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First observation of solution structures of bradykinin–penta-O-galloyl-d -glucopyranose complexes as determined by NMR and simulated annealing
Biochimica et Biophysica Acta 1571 (2002) 89 – 101 www.bba-direct.com
First observation of solution structures of bradykinin–penta-O-galloyl-d-glucopyranose complexes as determined by NMR and simulated annealing Sarah Verge´ a, Tristan Richard a, Serge Moreau b, Alain Nurich a, Jean-Michel Merillon a, Joseph Vercauteren a, Jean-Pierre Monti a,* a
GESNIT EA 491, Faculte´ des Sciences Pharmaceutiques, Universite´ de Bordeaux 2, 146 rue Le´o Saignat, 33076 Bordeaux cedex, France Laboratoire de Biophysique Mole´culaire, INSERM U386, Universite´ de Bordeaux 2, 146 rue Le´o Saignat, 33076 Bordeaux cedex, France
b
Received 10 October 2001; received in revised form 1 February 2002; accepted 19 February 2002
Abstract Polyphenols (tannins) are known for their high propensity to precipitate proteins. They bind most strongly to proteins with a high proline content. Understanding the mechanism of this association is of prime interest because this interaction might induce protein conformational changes that may modify their biological activity. To investigate the interaction, an NMR study was carried out on the binding of a representative polyphenol, penta-O-galloyl-D-glucopyranose, to a nonapeptide hormone, bradykinin (BDK), where proline accounts for 30% of residues. Series of 1D and 2D-NMR experiments were performed. For the first time, a three-dimensional structure of complexes was determined using 2D-NMR experiments and molecular modeling. These structure calculations are a potent tool to understand how the association arises. They clearly show that the interaction is a complex phenomenon where several parameters are involved. The PGG/BDK complexes are formed by multiple weak interactions between peptide side chains and galloyl rings. Proline and arginine are good anchoring points and the glycine gives a certain flexibility in the peptide backbone that allows the polyphenol to approach and interact. Therefore, it is not only the hydrophobic stackings between galloyl rings and proline and hydrogen bonding involving arginine and aromatic rings which are important. The residue sequence and the side chain steric bulk also intervene. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Bradikynin; Penta-O-galloyl-D-glucopyranose; NMR
1. Introduction Natural polyphenols or vegetable tannins are present in many higher plants and are consumed by humans in food and beverages. The biological effects of tannins are wideranging and one of their major properties is to form complexes with proteins, a process probably fundamental Abbreviations: BDK, Bradykinin; B3, dimeric proanthocyanidin (+)catechin-(4a!8)-(+)-catechin; B2, dimeric proantho-cyanidin ()-epicatechin-(4h!8)-()-epicatechin; PGG, h-1,2,3,4,6-penta-O-galloyl-D-glucopyranose; 2D-NMR, two-dimensional nuclear magnetic resonance; 3D, three-dimensional; SA, simulated annealing; DMSO, (2H6)-dimethyl sulphoxide; TMSP, trimethylsilyl-3 propionic acid d4-2,2,3,3 sodium salt; TOCSY, total correlation spectroscopy; ROESY, rotating frame Overhauser spectroscopy; HMBC, Heteronuclear Multiple Bond Correlation; PRP, proline-rich protein; RMSD, root mean square deviation * Corresponding author. Tel.: +33-5-5757-1792; fax: +33-5-5757-4563. E-mail address: [email protected] (J.-P. Monti).
to many biological effects observed [1,2]. Therefore, to understand the effect of polyphenols on human health, it is necessary to accurately assess the interactions between polyphenols of known structures and polypeptides involved in physiological processes whose biological activity might be modified by these interactions [3]. The intermolecular complexation seems to involve hydrophobic interactions and hydrogen bonding. Therefore, proteins or peptides with proline and/or arginine residues, such as bradykinin (BDK) (H-Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9-OH), represent good candidates for complexation [1,4 – 8]. This linear nonapeptide hormone, where proline and arginine account for 30% and 20% of residues, respectively, is involved in a variety of physiological processes and in a multitude of pathophysiological disorders such as inflammation, pain or hypotension [9– 12]. In a recent work, we showed that in the presence of the dimeric proanthocyanidin (+)-catechin-(4a ! 8)-(+)-cate-
0304-4165/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 2 ) 0 0 1 8 3 - 6
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chin (B3), the BDK conformation is modified [13]. Indeed, the dimer leads to the formation of a large flexible turn between the 6 – 9 residues. No intermolecular ROEs between protons of BDK and protons of B3 were observed. These results were in agreement with the weak value of the association constant calculated for the analogous dimeric proanthocyanidin ( )-epicatechin-(4h ! 8)-( )-epicatechin (B2) [1]. However, with the polyphenol penta-Ogalloyl-D-glucopyranose (PGG), the value of the association constant with BDK is higher than that with B2. Moreover, this constant value is of the same order of magnitude as that of a model peptide of proline-rich proteins (PRPs) in which intermolecular ROEs between protons of the peptide and protons of PGG were observed [14]. Thus, in this paper, we report the first observation of complex structures between a protein model and a polyphenol. The BDK model used in this investigation presents a large biological activity and the polyphenol PGG, which is a representative member of the hydrolysable tannin family, is well known to bind to a variety of substrates [1,4 – 6]. Using two-dimensional nuclear magnetic resonance (2DNMR) and molecular modeling, a three-dimensional (3D) structure of the complexes has been determined for the first time. The experimental distances calculated from NOE spectra are used in a simulated annealing (SA) protocol to generate families of 3D structures in agreement with NMR data. Conclusions are drawn to explain the involvement of the various chemical groups in the tannin –protein complexation and the Haslam hypotheses concerning the interactions are discussed [1,15].
2. Materials and methods 2.1. Materials BDK salt was purchased from Sigma Company and was used without further purification. The synthesis of PGG was done in two steps by galloylation of h-D-glucose in a 83% overall yield [16]. 2.2. Titration experiments For titration experiments, two identical 1-ml (5 mM) BDK samples, A and B, were prepared, as well as a 200 mM solution of PGG in (2H6)-dimethyl sulphoxide (DMSO) solvent. In sample A, 10-Al aliquots up to a total added volume of 50 Al, and a final 50-Al aliquot of the 200 mM PGG solution were added, giving the PGG/BDK molar ratios of 0:1, 0.4:1, 0.8: 1, 1.2:1, 1.6:1, 2.0:1 and 4.0:1. In sample B, the same volumes of DMSO were added, giving a peptide chemical shift reference solution. During titration, it was verified that the pH did not change. Moreover, by titrating DMSO in a mixture of BDK and PGG, we checked that any possible effect of the solvent polarity on the complex structure and/or formation was negligible.
2.3. Spectroscopy experiments All NMR spectra were referenced to internal trimethylsilyl-3 propionic acid d4-2,2,3,3 sodium salt (TMSP) and were recorded at 303 K, apart from the temperature dependence experiments. 1H NMR experiments were conducted on a Bruker AVANCE-500 NMR spectrometer. Solvent suppression was accomplished by use of a WATERGATE sequence with ‘‘3-9-19-19-9-3’’ 180j selective composite pulse applied at the 1H2O frequency [17]. The titration of PGG into BDK was monitored using one-dimensional experiments. The solvent and dilution effects on the BDK chemical shifts, during the titration, were corrected by subtracting the reference solution chemical shifts from PGG/BDK mixture chemical shifts. To study the complexes, the solvent mixture 90% H2O:10% DMSO was used for all experiments. NMR data were recorded on a 20 mM solution of PGG/BDK mixture in the molar ratio 1:1 (solution A) and on a 20 mM:10 mM solution of PGG/BDK mixture in the molar ratio 2:1 (solution B) to study complexes 1:1 (duplex) and 2:1 (triplex), respectively. This solvent mixture is a suitable solvent to solubilize the complexes as previously demonstrated [14]. To get closer to physiological conditions, the study was done at a pH value equal to 6.4 F 0.1. Total correlation (TOCSY) [18,19] and rotating frame Overhauser enhancement (OffResonance ROESY) [20,21] spectra were recorded in the phase-sensitive mode using the States-TPPI method. Water suppression was accomplished by use of a WATERGATE sequence. TOCSY were acquired with a total spin-lock mixing time of 90 ms using a MLEV-17 mixing sequence with a 2.5 ms trim pulse at the beginning and end of the sequence. ROESY spectra were acquired with mixing pulses of 200 and 400 ms, respectively. The spectral width was 5050.50 Hz at a digital resolution of 2.46 Hz per point. A total of 2048 time-domain data points for 256 t1 values of 64 scans each were acquired and then zero filled to 2048 512 followed by multiplication with a shifted sine bell window function. Temperature coefficients (Dd/DT) were obtained with 1D spectra recorded at 297, 302, 307, 312 and 317 K. They were calculated from plots of chemical shifts vs. temperature which were linear for all amide protons. Heteronuclear Multiple Bond Correlation (HMBC) spectra were optimised on long range couplings with a low-pass J-filter to suppress one-bond correlations, without any decoupling during acquisition and by using gradient pulses for selection [22 –24]. The spectral widths were 5050.50 Hz in F2 and 27670.4 Hz in F1 at a digital resolution of 2.46 and 108.1 Hz per point, respectively. A total of 2048 timedomain data points for 256 t1 values of 64 scans each were acquired and then zero filled to 2048 512 followed by multiplication with a shifted sine bell window in F1 and an exponential window function in F2. Data were processed with UX-NMR from Bruker SA and Gifa 4.2 software developed by Pons et al. (Faculte´ de Pharmacie, Montpellier, France) [25].
