2 Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain; 3 Centre de Recherches sur les Macromolécules Végétales (CERMAV-CNRS), Affiliated with Université Joseph Fourier, BP 53, 38041 Grenoble cedex 9, France; and 4 Centro de Apoyo a la Investigación, RMN, Facultad de Quimicas, Universidad Complutense de Madrid, 28040 Madrid, Spain
Received on October 16, 2003; revised on November 14, 2003; accepted on November 18, 2003
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Abstract |
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Key words: chitooligosaccharides / diffusion ordered spectroscopy / hevein / molecular weight standard / proteincarbohydrate interactions
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Introduction |
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Diffusion ordered spectroscopy (DOSY) (Stilbs, 1981) provides a method of molecular size determination through the measurement of diffusion coefficients (logD). This NMR method has found a number of applications. In the study of drug-receptor interactions (Cameron and Fielding, 2002
), the logD values of drugs were measured in the presence and absence of a small amount of receptor. The measured logD values were a weighted average of receptor-bound and free ligand. Therefore, ligands whose NMR signals increased in apparent molecular weight (MW) signified a receptor-bound population, whereas ligands that did not bind to the receptor experienced no change in logD. DOSY is also useful in resolving chemical mixtures and can provide the 1D 1H NMR spectra and the logD of individual components (Diaz and Berger, 2000
; Pelta et al., 2002
).
The measurement of accurate diffusion coefficients is prone to several potential errors. The gradients must be correctly calibrated, and temperature convection inside the NMR tube and sample-induced changes in viscosity must be avoided (Sorland and Aksnes, 2002). Herein, we report a simple strategy to overcome these drawbacks using standard equipment based on similar protocols to those developed for the pharmaceutical industry (Kerssebaum, 2002
). We also produce novel representations of DOSY data that are more readily interpreted.
Hevein is a small chitin-binding protein. The resolution of the 3D structure of hevein and its interactions with N-acetyl-chitooligosaccharides has been the focus of our attention and of others (Asensio et al., 1995, 1998
, 2000a
,b
; Siebert et al., 2000
) because it provides a model for the study of carbohydrateprotein interactions. The NMR structures of a variety of hevein complexes suggest that these protein domains efficiently bind N-acetyl-chitooligosaccharides, with (GlcNAc)3 moieties providing the basic interactions for the molecular recognition processes (Asensio et al., 2000a
,b
). Moreover, the recognition process is multivalent, because analytical ultracentrifugation measurements have indicated that two hevein molecules can bind to (GlcNAc)8, particularly when the hevein concentration exceeds the (GlcNAc)8 concentration (Asensio et al., 2000a
). In addition, isothermal microcalorimetry data for hevein binding to (GlcNAc)5 could not be fitted to either a 1:1 or 2:1 model (Asensio et al., 2000a
), suggesting a mixture of 1:1 and 2:1 complex species.
We have chosen the system provided by (GlcNAc)n and hevein to illustrate the application of DOSY in characterizing the size of different N-acetyl-chitooligosaccharides and their complexes to protein receptors. Thus the stoichiometry of the complexes formed by hevein with (GlcNAc)6 have been evaluated by DOSY as a complementary method to other currently used physical biochemical methods, such as analytical ultracentrifugation and size exclusion chromatography (SEC).
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Results |
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Figure 3B provides an alternative representation of the data presented in Figure 3A. Narrow strips from the 2D DOSY spectrum (as indicated by the bold boxes in Figures 2A and 2B) are taken and displayed side by side. The resulting figure can be interpreted the same way as the bands/spots obtained in a polyacrylamide gel. Smaller oligosaccharides are found at the bottom, and larger saccharides toward the top of the strips. The DOSY results of 1 and 2 are also presented in Figure 3B, lanes 7 and 8, respectively. It can be quickly and readily deduced that these molecules contain the correct number of sugar units by comparison to the other strips in the panel.
