues in the C- terminus. Care should be taken to invoke the participation of a certain residue in the active site from mutagenesis work. van Dijk et al. (1991) showed that replacement of Glu68 by an alanine lowered the phosphoryl group potential of P-HPr relative to wild-type P-HPr. Our structure shows that this residue is positioned on the opposite side of the molecule. A global structural change probably causes the decrease in phosphoryl group potential. van Dijk et al. (1990) have delineated the H-bond structure at the His15 residue by "N-NMR on "N-histidine enriched HPr. They concluded that the N' proton on the histidine is strongly H-bonded when the histidine is in its neutral form. Presumably this enhances the nucleophilicity of the N" nitrogen for its subsequent phosphorylation. A possible candidate for the H-bond acceptor is Asnl2: NOE were observed between its Cp protons and the C5 proton of the His15 imidazole ring. This is in agreement with the position of Serl2 in B. subtilis HPr (Herzberg et al., 1992) where a hydrogen bond is formed between the Serl2 Oy atom and the His15 N' atom. Further structural refinement and NMR studies on phosphorylated HPr are in progress to obtain a detailed picture of the His15 environment. This research was supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for Scientific Research (NWO). J. G. was supported by

12 89 1 the BRDGE program of the European Community (BLOT ). The SNARF program, written by F. H. J. van Hoesel, was used for processing, visualizing and analyzing all NMR data sets. We thank Varian for providing us with the triple resonance probe. We thank A. A. van Dijk for useful discussions, J. W. Meijberg and B. E. Maas for working on the exchange experiment and T. Nowak for assistance in acquiring the (13C6)glucose. REFERENCES Anderson, J. W., Bhanot, P., Georges, F., Klevit, R. E. & Waygood, E. B. (1991) Biochemistry 30, Bax, A. & Subramanian, S. (1986) J. Magn. Reson. 67, Bax, A., Clore, G. M., Driscoll, P. C., Gronenborn, A. M., kura, M. & Kay, L. E. (1990a) J. Magn. Reson. 87, Bax, A., Clore, G. M. & Gronenborn, A. M. (1990b) J. Magn. Reson. 88, Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A. & Haak, J. R. (1984) J. Chem. Phys. 8Z, Clore, G. M., Bax, A., Driscoll, P. C., Wingfield, P. T. & Gronenborn, A. M. (1990) Biochemistry 29, Crippen, G. M. & Havel, T. F. (1978) Actu Crystallogr. A34, De Vlieg, J., Boelens, R., Scheek, R. M., Kaptein, R. &van Gunsteren, W. F. (1986) sr. J. Chem. 27, El-Kabbani, 0. A. L., Waygood, E. B. & Delbaere, L. T. J. (1987) J. Biol. Chem. 262, Fesik, S. W. & Zuiderweg, E. R. P. (1988) J. Magn. Reson. 78, Hammen, P. K., Waygood, E. B. & Klevit, R. E. (1991) Biochemistry 30, Havel, T. F., Kuntz,. D. & Crippen, G. M. (1983) Bull. Math. Bid. 45, Havel, T. H. (1991) Prog. Biophys. Mol. Biol. 56, Herzberg, O., Reddy, P., Sutrina, S., Saier, M. H., Reizer, J., & Kapafia, G. (1992) Proc. Nut1 Acad. Sci. USA 89, Hol, W. G. J., van Duijnen, P. Th. & Berendsen, H. J. C. (1978) Nature 273, Hol, W. G. J., Halie, L. M. & Sander, C. (1981) Nature 294, kura, M., Kay, L. E. & Bax, A. (1990a) Biochemistry 29, kura, M., Kay, L. E. Tschudin, R. & Bax, A. (1990b) J. Magn. Reson. 86, kura, M., Spera, S., Barbato, G., Kay, L. E., Krinks, M. & Bax, A. (1991) Biochemistry 30, Kaptein, R., Boelens, R., Scheek, R. M. & van Gunsteren, W. F. (1988) Biochemistry 27, Kay, L. E., kura, M., Tschudin, R. & Bax, A. (1990a) J. Magn. Reson. 89, Kay, L. E., kura, M. & Bax, A. (1990b) J. Am. Chem. Soc. 112, Klevit, R. E., Drobny, G. P. & Waygood, E. B. (1986) Biochemistry 25, Klevit, R. E. & Drobny, G. P. (1986) Biochemistry 25, Klevit, R. E. & Waygood, E. B. (1986) Biochemistry 25, Live, D. H., Davis, D. G., Agosta, W. C. & Cowburn, D. (1984) J. Am. Chem. Soc. 106, Marion, D., Driscoll, P. C., Kay, L. E., Wingfield, P. T., Bax, A., Gronenborn, A. M. & Clore, G. M. (1989a) Biochemistry 28, Marion, D., Kay, L. E., Sparks, S. W., Torchia, D. A. & Bax, A. (1989b) J. Am. Chem. Soc. 111, Richardson, J. (1981) Adv. Protein Chem. 20, Scheek, R. M., van Gunsteren, W. F. & Kaptein, R. (1989) Methods Enzymol. 177, Shaka, A. J., Barker, P. B. & Pines, A. (1988) J. Magn. Res. 77, Spera, S. & Bax, A. (1991) J. Am. Chem. Soc. 113, States, D. J., Haberkorn, R. A. & Ruben, D. J. (1982) J. Magn. Res. 48, Torda, A. E., Scheek, R. M. & van Gunsteren, W. F. (1990) J. Mol. Biol. 214, van Dijk, A. A., de Lange, L. C. M., Bachovchin, W. W. & Robillard, G. T. (1990) Biochemistry 29, van Dijk, A. A., Eisermann, R., Hengstenberg, W. & Robillard, G. T. (1991) Biochemistry 30, van Nuland, N. A. J., van Dijk, A. A,, Dijkstra, K., van Hoesel, F. H. J., Scheek, R. M. & Robillard, G. T. (1992) Eur. J. Biochem. 203, Waygood, B. E., Erickson, E., El-Kabbani, 0. A. L. & Delbaere, L. T. J. (1985) Biochemistry 24, Weber, P. L., Morrison, R. & Hare, D. (1988) J. Mol. Biol. 204, Weigel, N., Powers, D. A. & Roseman, S. (1982a) J. Biol. Chem. 257, Weigel, N., Waygood, E. B., Kukuruzinska, M. A,, Nakazawa, A. & Roseman, S. (1982b) J. Biol. Chem. 257, Williamson, M. P. & Madison, V. S. (1990) Biochemistry 29, Wishart, D. S., Sykes, B. D. & Richards, F. M. (1991) J. Mol. Biol. 222, Wishart, D. S., Sykes, B. D. & Richards, F. M. (1992) Biochemistry 31, Wittekind, M., Reizer, J. & Klevit, R. E. (1990) Biochemistry 29, Wiithrich, K. (1976) NMR in biologica1research:peptides andproteins, North-Holland Publishing Co., Amsterdam. Wiithrich, K. (1986) NMR of proteins and nucleic acids, Wiley, New York. Zuiderweg, E. R. P., Mcntosh, L. P., Dahlquist, F. W. & Fesik, S. W. (1990) J. Mugn. Reson. 86,