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A complex study with mass spectrometry by using electrospray positive ionization experiments was conducted on a Perkin-Elmer Sciex API 365 (Universite´ Paul Sabatier, Toulouse, France). 2.4. Molecular modeling Molecular modeling was conducted on a Silicon Graphics O2 workstation using the Molecular Simulation Incorporated (MSI) company software package. SA and energy minimization were done with Discover and NMRRefine using the consistent-valence force field (cvff) model. Interproton distances were obtained from ROESY spectra. Quantitative determination of cross-peak intensities was based on the number of the contour levels and was calibrated by referencing with the ROE between the h-methylene protons of prolines [26]. The ROE-distance constraints were estimated using a relation of the Braun model [27]. The proline cis/trans isomerization was assigned from diagnostic Hai/Hyi + 1 cross-peaks between the proline residues and the preceding amino acids in the ROESY spectra. In the principal BDK isomer, the three prolines are in a trans conformation. Consequently, all residue bonds were assigned as trans in the molecular simulation. The simulation annealing calculation protocol involved 14 steps as described in a previous work [13]. The distance constraint and the dihedral constraint forces were kept to 30 kcal ˚ 2 and 30 Kcal mol 1 rad 2, respectively. mol 1 A
3. Results and discussion 3.1. Complex stoichiometry The unambiguous assignment of the BDK protons was carried out using the standard technique developed by Wu¨trich [28] (Table 1s of Appendix A). For the polyphenol protons it was done by comparison with literature references [14] and by using the HMBC sequence (Table 2s of Appendix A). As the PGG solution was added to the solution of BDK, the observation of a cloudy precipitate, which progressively disappeared, indicated that a binding interaction occurred. The complex stoichiometry was observed by using the chemical shift variations of amide protons (Fig. 1). These data are in agreement with previous studies [14,29,31]. Fig. 1 clearly suggests the formation of two complexes in fast equilibrium with the separate components and having two slope changes, one with a 1:1 binding interaction, the other with a 2 PGG:1 BDK binding interaction. The weaker effect for Arg9 is due to the terminal position of this amino-acid and thus to its high flexibility. The pH and DMSO controls during the titration experiments ruled out any contribution of these parameters to chemical shift variations. The mass spectrometry experiments are in agreement with these data while the singly and doubly protonated
Fig. 1. Relative chemical shift variations of peptide amide protons with increasing PGG/BDK molar ratio.
complex species were detected: m/z 2001.7 and 2941.2 and m/z 1001.0 and 1471.7, respectively. Additional experiments ruled out a simple cationization reaction (Tetrahedron Letters 2002, 43, 2363– 2366). Moreover, the complex stoichiometry and the binding constant determination were realized by fitting the chemical shift changes using the technique developed by Baxter et al. [29] and Charlton et al. [30]. The values obtained for the first and the second dissociation constant were 4 10 5 and 2.5 10 3 mol l 1, respectively. These constant values and the concentrations used in the complex NMR study led to a duplex/triplex ratio equal to f 3.6:1 and f 1:4 in solutions A and B, respectively. These ratios were in agreement with NMR features. In the following work, a discussion about the nature of complexes is essential because this study is dependent on it. Indeed, the authors of Refs. [14,29,31] present NMR data in agreement with the equation of complex 1:1 but suggest that the latter could be a multivalent binding equilibrium, i.e. several polyphenols binding the same peptide. Thus, in this case, the polyphenol binds the peptide alternately to follow the equation of complex 1:1. This hypothesis is in agreement with the numerous intermolecular NOE cross-peaks presented and with the peptide structure in the random coil [14,29]. Moreover, the peptides are PRPs longer than BDK (19 and 22 amino acids) and evidently richer in prolines. BDK has fewer binding sites, the NMR spectra show a relatively low number of intermolecular NOE cross-peaks, and particularly the structure is ordered (see above); this is in disagreement with a multivalent binding equilibrium. Thus, it is reasonable to postulate that the complexes observed are classical complexes. Note that for a short peptide, complex 1:1 in a mono binding equilibrium has been postulated elsewhere [2]. Lastly, to control for a possible evolution of the complexes, two 1D NMR spectra were done at the beginning and the end of the NMR experiments. The identical chem-
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Table 1 Twelve duplex intermolecular ROEs observed between protons of PGG and protons of BDK BDK Residue 3
Pro Gly4 Gly4 Phe5 Pro7 Pro7 Pro7 Arg9 Arg9 Arg9 Arg9 Arg9
PGG Proton
Galloyl ring protons
Relative intensity
Hy Ha Ha Hh Hg Hy Hy Ha Hh Hh Hg Hy
3 3 2 3 2 2 3 4 4 6 6 6
WW WW WW WW WW WW WW WW WW WW WW WW
(*) (*) (*)
(*) (*) (*)
All relative intensities are very weak corresponding to an upper constraint ˚. limit of 6.0 A (*) The first six unambiguous intermolecular connectivities (see text).
ical shifts and integrations showed that no evolution of the complexes during the experimental time had occurred. 3.2. 3D structure of BDK in the complexes To verify whether the structure of BDK in the complexes was ordered, only peptide ROE cross-peaks were considered in a first step. For example, in the complex 1:1, in addition to the classical sequential daN(i,i + 1) and dNN(i,i + 1) and intra-residue ROE connectivities (67 ROEs are counted), the ROESY spectra exhibited five interaction cross-peaks
d(i,i + 2) between Phe5 and Pro7, Ser6 and Phe8, Pro7 and Arg9, three d(i,i + 3) between Pro2 and Phe5, Ser6 and Arg9, and one d(i,i + 4) between Arg1 and Phe5. In the SA protocol, a total of 100 structures were calculated using the experimental distances determined from ROE spectra. Structural statistics for the converged structures were evaluated in terms of root mean square deviations (RMSDs) between the refined structures and their average structure. A 34-molecule family of low total energy was characterized by relative violations of the distance restraints ˚ upper limit is violated below 5% (i.e. for example, a 2.7 A ˚ below 0.14 A) and by torsion angle violations smaller than 5j. The Ramachandran plot shows that the backbone dihedral angles fall into peptide allowed regions (ProCheck-nmr control: http://pdb.rutgers.edu/validate/). According to the low RMSD value found for these 34 ˚ ) after backbone 2 – 8 residue structures (0.24 F 0.11 A superimposition, it is obvious that the peptide is ordered in section 2 –8. This result is confirmed by the average RMSDs between the 34 structures as a function of the residue number (Fig. 1s of Appendix A). The BDK presents a folded structure with two bends and with the lowest average RMSDs in the central region. On the contrary, the N- and C-terminal regions have higher RMSDs, which suggests a conformational averaging. Thus, for the BDK in the complex, no secondary structure such as helix or strand was detected because no characteristic NOE patterns were observed [28]. Nevertheless, the complex formation with the PGG involves a 3D ordered structure. A similar result with another ordered structure was obtained in the
Fig. 2. Thirteen superimposed structures of the complex between PGG and BDK. For clarity, only glucopyranose (Glc) rings are represented by balls for PGG and the peptide is represented by sticks. The complex is ordered and the peptide surrounds the glucopyranose molecule.
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complex 2:1. The comparison of the BDK mean structures between the duplex and the triplex shows that the peptide backbone globally keeps its structure in the central region, ˚ for the backbone Gly4 –Pro7 with an RMSD equal to 1.21 A domain (Fig. 2s of Appendix A). However, studies have shown that BDK dissolved in DMSO assumes a rigid structure with two bends in the N and C termini [32] or one turn in the C terminus [33]. To investigate the possible effect of DMSO on the BDK conformation in the solution 9:1 H2O/DMSO, we studied BDK alone in this solvent mixture. In addition to the classical sequential and intra-residue ROE connectivities, 3 d(i,i + 2) and 3 d(i,i + 3) cross-peaks were observed involving an ordered structure in the C-terminal region as determined by the SA protocol (data not shown). Yet this structure was different from that obtained for BDK in the presence of PGG. Thus, the ordered structures observed in the duplex and the triplex are really due to the complexation of the peptide hormone with the polyphenol.
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3.3. 3D structure of the complexes and modes of complexation of polyphenols with peptides and proteins 3.3.1. Duplex structure The temperature dependence of the amide proton chemical shift indicates whether the amide proton is protected from exchange with a solvent [34]. Dd/DT values for amide and guanidinium protons under the threshold showed the absence of intra-molecular hydrogen bonds, except for the NHq of Arg9 residue, which had a value of 4 ppb/K (Table 1s of Appendix A). However, this distance constraint was not included in the SA protocol because it was difficult to attribute without ambiguity. The PGG exhibits 13 intra-residue ROE connectivities. We observed 12 intermolecular ROEs between the protons of PGG and protons of BDK (Table 1 and Fig. 3s of Appendix A). In a first step in the SA protocol, we used only the six unambiguous intermolecular ROEs between the protons of galloyls 3 and 4 and those of BDK (Table 1).
Fig. 3. Representations of the closest PGG/BDK complex to the mean structure after SA. The galloyl (gall) rings are represented by large balls and small balls indicate the peptide residues involved in the interactions. The whole complex is presented in A; hydrophobic stacking between galloyl ring 3 and proline-3 in B; edge-to-face interaction between galloyl ring 2 and proline-7 in C; interactions between galloyl rings 4 and 6 and the BDK C-terminus region in D.