Passing to saccharide binding to receptors and choosing hevein as a model, Figure 4A shows fluorescence spectroscopy data for hevein binding to (GlcNAc)6 that can be fitted to an equation assuming 1:1 stoichiometry when determined at low, micromolar protein concentration (K = 6.0 ± 1.0 x 105 M1). However, it has been demonstrated that multivalent effects may take place for (GlcNAc)5 and (GlcNAc)8 at millimolar concentrations (Asensio et al., 2000a). Therefore we also decided to employ DOSY as an alternative to determine the stoichiometry of the hevein:(GlcNAc)6 complex. Six hevein resonances, which do not overlap with ligand resonances, were followed by DOSY during a titration with (GlcNAc)6. Changes in logD are plotted against the protein:ligand stoichiometry. In this case, a calibration curve formed by five proteins of MW between 6.5 and 66 kDa (not shown, logD = 0.427 logMW 8.231, r2 = 0.983) was used. A hevein concentration of 0.3 mM, together with the measured binding constant from the fluorescence experiments, means that stoichiometric binding of (GlcNAc)6 should be observed as indicated by the theoretical plot given in Figure 4B. The expected values of the 1:1 and 2:1 protein:ligand complexes are also indicated to the right of Figure 4B. Titration of up to 0.5 equivalents of ligand results in a sharp increase in the logD values in excess of that expected for 1:1 stoichiometry, approaching that for 2:1 stoichiometry. The titration between 0.5 and 1.0 equivalents of ligand results in a decrease in logD values back toward that expected for a 1:1 complex. The logD value is constant between 1.0 and 3.0 equivalents of added ligand and slightly elevated compared to that expected for a 1:1 complex. Therefore the DOSY experiment for this system easily allows the detection of a significant proportion of 2:1 protein:ligand complex for protein:ligand ratios higher than 1:1.
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Discussion |
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The Stokes-Einstein equation that describes diffusion phenomena and from which the analysis of DOSY data is derived, holds for spherical molecules. Therefore it is interesting that Figure 3A yields a linear correlation, even for relatively long N-acetyl-chitooligosaccharides, which are expected to adopt nonspherical structures (González et al., 1999). Such a linear correlation of logD versus logMW has also been observed with ß-amyloid-derived polypeptides (Danielsson et al., 2002
), proteins (Nesmelova et al., 2002
; Wilkins et al., 1999
), as well as cyclodextrins and their inclusion complexes (Cameron and Fielding, 2002
).
Figure 3A can be compared with SEC calibration plots of logVe/V0 against logMW generated from the relative mobility of MW standards. These plots circumvent systematic errors that otherwise affect size determination by SEC (accurate determination of pump rates, total and excluded column volumes). In the same way, Figure 3A may surmount some of the systematic errors associated with DOSY (gradient calibration, temperature convection) (Sorland and Aksnes, 2002), even if some of these errors, such as gradient calibration (Damberg et al., 2001
), can be significantly corrected. Access to a series of compounds allows a calibration curve be easily constructed using the same solvent or buffer as for the sample. However, as it has been found that the relationship between logD and logMW for a series of proteins depends on whether the proteins are folded or denatured (Wilkins et al., 1999
), it is clear that the choice of reference compounds is important. Nonetheless, the reference curve allows a more reliable MW to be obtained for the unknown. In principle, the calculated MW should give a consistent value when determined on any NMR instrument that provides a good calibration plot with suitable reference compounds.
The advantages of DOSY over SEC in the carbohydrate field include an ease of detection (all carbohydrates contain protons that can be detected by NMR), compatibility with a wide range of solvents, and the fact that experiments are essentially performed under equilibrium conditions. Disadvantages include the existence of overlapping NMR resonancesparticularly a concern for saccharide mixtures, that might have similar proton signals. Nevertheless, analytical algorithms are being improved (Armstrong et al., 2003; Mouro et al., 2002
) and extension to 3D NMR DOSY experiments may also alleviate the overlapping problem.
The representation of DOSY data need not be as shown in Figure 2, although this is the best representation to confirm the absence of overlapping signals from solvents or chemicals used in synthesis. With Figure 3A, the quality of the NMR data cannot be evaluated. For this reason, Figure 3B might be favored, because it provides part of the NMR data that can be easily evaluated by readers. Calculated MWs can be quoted as based on the linear fit provided by Figure 3A.