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Indeed, the protons of galloyls 2 and 6 overlapped and this first simulation explored the conformational spaces of galloyls 2 and 6. From these data, four supplementary cross-peaks concerning these galloyls could be unambiguously introduced in a new SA. At the end of this simulation, the results obtained allowed us to attribute the two last intermolecular ROEs. In the final SA, 100 structures were calculated and 13 low total energy structures were detected with an RMSD, between the refined structures and the mean ˚ for the backbone conformation, equal to 0.41 F 0.10 A 2 8 Pro –Phe domain and the heavy atoms of the glucopyranose molecule (Fig. 2). The Ramachandran plot shows that the backbone dihedral angles fall into peptide allowed regions (Fig. 4s, in supplementary materials) and the use of Swiss-PdbViewer http://www.expasy. ch/spdbv/) and ProCheck-nmr http://pdb.rutgers.edu/validate/) show that no protein problem was detected. Concerning the 156 intermolecular NOE constraints used (12 interNOE 13 structures) we have: 78 nonviolated, 66 violated i1%, 9 violated i2%, and 3 violated i3%. This result can be considered as an indirect proof that the hypothesis of a mono binding equilibrium is reasonable. Even though the BDK and PGG are flexible molecules, it would be surprising not to observe either some large distance violations or BDK structure violations of the Ramachandran plot. To present the complex formed between BDK and PGG, Fig. 3A shows the closest structure to the SA mean structure. Our results clearly show that the anchoring points of PGG are principally the galloyl groups 2, 3 and 6. The 2 and 3 galloyl rings have three and four intermolecular connectivities with the peptide, respectively. This leads to RMSD values for heavy atoms of galloyl rings equal to ˚ , respectively, implying a 2.36 F 0.33 and 1.49 F 0.48 A weak degree of freedom for these rings. The RMSD value ˚ for galloyl 6 is in agreement with the equal to 3.92 F 1.09 A conformational averaging of the peptide C terminus because the three intermolecular connectivities are with the Arg9 residue. Lastly, no intermolecular cross-peak was detected with galloyl 1. The latter can occupy a large conformational space, which is expressed by a high RMSD value for heavy ˚ . Concerning the atoms of this galloyl ring: 4.66 F 1.75 A anchoring points of BDK, the residues Pro and Arg9 are involved as shown in Fig. 3. The presence of the Gly4 residue, which has the less bulky side chain, breaks the rigidity of the backbone due to proline residues and gives a certain flexibility. This allows the PGG galloyl rings to come near to the central region of the peptide and to interact with the Pro residues on both sides of this position 4. In the literature, various hypotheses based on proton chemical shift changes were made: k k stacking or k j attraction for phenylalanine residues, hydrophobic stacking for the proline rings and/or hydrogen bonding for arginine guanidinium [1,35]. As shown in Fig. 3B, the galloyl ring 3 presents a hydrophobic stacking with the Pro3 ring while the Pro2 ring is nearly perpendicular to the
galloyl ring (Fig. 3B). The rings of galloyl 2 and Pro7 are nearly perpendicular (Fig. 3C). Indeed, the presence of Phe5 and Phe8 side chains prevents a parallel positioning of the galloyl 2 and Pro7 rings. The space of galloyl ring 4 is bounded by one or two hydrogen bonds obtained from the SA protocol. The NHq and one NHD of the Arg9 residue are perpendicular to galloyl 4 and thus might create a hydrogen bond with the k system of the galloyl ring (Fig. 3D). This SA result is in agreement with the experimental data because a value of 4 ppb/K was obtained for the Arg9 NHq (Table 1s). Moreover, it should be underlined that the k system of Phe8 is positioned to favour an edge-to-face k k interaction with the hydrogen of a galloyl 4 hydroxyl group [36,37]. As shown in Fig. 3, the position of the three galloyls 2, 3 and 4, in regard to the peptide backbone, means that galloyls 1 and 6 are situated on the other side, so it was surprising to observe ROESY connectivities with galloyl 6. However, a careful examination showed that the turn imposed by prolines 2 and 3 prevented an approach of the Arg1 residue to galloyl 1, while the spatial position of galloyl 6 is different. The presence of a supplementary CH2 in the chain implies a Table 2 Twenty seven triplex intermolecular ROEs observed between protons of PGG and protons of BDK BDK Residue 1
Arg Arg1 Gly4 Gly4 Phe5 Phe5 Phe5 Phe5 Phe5 Ser6 Pro7 Pro7 Pro7 Phe8 Arg9 Arg9
PGG1 Proton
Galloyl ring protons
Relative intensity
Hh Hy Ha Ha Ha Hh Hh Hq Hq Hh Ha Hy Hy Hq Hy Hg
4 4 2 3 2 3 4 4 H4 gluco-pyranose 2 2 2 1 6 6 6
W W WW WW WW WW WW W WW WW W WW W WW WW WW
(*) (*) (*)
(*)
(*) (*)
PGG2 3
Pro Ser6 Ser6 Phe8 Phe8 Arg9 Arg9 Arg9 Arg9 Arg9 Arg9
Hy Hh Hh Hh Hh Ha Ha Ha Ha Hy Hg
4 1 4 1 6 H1 gluco-pyranose H3 gluco-pyranose 3 6 4 4
WW WW WW W W W WW WW WW W W
All relative intensities are weak or very weak corresponding to an upper ˚ , respectively. constraint limit of 5.0 or 6.0 A (*) The first six intermolecular connectivities attributed to the PGG1 (see text).
S. Verge´ et al. / Biochimica et Biophysica Acta 1571 (2002) 89–101 Table 3 Triplex structural data for the 10 refined structures in agreement with NMR restraints Distances and torsion angle restraints Intra-residue Inter-residue V
45 44 8
PGG* 22 Inter-molecule 27
Number of relative NOE violations
Not-violated 0 – 1% 2 – 4% 5 – 7%
All 1600 restraints (160 restraints 10 molecules)
270 inter-molecular restraints (27 restraints 10 molecules)
1270 207 120 3
106 90 71 3
79.4% 12.9% 7.5% 0.2%
39.3% 33.3% 26.3% 1.1%
Atomic RMS values for converged structures vs. their geometric average All heavy atoms Backbone + GLC Backbone Backbone (2 – 8)
1.36 F 0.21 0.45 F 0.10 0.42 F 0.10 0.35 F 0.10
(*) The cross-peaks were attributed to both molecules since both PGGs being identical (fast equilibrium).
larger freedom of positioning for this galloyl ring, and the presence of guanidinium of Arg9 residue in a spatial proximity leads to a hydrogen bond between these groups. 3.3.2. Triplex structure In the molar ratio 2 PGG:1 BDK solution, we observed 27 intermolecular ROEs between the protons of PGGs and
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those of BDK (Table 2). From the NMR spectra, it is clear that the complex is in fast exchange with the separate components, over the NMR time scale. Particularly, both PGG are in fast equilibrium. Therefore, it is important to explain the way both PGGs were attributed in SA because the following study is dependent on it. We considered that the first PGG molecule present in complex 1:1 occupied globally the same spatial environment as in complex 2 PGG:1 BDK. This hypothesis is in agreement with the results since six ROESY cross-peaks were already observed in the complex 1:1 spectra and were present in the complex 2 PGG:1 BDK spectra. Thus, in a first SA protocol, we attributed these cross-peaks only to the first PGG molecule (PGG1). After analysis, the model demonstrated the limits of the conformational space for the second PGG molecule (PGG2), i.e. by measuring the inter-molecular proton distances and by using the spatial geometries of the peptide and PGG1, six ROESY cross-peaks could be attributed to PGG2. At the end of the second SA protocol and after analysis, we had more accurate conformational spaces for both PGGs. The protocol was repeated about 30 times with the gradual insertion of all cross-peaks. All large violations of distance and/or all Ramachandran plot violations revealed an inaccurate attribution so we did it again. At the end of these simulations, the results obtained allowed us to attribute 16 and 11 intermolecular ROEs to the PGG1 and PGG2, respectively (Table 2). Moreover, no intramolecular hydrogen bond distance constraint was included in the SA protocol. In the final SA, 100 structures were calculated and 10 low total energy structures were detected
Fig. 4. Ten superimposed molecules for 2 PGG:1 BDK triplex. The peptide is clearly structured. The two molecules of PGG form a cage around it.
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with an RMSD, between the refined structures and the mean ˚ for the conformation, which was equal to 0.45 F 0.10 A backbone domain and the heavy atoms of the glucopyranose core. Table 3 summarizes the structural data for this set of refined molecules and Fig. 4 shows the triplex structures. To present the triplex formed between BDK and PGGs, Fig. 5 shows the closest structure to the SA mean structure. What was interesting when we analyzed the 3D-structure was the presence of common anchoring points between PGGs and BDK. Indeed, Pro7 interacts with galloyl 2 of PGG1 and galloyl 1 of PGG2 and Arg9 interacts with galloyl 6 of PGG1 and galloyl 4 of PGG2. It should be noted that for PGG1, the same anchoring points had been evidenced in complex 1:1. As shown in Fig. 5A, the galloyl ring 2 of PGG1 presents a hydrophobic stacking with the Pro7 with rings nearly parallel, while the same Pro7 ring is nearly perpendicular to the galloyl ring 1 of PGG2. Thus, Pro7 leads to such a spatial proximity of both PGGs that they could interact together. In fact, a hydroxyl group of galloyl 2 in PGG1 and that of galloyl 1 in PGG2 are orientated towards each other, allowing a possible H bond between them. Thus, both galloyl rings attached by this H bond might bound a hydrophobic pocket which might surround the Pro7 residue.
Concerning Arg9 and PGG1, a first H bond is possible between a hydroxyl oxygen of galloyl 6 and a hydrogen of the guanidinium group. Moreover, a second possible H bond between HD of Phe8 and a hydroxyl oxygen of galloyl 6 would allow a better stabilization of this structure (Fig. 5B). With PGG2, a hydrogen bond could be created between a hydroxyl proton of galloyl 4 and an oxygen atom of the Arg9 carboxyl terminal (Fig. 5B). Therefore, it appears that Arg9 and Pro7 are the important residues for the structure. They are the meeting points of PGGs, and in the case of Pro7 are able to cooperate to form a hydrophobic pocket. However, the stabilization of the triplex is also due to a set of other anchoring points. For PGG1, Phe5 ring is sandwiched between both rings of galloyls 3 and 4 by a double k k stacking (Fig. 5C). Moreover, the NHq of the Arg1 residue might create an H bond with a hydroxyl oxygen of galloyl 4, which would be in agreement with the NMR temperature coefficients (Fig. 5D). Thus, it seems that compared to the duplex, PGG1 is shifted from the C to the N termini probably because of the presence of the second PGG, which is positioned on the other side of the peptide and could reduce the freedom of positioning. Nevertheless, the anchoring of PGG1 from the N to the C termini is made possible, thanks to the backbone flexibility (Gly4 and Ser6 presence in the sequence), to the long and flexible
Fig. 5. Closest structure to the SA mean structure. Galloyls are represented by balls and BDK bakbone is represented by large sticks. (A – E) Galloyl rings are represented by large balls and small balls indicate the peptide residues involved in the interactions. (A) Hydrophobic stacking between Pro7 and galloyl 2 of PGG1 and perpendicular positionning of Pro7 and galloyl 1 of PGG2. The Pro7 is an anchoring point between PGG1 and PGG2. (B) Arg9 as another anchoring point between PGG1 and PGG2. (C) Double stacking between galloyl 3 and 4 of PGG1 and Phe5. (D) Possible H bond between the NHq of Arg1 and galloyl 4 of PGG1. (E) Perpendicular positionning of Pro3 and galloyl 4 of PGG2.
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Fig. 5 (continued ).
side chain of arginine residues and also to the articulated arms of the PGG molecule. Lastly, the distance between the nitrogen atom of Ser6 and a hydroxyl oxygen of galloyl 2 is in agreement with the possibility of an H bond. The latter could be created with the amide proton and the oxygen of galloyl 2. However, the hypothesis is not in agreement with the NMR temperature coefficients. For PGG2, the k system of galloyl 6 is positioned in such a way that it favors an edge-to-face k k interaction with a hydrogen atom of the Phe8 ring [36,37]. Moreover, the positioning of the Pro3 ring nearly perpendicular to the galloyl ring 4 could allow hydrophobic interaction (Fig. 5E). Thus, the PGG2 is anchored from the central domain to the C terminus on the other side of the peptide vs. PGG1. The impossibility for this PGG to anchor the N terminus is due to the bend imposed by the Pro2 – Pro3 sequence, which positions the Arg1 side chain on the side of the PGG1. Lastly, we observed ROESY cross-peaks between peptide protons and gluropyranose ring protons (Table 2). The gluropyranose ring and the BDK moved closer together, and this was more pronounced for PGG2 than for PGG1 because the ROESY connectivities involved the backbone protons for the former and the side chain for the latter.
4. Conclusion In summary, the results of this investigation suggest that the PGG/BDK complexes are formed by multiple weak interactions between peptide side chains and galloyl rings. It appears that the residue sequence, and in particular the side chain steric bulk, acts on the interaction mechanism. The goal of this work was to obtain a complex model for tannin– protein interactions. And even though this interaction might be important at the biological activity level [35], it is extremely difficult to extrapolate this in vitro model to human physiology, if only for in vivo concentration values.
Acknowledgements We thank Dr. Richelme-David S. for her technical assistance and helpful discussions (Service de Spectrome´trie de masse, FR 1744, Universite´ Paul Sabatier, Toulouse, France). This work was supported in part by the Conseil Re´gional d’Aquitaine (grant 990305002).
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Appendix A Table 1s. Chemical shifts of the assigned 1H-NMR resonances in 1H2O/DMSO (9:1), pH 6.5, in molar ratio solutions 1 PGG:1 BDK, 2 PGG:1 BDK (in italic) and in absence of PGG (in brackets). NH
Ha
Hh
Others
1
Arg
–
Pro2
–
Pro3
–
1.97 1.98 (1.87) 2.40/1.91 2.38/1.90 (2.43/1.87) 2.34/1.97 2.34/1.96 (2.30/1.90)
Hg 1.77; 1.79 (1.69) Hg 2.02; 2.00 (2.02) Hg 2.10; 2.09 (2.06)
Gly4
8.37 8.37 (8.34) 8.06 8.06 (8.00) 8.16 8.16 (8.11) –
4.38 4.39 (4.28) 4.77 4.73 (4.79) 4.49 4.51 (4.43) 3.98 4.01 (3.91) 4.65 4.70 (4.60) 4.73 4.75 (4.70) 4.40 4.42 (4.33) 4.69 4.73 (4.63) 4.26 4.28 (4.15)
Phe5
Ser6
Pro7
Phe8
Arg9
7.96 7.94 (7.94) 7.76 7.77 (7.66)
Dd/DT, ppb/K Hy 3.22 3.22 (3.15) Hy 3.75/3.51 3.74/3.50 (3.75/3.50) Hy 3.84/3.68 3.83/3.67 (3.83/3.68)
–
–
–
10 10 3.08 3.09 (3.05) 3.83 3.86 (3.75) 2.16/1.67 2.16/1.72 (2.15/1.67) 3.27/3.00 3.28/2.98 (3.22/2.95) 1.91/1.78 1.92/1.80 (1.81/1.69)
Hy 7.22; 7.20 (7.23)
Hq 7.31; 7.31 (7.35)
HD 7.28 7.29 (7.30)
8 8 9 9
Hg 1.87; 1.85/1.67 (1.88/1.67) Hy 7.27; 7.27 (7.27) Hg 1.62; 1.64 (1.54) NHq 7.26; 7.27 (7.19)
Hy 3.60 3.60 (3.58) Hq 7.35; 7.35 (7.38) Hy 3.23 3.23 (3.18); NHD 6.75 6.74 (6.66)
–
HD 7.30 7.30 (7.33)
8 8 6 7 NHq 4; 3
NHD 10 11
Table 2s. Chemical shifts of the assigned 1H-NMR resonances for PGG in 1H2O/DMSO (9:1), pH 6.5, in the molar ratio solutions 1 PGG:1 BDK, 2 PGG:1 BDK (in italic). Glucopyranose ring protons
Galloyl ring protons
Position
d (ppm)
Position
d (ppm)
H-1
6.30 6.33
1
7.20 7.23
H-2
5.70 5.73
2
7.12 7.15
H-3
5.96 5.98
3
7.05 7.14
H-4
4.68 4.69
4
7.07 7.07
H-5
4.50 6
7.12 7.08
4.51 H-6
4.60 4.61
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Fig. 1s. Thirty-four superimposed structures for the BDK in the duplex and RMS deviations per residue obtained using only ROE nonapeptide constraints. The peptide is clearly structured between residues 2 and 8.
Fig. 2s. Comparison of the BDK mean structure in the duplex and in the triplex. The structure is globally kept in the central region Gly4 – Pro7, but a re-orientation is observed in the C and N termini.
100
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Fig. 3s. Galloyl (gall) region of the duplex PGG/BDK 20 mM solution 400 ms ROESY spectrum in the solvent mixture H2O/DMSO (9:1). This region contains cross-peaks between galloyl ring resonances and peptide protons. These connectivities are also present in the 200-ms ROESY spectrum with a less favorable signal-to-noise ratio.
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101
Fig. 4s. Ramachandran plot of the 13 structures of the BDK in the duplex. Black circles stand for the torsion angle (U, W) values, except for Gly residues symbolized by black squares. The large circles stand for the areas of ideal turns ( F 20j).