Multiple hevein binding to oligosaccharides is not directly detected by fluorescence. This might be due to the relatively low affinity of the ligand for the protein at the 1 µM protein concentration used in the titrations and the fact that only 2 of the 18 titration points are collected below a 2:1 protein:(GlcNAc)6 stoichiometry. The fact that the a full 2:1 stoichiometry is not observed in Figure 4B and that the peak at 2:1 stoichiometry is symmetrical in the DOSY experiment suggests that the hexasaccharide provides two binding sites, in a case of multivalency, but with either a lack of cooperativity or even with steric hindrance between the two sites. It is also possible that hevein has a preference to bind to the middle portion of the hexasaccharide, as also supported by earlier studies (Asensio et al., 2000a), which would hinder the binding of further protein molecules to the available mono- or disaccharide ends of the hexasaccharide.
To summarize, DOSY is a powerful NMR experiment that can help characterize an oligosaccharide by confirming the absence of small chemicals used in synthesis and, when measured against appropriated mass standards, its correct molecular size (to a practical limit of octasaccharide, Figure 3B). The calculated masses of chitooligosaccharides 1 and 2 are within 10% of their calculated values, which were confirmed by mass spectrometry. Because we applied a readily available protocol (Kerssebaum, 2002) without implementing improvements (Armstrong et al., 2003
; Damberg et al., 2001
; Mouro et al., 2002
), it should be possible to improve the accuracy of DOSY measurements. DOSY might be employed as a complementary tool to SEC in studying intermolecular interactions, as demonstrated here for the hevein:(GlcNAc)6 complex.
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Materials and methods |
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Chitooligosaccharide expression and purification
Chitooligosaccharides 1 and 2 were obtained from metabolically engineered E. coli, as described (Samain et al., 1997, 1999
). Both compounds were further purified by silica gel chromatography. Compound 1 was purified on Merck Silica gel 4063 µm with a acetonitrile/water/ammonia gradient (70:30:1 to 60:40:1) with 89% recovery of the material. The mass spectrum (matrix-assisted laser desorption/ionization time-of-flight [MALDI-TOF]) of 1 gave m/z 1014 (M + Na+). Compound 2 (CO-IV(S, NH2) was purified on Merck Silica gel 63200 µm with a chloroform/methanol/water gradient (40:30:1 to 35:40:5) with 33% recovery of the material. The mass spectrum (MALDI-TOF) of 2 gave m/z 913 (M + Na+). MALDI-TOF measurements were performed on a Bruker Daltonics Autoflex apparatus.
NMR spectroscopy
All samples were prepared (1 mM concentration) in 20 mM phosphate buffer, 100 mM NaCl, pH 5.6, 100% D2O. The standard Bruker DOSY protocol was used at 298 K on an Advance 500 MHz (Kerssebaum, 2002
) equipped with a broad-band z-gradient probe. Thirty-two 1D 1H spectra were collected with a gradient duration of
= 2 ms and an echo delay of
= 100 ms for oligosaccharides, or 250 ms for the hevein complexes. Acquisition times of 8 and 32 min were required for the oligosaccharide samples and hevein complexes, respectively. Samples of aprotonin (6.5 kDa),
-lactalbumin (14 kDa), carbonic anhydrase (29 kDa), ovalbumin (44 kDa), and bovine serum albumin were used to calibrate the hevein/(GlcNAc)6 titration (logD = 0.427 logMW 8.231, r2 = 0.983) in a similar way as described for Figure 3A. The ledbpg2 s pulse sequence, with stimulated echo, longitudinal eddy current compensation, bipolar gradient pulses, and two spoil gradients, was run with a linear gradient (53.5 G cm1) stepped between 2% and 95%. The 1D 1H spectra were processed and automatically baseline corrected. The diffusion dimension, zero-filled to 1 k, was exponentially fitted according to preset windows for the diffusion dimension (8.5 < logD < 10.0 [oligosaccharides] or 10.5 [hevein complexes]).
Fluorescence spectroscopy
Fluorescence spectroscopy was performed on a Hitachi F-2500 spectrometer. Experiments were performed in a 20 mM phosphate, 100 mM NaCl, pH 5.6 buffer at 25.0°C maintained by a Julaba F-12 temperature control unit. Data were collected with 1 scan at 1 nm/s with excitation and emission slits of 5 nm. The maximum change in hevein emission fluorescence (335 nm) was followed as a function of added (GlcNAc)6 after excitation at 280 nm. The binding curve was least squares fitted to an equation assuming 1:1 ligand:host interactions using SigmaPlot (SPSS, Chicago, Il).
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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