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[20] A.A. Bothner-By, R.L. Stephens, J. Lee, C.D. Warren, R.W. Jeanloz, J. Am. Chem. Soc. 106 (1984) 811 – 813. [21] H. Desvaux, P. Berthault, N. Birlirakis, M. Goldman, M. Piotto, Magn. Reson., A 113 (1995) 47 – 52. [22] A. Bax, M.F. Summers, J. Am. Chem. Soc. 108 (1986) 2093 – 2094. [23] W. Willker, D. Leibfritz, R. Kerssebaum, W. Bermel, Magn. Reson. Chem. 31 (1993) 287 – 292. [24] J. Ruiz-Cabello, G.W. Vuister, C.T.W. Moonen, P. van Gelderen, J.S. Cohen, P.C.M. van Zijl, J. Magn. Reson. 100 (1992) 282 – 303. [25] J.L. Pons, T.E. Malliavin, M.A. Delsuc, J. Biomol. NMR 8 (1996) 445 – 452. [26] M. Pellegrini, S. Mammi, E. Peggion, F. Mierke, J. Med. Chem. 40 (1997) 92 – 98. [27] W. Braun, C. Bo¨sch, L.R. Brown, N. Go, K. Wuthrich, Biochim. Biophys. Acta 667 (1981) 377 – 396. [28] K. Wu¨thrich, NMR of Proteins and Nucleic Acids, Wiley, New-York, 1986. [29] N.J. Baxter, T.H. Lilley, E. Haslam, M.P. Williamson, Biochemistry 36 (1997) 5566 – 5577. [30] A.J. Charlton, A.L. Davis, D.P. Jones, J.R. Lewis, A.P. Davies, E. Haslam, M.P. Williamson, J. Chem. Soc., Perkin Trans. 2 (2000) 317 – 322. [31] A.J. Charlton, N.J. Baxter, T.H. Lilley, E. Haslam, C.J. McDonald, M.P. Williamson, FEBS Lett. 382 (1996) 289 – 292. [32] S.R. Mirmira, S. Durani, S. Srivastava, R.S. Phadke, Magn. Reson. Chem. 28 (1990) 587 – 593. [33] J.K. Young, R.P. Hicks, Biopolymers 34 (1994) 611 – 623. [34] G. Kotovych, J.R. Cann, J.M. Stewart, H. Yamamoto, Biochem. Cell. Biol. 76 (1998) 257 – 266. [35] E. Haslam, Practical Polyphenolics, Cambridge Univ. Press, Cambridge, 1998. [36] L.R. Hanton, C.A. Hunter, D.H. Purvis, J. Chem. Soc., Chem. Commun. (1992) 1134 – 1136. [37] K. Mu¨ller-Dethlefs, P. Hobza, Chem. Rev. 100 (2000) 143 – 167.
First observation of solution structures of bradykinin–penta-O-galloyl-d-glucopyranose complexes as determined by NMR and simulated annealing Sarah Verge´ a, Tristan Richard a, Serge Moreau b, Alain Nurich a, Jean-Michel Merillon a, Joseph Vercauteren a, Jean-Pierre Monti a,* a
GESNIT EA 491, Faculte´ des Sciences Pharmaceutiques, Universite´ de Bordeaux 2, 146 rue Le´o Saignat, 33076 Bordeaux cedex, France Laboratoire de Biophysique Mole´culaire, INSERM U386, Universite´ de Bordeaux 2, 146 rue Le´o Saignat, 33076 Bordeaux cedex, France
b
Received 10 October 2001; received in revised form 1 February 2002; accepted 19 February 2002
Abstract Polyphenols (tannins) are known for their high propensity to precipitate proteins. They bind most strongly to proteins with a high proline content. Understanding the mechanism of this association is of prime interest because this interaction might induce protein conformational changes that may modify their biological activity. To investigate the interaction, an NMR study was carried out on the binding of a representative polyphenol, penta-O-galloyl-D-glucopyranose, to a nonapeptide hormone, bradykinin (BDK), where proline accounts for 30% of residues. Series of 1D and 2D-NMR experiments were performed. For the first time, a three-dimensional structure of complexes was determined using 2D-NMR experiments and molecular modeling. These structure calculations are a potent tool to understand how the association arises. They clearly show that the interaction is a complex phenomenon where several parameters are involved. The PGG/BDK complexes are formed by multiple weak interactions between peptide side chains and galloyl rings. Proline and arginine are good anchoring points and the glycine gives a certain flexibility in the peptide backbone that allows the polyphenol to approach and interact. Therefore, it is not only the hydrophobic stackings between galloyl rings and proline and hydrogen bonding involving arginine and aromatic rings which are important. The residue sequence and the side chain steric bulk also intervene. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Bradikynin; Penta-O-galloyl-D-glucopyranose; NMR
1. Introduction Natural polyphenols or vegetable tannins are present in many higher plants and are consumed by humans in food and beverages. The biological effects of tannins are wideranging and one of their major properties is to form complexes with proteins, a process probably fundamental Abbreviations: BDK, Bradykinin; B3, dimeric proanthocyanidin (+)catechin-(4a!8)-(+)-catechin; B2, dimeric proantho-cyanidin ()-epicatechin-(4h!8)-()-epicatechin; PGG, h-1,2,3,4,6-penta-O-galloyl-D-glucopyranose; 2D-NMR, two-dimensional nuclear magnetic resonance; 3D, three-dimensional; SA, simulated annealing; DMSO, (2H6)-dimethyl sulphoxide; TMSP, trimethylsilyl-3 propionic acid d4-2,2,3,3 sodium salt; TOCSY, total correlation spectroscopy; ROESY, rotating frame Overhauser spectroscopy; HMBC, Heteronuclear Multiple Bond Correlation; PRP, proline-rich protein; RMSD, root mean square deviation * Corresponding author. Tel.: +33-5-5757-1792; fax: +33-5-5757-4563. E-mail address: [email protected] (J.-P. Monti).
to many biological effects observed [1,2]. Therefore, to understand the effect of polyphenols on human health, it is necessary to accurately assess the interactions between polyphenols of known structures and polypeptides involved in physiological processes whose biological activity might be modified by these interactions [3]. The intermolecular complexation seems to involve hydrophobic interactions and hydrogen bonding. Therefore, proteins or peptides with proline and/or arginine residues, such as bradykinin (BDK) (H-Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9-OH), represent good candidates for complexation [1,4 – 8]. This linear nonapeptide hormone, where proline and arginine account for 30% and 20% of residues, respectively, is involved in a variety of physiological processes and in a multitude of pathophysiological disorders such as inflammation, pain or hypotension [9– 12]. In a recent work, we showed that in the presence of the dimeric proanthocyanidin (+)-catechin-(4a ! 8)-(+)-cate-
0304-4165/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 2 ) 0 0 1 8 3 - 6
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chin (B3), the BDK conformation is modified [13]. Indeed, the dimer leads to the formation of a large flexible turn between the 6 – 9 residues. No intermolecular ROEs between protons of BDK and protons of B3 were observed. These results were in agreement with the weak value of the association constant calculated for the analogous dimeric proanthocyanidin ( )-epicatechin-(4h ! 8)-( )-epicatechin (B2) [1]. However, with the polyphenol penta-Ogalloyl-D-glucopyranose (PGG), the value of the association constant with BDK is higher than that with B2. Moreover, this constant value is of the same order of magnitude as that of a model peptide of proline-rich proteins (PRPs) in which intermolecular ROEs between protons of the peptide and protons of PGG were observed [14]. Thus, in this paper, we report the first observation of complex structures between a protein model and a polyphenol. The BDK model used in this investigation presents a large biological activity and the polyphenol PGG, which is a representative member of the hydrolysable tannin family, is well known to bind to a variety of substrates [1,4 – 6]. Using two-dimensional nuclear magnetic resonance (2DNMR) and molecular modeling, a three-dimensional (3D) structure of the complexes has been determined for the first time. The experimental distances calculated from NOE spectra are used in a simulated annealing (SA) protocol to generate families of 3D structures in agreement with NMR data. Conclusions are drawn to explain the involvement of the various chemical groups in the tannin –protein complexation and the Haslam hypotheses concerning the interactions are discussed [1,15].
2. Materials and methods 2.1. Materials BDK salt was purchased from Sigma Company and was used without further purification. The synthesis of PGG was done in two steps by galloylation of h-D-glucose in a 83% overall yield [16]. 2.2. Titration experiments For titration experiments, two identical 1-ml (5 mM) BDK samples, A and B, were prepared, as well as a 200 mM solution of PGG in (2H6)-dimethyl sulphoxide (DMSO) solvent. In sample A, 10-Al aliquots up to a total added volume of 50 Al, and a final 50-Al aliquot of the 200 mM PGG solution were added, giving the PGG/BDK molar ratios of 0:1, 0.4:1, 0.8: 1, 1.2:1, 1.6:1, 2.0:1 and 4.0:1. In sample B, the same volumes of DMSO were added, giving a peptide chemical shift reference solution. During titration, it was verified that the pH did not change. Moreover, by titrating DMSO in a mixture of BDK and PGG, we checked that any possible effect of the solvent polarity on the complex structure and/or formation was negligible.
2.3. Spectroscopy experiments All NMR spectra were referenced to internal trimethylsilyl-3 propionic acid d4-2,2,3,3 sodium salt (TMSP) and were recorded at 303 K, apart from the temperature dependence experiments. 1H NMR experiments were conducted on a Bruker AVANCE-500 NMR spectrometer. Solvent suppression was accomplished by use of a WATERGATE sequence with ‘‘3-9-19-19-9-3’’ 180j selective composite pulse applied at the 1H2O frequency [17]. The titration of PGG into BDK was monitored using one-dimensional experiments. The solvent and dilution effects on the BDK chemical shifts, during the titration, were corrected by subtracting the reference solution chemical shifts from PGG/BDK mixture chemical shifts. To study the complexes, the solvent mixture 90% H2O:10% DMSO was used for all experiments. NMR data were recorded on a 20 mM solution of PGG/BDK mixture in the molar ratio 1:1 (solution A) and on a 20 mM:10 mM solution of PGG/BDK mixture in the molar ratio 2:1 (solution B) to study complexes 1:1 (duplex) and 2:1 (triplex), respectively. This solvent mixture is a suitable solvent to solubilize the complexes as previously demonstrated [14]. To get closer to physiological conditions, the study was done at a pH value equal to 6.4 F 0.1. Total correlation (TOCSY) [18,19] and rotating frame Overhauser enhancement (OffResonance ROESY) [20,21] spectra were recorded in the phase-sensitive mode using the States-TPPI method. Water suppression was accomplished by use of a WATERGATE sequence. TOCSY were acquired with a total spin-lock mixing time of 90 ms using a MLEV-17 mixing sequence with a 2.5 ms trim pulse at the beginning and end of the sequence. ROESY spectra were acquired with mixing pulses of 200 and 400 ms, respectively. The spectral width was 5050.50 Hz at a digital resolution of 2.46 Hz per point. A total of 2048 time-domain data points for 256 t1 values of 64 scans each were acquired and then zero filled to 2048 512 followed by multiplication with a shifted sine bell window function. Temperature coefficients (Dd/DT) were obtained with 1D spectra recorded at 297, 302, 307, 312 and 317 K. They were calculated from plots of chemical shifts vs. temperature which were linear for all amide protons. Heteronuclear Multiple Bond Correlation (HMBC) spectra were optimised on long range couplings with a low-pass J-filter to suppress one-bond correlations, without any decoupling during acquisition and by using gradient pulses for selection [22 –24]. The spectral widths were 5050.50 Hz in F2 and 27670.4 Hz in F1 at a digital resolution of 2.46 and 108.1 Hz per point, respectively. A total of 2048 timedomain data points for 256 t1 values of 64 scans each were acquired and then zero filled to 2048 512 followed by multiplication with a shifted sine bell window in F1 and an exponential window function in F2. Data were processed with UX-NMR from Bruker SA and Gifa 4.2 software developed by Pons et al. (Faculte´ de Pharmacie, Montpellier, France) [25].
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A complex study with mass spectrometry by using electrospray positive ionization experiments was conducted on a Perkin-Elmer Sciex API 365 (Universite´ Paul Sabatier, Toulouse, France). 2.4. Molecular modeling Molecular modeling was conducted on a Silicon Graphics O2 workstation using the Molecular Simulation Incorporated (MSI) company software package. SA and energy minimization were done with Discover and NMRRefine using the consistent-valence force field (cvff) model. Interproton distances were obtained from ROESY spectra. Quantitative determination of cross-peak intensities was based on the number of the contour levels and was calibrated by referencing with the ROE between the h-methylene protons of prolines [26]. The ROE-distance constraints were estimated using a relation of the Braun model [27]. The proline cis/trans isomerization was assigned from diagnostic Hai/Hyi + 1 cross-peaks between the proline residues and the preceding amino acids in the ROESY spectra. In the principal BDK isomer, the three prolines are in a trans conformation. Consequently, all residue bonds were assigned as trans in the molecular simulation. The simulation annealing calculation protocol involved 14 steps as described in a previous work [13]. The distance constraint and the dihedral constraint forces were kept to 30 kcal ˚ 2 and 30 Kcal mol 1 rad 2, respectively. mol 1 A
3. Results and discussion 3.1. Complex stoichiometry The unambiguous assignment of the BDK protons was carried out using the standard technique developed by Wu¨trich [28] (Table 1s of Appendix A). For the polyphenol protons it was done by comparison with literature references [14] and by using the HMBC sequence (Table 2s of Appendix A). As the PGG solution was added to the solution of BDK, the observation of a cloudy precipitate, which progressively disappeared, indicated that a binding interaction occurred. The complex stoichiometry was observed by using the chemical shift variations of amide protons (Fig. 1). These data are in agreement with previous studies [14,29,31]. Fig. 1 clearly suggests the formation of two complexes in fast equilibrium with the separate components and having two slope changes, one with a 1:1 binding interaction, the other with a 2 PGG:1 BDK binding interaction. The weaker effect for Arg9 is due to the terminal position of this amino-acid and thus to its high flexibility. The pH and DMSO controls during the titration experiments ruled out any contribution of these parameters to chemical shift variations. The mass spectrometry experiments are in agreement with these data while the singly and doubly protonated
Fig. 1. Relative chemical shift variations of peptide amide protons with increasing PGG/BDK molar ratio.
complex species were detected: m/z 2001.7 and 2941.2 and m/z 1001.0 and 1471.7, respectively. Additional experiments ruled out a simple cationization reaction (Tetrahedron Letters 2002, 43, 2363– 2366). Moreover, the complex stoichiometry and the binding constant determination were realized by fitting the chemical shift changes using the technique developed by Baxter et al. [29] and Charlton et al. [30]. The values obtained for the first and the second dissociation constant were 4 10 5 and 2.5 10 3 mol l 1, respectively. These constant values and the concentrations used in the complex NMR study led to a duplex/triplex ratio equal to f 3.6:1 and f 1:4 in solutions A and B, respectively. These ratios were in agreement with NMR features. In the following work, a discussion about the nature of complexes is essential because this study is dependent on it. Indeed, the authors of Refs. [14,29,31] present NMR data in agreement with the equation of complex 1:1 but suggest that the latter could be a multivalent binding equilibrium, i.e. several polyphenols binding the same peptide. Thus, in this case, the polyphenol binds the peptide alternately to follow the equation of complex 1:1. This hypothesis is in agreement with the numerous intermolecular NOE cross-peaks presented and with the peptide structure in the random coil [14,29]. Moreover, the peptides are PRPs longer than BDK (19 and 22 amino acids) and evidently richer in prolines. BDK has fewer binding sites, the NMR spectra show a relatively low number of intermolecular NOE cross-peaks, and particularly the structure is ordered (see above); this is in disagreement with a multivalent binding equilibrium. Thus, it is reasonable to postulate that the complexes observed are classical complexes. Note that for a short peptide, complex 1:1 in a mono binding equilibrium has been postulated elsewhere [2]. Lastly, to control for a possible evolution of the complexes, two 1D NMR spectra were done at the beginning and the end of the NMR experiments. The identical chem-
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Table 1 Twelve duplex intermolecular ROEs observed between protons of PGG and protons of BDK BDK Residue 3
Pro Gly4 Gly4 Phe5 Pro7 Pro7 Pro7 Arg9 Arg9 Arg9 Arg9 Arg9
PGG Proton
Galloyl ring protons
Relative intensity
Hy Ha Ha Hh Hg Hy Hy Ha Hh Hh Hg Hy
3 3 2 3 2 2 3 4 4 6 6 6
WW WW WW WW WW WW WW WW WW WW WW WW
(*) (*) (*)
(*) (*) (*)
All relative intensities are very weak corresponding to an upper constraint ˚. limit of 6.0 A (*) The first six unambiguous intermolecular connectivities (see text).
ical shifts and integrations showed that no evolution of the complexes during the experimental time had occurred. 3.2. 3D structure of BDK in the complexes To verify whether the structure of BDK in the complexes was ordered, only peptide ROE cross-peaks were considered in a first step. For example, in the complex 1:1, in addition to the classical sequential daN(i,i + 1) and dNN(i,i + 1) and intra-residue ROE connectivities (67 ROEs are counted), the ROESY spectra exhibited five interaction cross-peaks
d(i,i + 2) between Phe5 and Pro7, Ser6 and Phe8, Pro7 and Arg9, three d(i,i + 3) between Pro2 and Phe5, Ser6 and Arg9, and one d(i,i + 4) between Arg1 and Phe5. In the SA protocol, a total of 100 structures were calculated using the experimental distances determined from ROE spectra. Structural statistics for the converged structures were evaluated in terms of root mean square deviations (RMSDs) between the refined structures and their average structure. A 34-molecule family of low total energy was characterized by relative violations of the distance restraints ˚ upper limit is violated below 5% (i.e. for example, a 2.7 A ˚ below 0.14 A) and by torsion angle violations smaller than 5j. The Ramachandran plot shows that the backbone dihedral angles fall into peptide allowed regions (ProCheck-nmr control: http://pdb.rutgers.edu/validate/). According to the low RMSD value found for these 34 ˚ ) after backbone 2 – 8 residue structures (0.24 F 0.11 A superimposition, it is obvious that the peptide is ordered in section 2 –8. This result is confirmed by the average RMSDs between the 34 structures as a function of the residue number (Fig. 1s of Appendix A). The BDK presents a folded structure with two bends and with the lowest average RMSDs in the central region. On the contrary, the N- and C-terminal regions have higher RMSDs, which suggests a conformational averaging. Thus, for the BDK in the complex, no secondary structure such as helix or strand was detected because no characteristic NOE patterns were observed [28]. Nevertheless, the complex formation with the PGG involves a 3D ordered structure. A similar result with another ordered structure was obtained in the
Fig. 2. Thirteen superimposed structures of the complex between PGG and BDK. For clarity, only glucopyranose (Glc) rings are represented by balls for PGG and the peptide is represented by sticks. The complex is ordered and the peptide surrounds the glucopyranose molecule.
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complex 2:1. The comparison of the BDK mean structures between the duplex and the triplex shows that the peptide backbone globally keeps its structure in the central region, ˚ for the backbone Gly4 –Pro7 with an RMSD equal to 1.21 A domain (Fig. 2s of Appendix A). However, studies have shown that BDK dissolved in DMSO assumes a rigid structure with two bends in the N and C termini [32] or one turn in the C terminus [33]. To investigate the possible effect of DMSO on the BDK conformation in the solution 9:1 H2O/DMSO, we studied BDK alone in this solvent mixture. In addition to the classical sequential and intra-residue ROE connectivities, 3 d(i,i + 2) and 3 d(i,i + 3) cross-peaks were observed involving an ordered structure in the C-terminal region as determined by the SA protocol (data not shown). Yet this structure was different from that obtained for BDK in the presence of PGG. Thus, the ordered structures observed in the duplex and the triplex are really due to the complexation of the peptide hormone with the polyphenol.
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3.3. 3D structure of the complexes and modes of complexation of polyphenols with peptides and proteins 3.3.1. Duplex structure The temperature dependence of the amide proton chemical shift indicates whether the amide proton is protected from exchange with a solvent [34]. Dd/DT values for amide and guanidinium protons under the threshold showed the absence of intra-molecular hydrogen bonds, except for the NHq of Arg9 residue, which had a value of 4 ppb/K (Table 1s of Appendix A). However, this distance constraint was not included in the SA protocol because it was difficult to attribute without ambiguity. The PGG exhibits 13 intra-residue ROE connectivities. We observed 12 intermolecular ROEs between the protons of PGG and protons of BDK (Table 1 and Fig. 3s of Appendix A). In a first step in the SA protocol, we used only the six unambiguous intermolecular ROEs between the protons of galloyls 3 and 4 and those of BDK (Table 1).
Fig. 3. Representations of the closest PGG/BDK complex to the mean structure after SA. The galloyl (gall) rings are represented by large balls and small balls indicate the peptide residues involved in the interactions. The whole complex is presented in A; hydrophobic stacking between galloyl ring 3 and proline-3 in B; edge-to-face interaction between galloyl ring 2 and proline-7 in C; interactions between galloyl rings 4 and 6 and the BDK C-terminus region in D.
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Indeed, the protons of galloyls 2 and 6 overlapped and this first simulation explored the conformational spaces of galloyls 2 and 6. From these data, four supplementary cross-peaks concerning these galloyls could be unambiguously introduced in a new SA. At the end of this simulation, the results obtained allowed us to attribute the two last intermolecular ROEs. In the final SA, 100 structures were calculated and 13 low total energy structures were detected with an RMSD, between the refined structures and the mean ˚ for the backbone conformation, equal to 0.41 F 0.10 A 2 8 Pro –Phe domain and the heavy atoms of the glucopyranose molecule (Fig. 2). The Ramachandran plot shows that the backbone dihedral angles fall into peptide allowed regions (Fig. 4s, in supplementary materials) and the use of Swiss-PdbViewer http://www.expasy. ch/spdbv/) and ProCheck-nmr http://pdb.rutgers.edu/validate/) show that no protein problem was detected. Concerning the 156 intermolecular NOE constraints used (12 interNOE 13 structures) we have: 78 nonviolated, 66 violated i1%, 9 violated i2%, and 3 violated i3%. This result can be considered as an indirect proof that the hypothesis of a mono binding equilibrium is reasonable. Even though the BDK and PGG are flexible molecules, it would be surprising not to observe either some large distance violations or BDK structure violations of the Ramachandran plot. To present the complex formed between BDK and PGG, Fig. 3A shows the closest structure to the SA mean structure. Our results clearly show that the anchoring points of PGG are principally the galloyl groups 2, 3 and 6. The 2 and 3 galloyl rings have three and four intermolecular connectivities with the peptide, respectively. This leads to RMSD values for heavy atoms of galloyl rings equal to ˚ , respectively, implying a 2.36 F 0.33 and 1.49 F 0.48 A weak degree of freedom for these rings. The RMSD value ˚ for galloyl 6 is in agreement with the equal to 3.92 F 1.09 A conformational averaging of the peptide C terminus because the three intermolecular connectivities are with the Arg9 residue. Lastly, no intermolecular cross-peak was detected with galloyl 1. The latter can occupy a large conformational space, which is expressed by a high RMSD value for heavy ˚ . Concerning the atoms of this galloyl ring: 4.66 F 1.75 A anchoring points of BDK, the residues Pro and Arg9 are involved as shown in Fig. 3. The presence of the Gly4 residue, which has the less bulky side chain, breaks the rigidity of the backbone due to proline residues and gives a certain flexibility. This allows the PGG galloyl rings to come near to the central region of the peptide and to interact with the Pro residues on both sides of this position 4. In the literature, various hypotheses based on proton chemical shift changes were made: k k stacking or k j attraction for phenylalanine residues, hydrophobic stacking for the proline rings and/or hydrogen bonding for arginine guanidinium [1,35]. As shown in Fig. 3B, the galloyl ring 3 presents a hydrophobic stacking with the Pro3 ring while the Pro2 ring is nearly perpendicular to the
galloyl ring (Fig. 3B). The rings of galloyl 2 and Pro7 are nearly perpendicular (Fig. 3C). Indeed, the presence of Phe5 and Phe8 side chains prevents a parallel positioning of the galloyl 2 and Pro7 rings. The space of galloyl ring 4 is bounded by one or two hydrogen bonds obtained from the SA protocol. The NHq and one NHD of the Arg9 residue are perpendicular to galloyl 4 and thus might create a hydrogen bond with the k system of the galloyl ring (Fig. 3D). This SA result is in agreement with the experimental data because a value of 4 ppb/K was obtained for the Arg9 NHq (Table 1s). Moreover, it should be underlined that the k system of Phe8 is positioned to favour an edge-to-face k k interaction with the hydrogen of a galloyl 4 hydroxyl group [36,37]. As shown in Fig. 3, the position of the three galloyls 2, 3 and 4, in regard to the peptide backbone, means that galloyls 1 and 6 are situated on the other side, so it was surprising to observe ROESY connectivities with galloyl 6. However, a careful examination showed that the turn imposed by prolines 2 and 3 prevented an approach of the Arg1 residue to galloyl 1, while the spatial position of galloyl 6 is different. The presence of a supplementary CH2 in the chain implies a Table 2 Twenty seven triplex intermolecular ROEs observed between protons of PGG and protons of BDK BDK Residue 1
Arg Arg1 Gly4 Gly4 Phe5 Phe5 Phe5 Phe5 Phe5 Ser6 Pro7 Pro7 Pro7 Phe8 Arg9 Arg9
PGG1 Proton
Galloyl ring protons
Relative intensity
Hh Hy Ha Ha Ha Hh Hh Hq Hq Hh Ha Hy Hy Hq Hy Hg
4 4 2 3 2 3 4 4 H4 gluco-pyranose 2 2 2 1 6 6 6
W W WW WW WW WW WW W WW WW W WW W WW WW WW
(*) (*) (*)
(*)
(*) (*)
PGG2 3
Pro Ser6 Ser6 Phe8 Phe8 Arg9 Arg9 Arg9 Arg9 Arg9 Arg9
Hy Hh Hh Hh Hh Ha Ha Ha Ha Hy Hg
4 1 4 1 6 H1 gluco-pyranose H3 gluco-pyranose 3 6 4 4
WW WW WW W W W WW WW WW W W
All relative intensities are weak or very weak corresponding to an upper ˚ , respectively. constraint limit of 5.0 or 6.0 A (*) The first six intermolecular connectivities attributed to the PGG1 (see text).
S. Verge´ et al. / Biochimica et Biophysica Acta 1571 (2002) 89–101 Table 3 Triplex structural data for the 10 refined structures in agreement with NMR restraints Distances and torsion angle restraints Intra-residue Inter-residue V
45 44 8
PGG* 22 Inter-molecule 27
Number of relative NOE violations
Not-violated 0 – 1% 2 – 4% 5 – 7%
All 1600 restraints (160 restraints 10 molecules)
270 inter-molecular restraints (27 restraints 10 molecules)
1270 207 120 3
106 90 71 3
79.4% 12.9% 7.5% 0.2%
39.3% 33.3% 26.3% 1.1%
Atomic RMS values for converged structures vs. their geometric average All heavy atoms Backbone + GLC Backbone Backbone (2 – 8)
1.36 F 0.21 0.45 F 0.10 0.42 F 0.10 0.35 F 0.10
(*) The cross-peaks were attributed to both molecules since both PGGs being identical (fast equilibrium).
larger freedom of positioning for this galloyl ring, and the presence of guanidinium of Arg9 residue in a spatial proximity leads to a hydrogen bond between these groups. 3.3.2. Triplex structure In the molar ratio 2 PGG:1 BDK solution, we observed 27 intermolecular ROEs between the protons of PGGs and
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those of BDK (Table 2). From the NMR spectra, it is clear that the complex is in fast exchange with the separate components, over the NMR time scale. Particularly, both PGG are in fast equilibrium. Therefore, it is important to explain the way both PGGs were attributed in SA because the following study is dependent on it. We considered that the first PGG molecule present in complex 1:1 occupied globally the same spatial environment as in complex 2 PGG:1 BDK. This hypothesis is in agreement with the results since six ROESY cross-peaks were already observed in the complex 1:1 spectra and were present in the complex 2 PGG:1 BDK spectra. Thus, in a first SA protocol, we attributed these cross-peaks only to the first PGG molecule (PGG1). After analysis, the model demonstrated the limits of the conformational space for the second PGG molecule (PGG2), i.e. by measuring the inter-molecular proton distances and by using the spatial geometries of the peptide and PGG1, six ROESY cross-peaks could be attributed to PGG2. At the end of the second SA protocol and after analysis, we had more accurate conformational spaces for both PGGs. The protocol was repeated about 30 times with the gradual insertion of all cross-peaks. All large violations of distance and/or all Ramachandran plot violations revealed an inaccurate attribution so we did it again. At the end of these simulations, the results obtained allowed us to attribute 16 and 11 intermolecular ROEs to the PGG1 and PGG2, respectively (Table 2). Moreover, no intramolecular hydrogen bond distance constraint was included in the SA protocol. In the final SA, 100 structures were calculated and 10 low total energy structures were detected
Fig. 4. Ten superimposed molecules for 2 PGG:1 BDK triplex. The peptide is clearly structured. The two molecules of PGG form a cage around it.
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with an RMSD, between the refined structures and the mean ˚ for the conformation, which was equal to 0.45 F 0.10 A backbone domain and the heavy atoms of the glucopyranose core. Table 3 summarizes the structural data for this set of refined molecules and Fig. 4 shows the triplex structures. To present the triplex formed between BDK and PGGs, Fig. 5 shows the closest structure to the SA mean structure. What was interesting when we analyzed the 3D-structure was the presence of common anchoring points between PGGs and BDK. Indeed, Pro7 interacts with galloyl 2 of PGG1 and galloyl 1 of PGG2 and Arg9 interacts with galloyl 6 of PGG1 and galloyl 4 of PGG2. It should be noted that for PGG1, the same anchoring points had been evidenced in complex 1:1. As shown in Fig. 5A, the galloyl ring 2 of PGG1 presents a hydrophobic stacking with the Pro7 with rings nearly parallel, while the same Pro7 ring is nearly perpendicular to the galloyl ring 1 of PGG2. Thus, Pro7 leads to such a spatial proximity of both PGGs that they could interact together. In fact, a hydroxyl group of galloyl 2 in PGG1 and that of galloyl 1 in PGG2 are orientated towards each other, allowing a possible H bond between them. Thus, both galloyl rings attached by this H bond might bound a hydrophobic pocket which might surround the Pro7 residue.
Concerning Arg9 and PGG1, a first H bond is possible between a hydroxyl oxygen of galloyl 6 and a hydrogen of the guanidinium group. Moreover, a second possible H bond between HD of Phe8 and a hydroxyl oxygen of galloyl 6 would allow a better stabilization of this structure (Fig. 5B). With PGG2, a hydrogen bond could be created between a hydroxyl proton of galloyl 4 and an oxygen atom of the Arg9 carboxyl terminal (Fig. 5B). Therefore, it appears that Arg9 and Pro7 are the important residues for the structure. They are the meeting points of PGGs, and in the case of Pro7 are able to cooperate to form a hydrophobic pocket. However, the stabilization of the triplex is also due to a set of other anchoring points. For PGG1, Phe5 ring is sandwiched between both rings of galloyls 3 and 4 by a double k k stacking (Fig. 5C). Moreover, the NHq of the Arg1 residue might create an H bond with a hydroxyl oxygen of galloyl 4, which would be in agreement with the NMR temperature coefficients (Fig. 5D). Thus, it seems that compared to the duplex, PGG1 is shifted from the C to the N termini probably because of the presence of the second PGG, which is positioned on the other side of the peptide and could reduce the freedom of positioning. Nevertheless, the anchoring of PGG1 from the N to the C termini is made possible, thanks to the backbone flexibility (Gly4 and Ser6 presence in the sequence), to the long and flexible
Fig. 5. Closest structure to the SA mean structure. Galloyls are represented by balls and BDK bakbone is represented by large sticks. (A – E) Galloyl rings are represented by large balls and small balls indicate the peptide residues involved in the interactions. (A) Hydrophobic stacking between Pro7 and galloyl 2 of PGG1 and perpendicular positionning of Pro7 and galloyl 1 of PGG2. The Pro7 is an anchoring point between PGG1 and PGG2. (B) Arg9 as another anchoring point between PGG1 and PGG2. (C) Double stacking between galloyl 3 and 4 of PGG1 and Phe5. (D) Possible H bond between the NHq of Arg1 and galloyl 4 of PGG1. (E) Perpendicular positionning of Pro3 and galloyl 4 of PGG2.
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Fig. 5 (continued ).
side chain of arginine residues and also to the articulated arms of the PGG molecule. Lastly, the distance between the nitrogen atom of Ser6 and a hydroxyl oxygen of galloyl 2 is in agreement with the possibility of an H bond. The latter could be created with the amide proton and the oxygen of galloyl 2. However, the hypothesis is not in agreement with the NMR temperature coefficients. For PGG2, the k system of galloyl 6 is positioned in such a way that it favors an edge-to-face k k interaction with a hydrogen atom of the Phe8 ring [36,37]. Moreover, the positioning of the Pro3 ring nearly perpendicular to the galloyl ring 4 could allow hydrophobic interaction (Fig. 5E). Thus, the PGG2 is anchored from the central domain to the C terminus on the other side of the peptide vs. PGG1. The impossibility for this PGG to anchor the N terminus is due to the bend imposed by the Pro2 – Pro3 sequence, which positions the Arg1 side chain on the side of the PGG1. Lastly, we observed ROESY cross-peaks between peptide protons and gluropyranose ring protons (Table 2). The gluropyranose ring and the BDK moved closer together, and this was more pronounced for PGG2 than for PGG1 because the ROESY connectivities involved the backbone protons for the former and the side chain for the latter.
4. Conclusion In summary, the results of this investigation suggest that the PGG/BDK complexes are formed by multiple weak interactions between peptide side chains and galloyl rings. It appears that the residue sequence, and in particular the side chain steric bulk, acts on the interaction mechanism. The goal of this work was to obtain a complex model for tannin– protein interactions. And even though this interaction might be important at the biological activity level [35], it is extremely difficult to extrapolate this in vitro model to human physiology, if only for in vivo concentration values.
Acknowledgements We thank Dr. Richelme-David S. for her technical assistance and helpful discussions (Service de Spectrome´trie de masse, FR 1744, Universite´ Paul Sabatier, Toulouse, France). This work was supported in part by the Conseil Re´gional d’Aquitaine (grant 990305002).
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Appendix A Table 1s. Chemical shifts of the assigned 1H-NMR resonances in 1H2O/DMSO (9:1), pH 6.5, in molar ratio solutions 1 PGG:1 BDK, 2 PGG:1 BDK (in italic) and in absence of PGG (in brackets). NH
Ha
Hh
Others
1
Arg
–
Pro2
–
Pro3
–
1.97 1.98 (1.87) 2.40/1.91 2.38/1.90 (2.43/1.87) 2.34/1.97 2.34/1.96 (2.30/1.90)
Hg 1.77; 1.79 (1.69) Hg 2.02; 2.00 (2.02) Hg 2.10; 2.09 (2.06)
Gly4
8.37 8.37 (8.34) 8.06 8.06 (8.00) 8.16 8.16 (8.11) –
4.38 4.39 (4.28) 4.77 4.73 (4.79) 4.49 4.51 (4.43) 3.98 4.01 (3.91) 4.65 4.70 (4.60) 4.73 4.75 (4.70) 4.40 4.42 (4.33) 4.69 4.73 (4.63) 4.26 4.28 (4.15)
Phe5
Ser6
Pro7
Phe8
Arg9
7.96 7.94 (7.94) 7.76 7.77 (7.66)
Dd/DT, ppb/K Hy 3.22 3.22 (3.15) Hy 3.75/3.51 3.74/3.50 (3.75/3.50) Hy 3.84/3.68 3.83/3.67 (3.83/3.68)
–
–
–
10 10 3.08 3.09 (3.05) 3.83 3.86 (3.75) 2.16/1.67 2.16/1.72 (2.15/1.67) 3.27/3.00 3.28/2.98 (3.22/2.95) 1.91/1.78 1.92/1.80 (1.81/1.69)
Hy 7.22; 7.20 (7.23)
Hq 7.31; 7.31 (7.35)
HD 7.28 7.29 (7.30)
8 8 9 9
Hg 1.87; 1.85/1.67 (1.88/1.67) Hy 7.27; 7.27 (7.27) Hg 1.62; 1.64 (1.54) NHq 7.26; 7.27 (7.19)
Hy 3.60 3.60 (3.58) Hq 7.35; 7.35 (7.38) Hy 3.23 3.23 (3.18); NHD 6.75 6.74 (6.66)
–
HD 7.30 7.30 (7.33)
8 8 6 7 NHq 4; 3
NHD 10 11
Table 2s. Chemical shifts of the assigned 1H-NMR resonances for PGG in 1H2O/DMSO (9:1), pH 6.5, in the molar ratio solutions 1 PGG:1 BDK, 2 PGG:1 BDK (in italic). Glucopyranose ring protons
Galloyl ring protons
Position
d (ppm)
Position
d (ppm)
H-1
6.30 6.33
1
7.20 7.23
H-2
5.70 5.73
2
7.12 7.15
H-3
5.96 5.98
3
7.05 7.14
H-4
4.68 4.69
4
7.07 7.07
H-5
4.50 6
7.12 7.08
4.51 H-6
4.60 4.61
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Fig. 1s. Thirty-four superimposed structures for the BDK in the duplex and RMS deviations per residue obtained using only ROE nonapeptide constraints. The peptide is clearly structured between residues 2 and 8.
Fig. 2s. Comparison of the BDK mean structure in the duplex and in the triplex. The structure is globally kept in the central region Gly4 – Pro7, but a re-orientation is observed in the C and N termini.
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Fig. 3s. Galloyl (gall) region of the duplex PGG/BDK 20 mM solution 400 ms ROESY spectrum in the solvent mixture H2O/DMSO (9:1). This region contains cross-peaks between galloyl ring resonances and peptide protons. These connectivities are also present in the 200-ms ROESY spectrum with a less favorable signal-to-noise ratio.
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Fig. 4s. Ramachandran plot of the 13 structures of the BDK in the duplex. Black circles stand for the torsion angle (U, W) values, except for Gly residues symbolized by black squares. The large circles stand for the areas of ideal turns ( F 20j).
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