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Langmuir 2002, 18, 3396-3398
Direct Observation of Polylysine Side-Chain Interaction with Smectites Interlayer Surfaces through 1H-27Al Heteronuclear Correlation NMR Spectroscopy Re´gis D. Gougeon,*,†,‡ Marc Reinholdt,† Luc Delmotte,† Jocelyne Miehe´-Brendle´,† Jean-Michel Che´zeau,† Ronan Le Dred,† Richard Marchal,‡ and Philippe Jeandet‡ Laboratoire d’œnologie URVVC, Universite´ de Reims, UPRES EA 2069, Faculte´ des Sciences, Moulin de la Housse, BP 1039, 51687 Reims Cedex 2, France, and Laboratoire de Mate´ riaux Mine´ raux, Universite´ de Haute Alsace, UPRES-A-CNRS 7016, 3, Rue A. Werner, 68093 Mulhouse Cedex, France Received October 10, 2001. In Final Form: January 21, 2002
Interaction of polypeptides with clay minerals is at the basis of both natural biochemical processes1 and technological applications.2,3 Detailed descriptions of the organicinorganic interfacial structures are necessary to obtain valuable new clues for both the understanding of biochemical processes in ecosystems and the controlled design of either advanced hybrid materials4 or adsorbents with tailored specific properties. Solid-state NMR has emerged as a powerful technique for determining both the secondary structure and dynamics of surface-adsorbed proteins,5 two features that are considered of prime importance in the activity of biological systems.6,7 However, these studies have focused on the protein characteristics resulting from the adsorption, but little is known on the organic-inorganic interfacial structure. Clay minerals for instance possess a small external surface and a high internal specific surface area, onto which organic compounds can adsorb. This high internal area results from the regular arrangement of aluminosilicate layers, negatively charged due to intralayer substitutions. At pH below the isoelectric point (iep) of proteins, the hydrated cations such as sodium, which are originally located within the interlayer space of the clay mineral to balance the layer negative charge, are exchanged for the positively charged proteins. Their adsorption is basically seen as an electrostatic, hydrophobic, and/or hydrophilic interaction.8 However, a detailed and unambiguous description of which part of a polypeptide interacts with the clay mineral surface, and, ideally, at which distance, remains to be proposed, which cannot be obtained from * To whom correspondence may be addressed. Present address: Universite´ de Haute Alsace. † Laboratoire de Mate ´ riaux Mine´raux, Universite´ de Haute Alsace. ‡ Laboratoire d’œnologie URVVC, Universite ´ de Reims. (1) Quiquampoix, H. The Encyclopedia of Soil Science and Technology; Chapman & Hall: New York, 1994. (2) Causserand, C.; Jover, K.; Aimar, P.; Meireles, M. J. Membr. Sci. 1997, 137, 31-44. (3) Blade, H. W.; Boulton, R. Am. J. Enol. Vitic. 1988, 39, 193-199. (4) Giannelis, E. P. Adv. Mater. 1996, 8, 29-35. (5) (a) Fernandez, V. L.; Reimer, J. A., Denn, M. M. J. Am. Chem. Soc. 1992, 114, 9634-9642. (b) Shaw, W. J.; Long, J. R.; Dindot, J. L.; Campbell, A. A.; Stayton, P. S.; Drobny, G. P. J. Am. Chem. Soc. 2000, 122, 1709-1716. (6) Baron, M. H.; Revault, M.; Servagent-Noinville, S.; Abadie, J.; Quiquampoix, H. J. Colloid Interface Sci. 1999, 214, 319-332. (7) DeOliviera, D. B.; Laursen, R. A. J. Am. Chem. Soc. 1997, 119, 10627-10631. (8) Quiquampoix, H.; Ratcliffe, R. G. J. Colloid Interface Sci. 1992, 148, 343-352.
the sole description of the secondary structure and dynamics of the adsorbed protein. The identification of entities such as adsorbed moieties of proteins onto lamellar surfaces can be achieved through 1H-27Al heteronuclear correlation (HETCOR) experiments.9 The latter involve the transfer of polarization from the protons of the different interfacial species to the adjacent aluminum atoms, through 1H-27Al dipole-dipole couplings (see Supporting Information). Since these dipolar couplings have a strong 1/r3 dependence, r being the internuclear distance, only correlations between proximate atoms are observed. On that basis, correlation with octahedral aluminum atoms which are in the center of the clay mineral layers will only be observable with protons in the vicinity of the layers. For this study, poly-D-lysine (PL) was adsorbed in acidic medium10 onto a synthetic Na-containing montmorillonite (M) and a synthetic Na+/NH4+-beidellite (B).11 Montmorillonite and beidellite are 2:1 dioctahedral phyllosilicates which differ by the origin of the layer charges. Montmorillonite is an octahedrally substituted smectite, meaning that the layer charge is distributed over the complete oxygen framework, whereas beidellite is a tetrahedrally substituted smectite, which is characterized by a more localized charge distribution over the surface oxygen atoms of the layer. The observation of heteronuclear correlations in such two-dimensional experiments requires that both the proton and the aluminum species give rise to resolved signals in 1H magic angle spinning (MAS) and 1Hf27Al cross-polarization magic angle spinning (CP-MAS) onedimensional (1D) spectra, respectively. While the 1Hf27Al CP-MAS spectrum of a clay mineral shows resolved signals for both the octahedral and/or the tetrahedral aluminum signals, this is not the case for the different CH and CH2 groups of a polypeptide 1H MAS spectrum, owing to the strong proton-proton dipolar coupling. Furthermore, in the case of polypeptide adsorbed on clay minerals, the situation is complicated by the fact that the different OH groups of the latter give rise to 1H NMR signals in the same region as those from the polypeptide aliphatic carbons, i.e., between 0 and 4 ppm.12 This is illustrated in Figure 1 which exhibits the 1H MAS spectra of the montmorillonite and the beidellite before and after polylysine adsorption, at different spinning speeds. In such a situation where high spinning speeds only bring weakly enhanced resolution, multiple-pulse experiments may provide high-resolved 1H spectra. (9) Ernst, R. R.; Bodenhausen, G.; Wokaun, A. Principles of Nuclear Magnetic Resonance in One and Two Dimensions; Oxford University Press: New York, 1987. (10) Adsorption was realized by mixing under stirring at 10 °C, for 6 h, a solution of 30 mg of poly-D-lysine (PL) dissolved in 15 mL of an acidic aqueous solution buffered to pH 3.35 with a solution of 500 mg of montmorillonite (M) or beidellite (B) initially suspended for 5 days in 25 mL of the same acidic solution. After filtration through 0.45 µm filters and repeated washes with the buffer and deionized water, the M/PL and B/PL samples were dried overnight at room temperature in air and stored at 2-6 °C. 13C CP-MAS spectra confirmed the adsorption of polypeptides. PL (Mw ) 20900) was purchased from Sigma Aldrich Chimie and used as received. At pH 3.35, PL displays (CH2)4NH3+ amino side chains (iep ∼ 10). (11) (a) Huve, L.; Le Dred, R.; J.; Saehr, Baron, D. In Synthesis of Microporous Materials; Occelli, M. L., Robson, H. E., Eds.; Van Nostrand Reinhold: New York, 1992; Vol. II, p 207. (b) Reinholdt, M.; Miehe´Brendle´, J.; Delmotte, L.; Tuilier, M. H.; Le Dred, R.; Corte`s, R.; Flank, A. M. Eur. J. Inorg. Chem. 2001, 2831-2841. (12) Alba, M. D.; Becerro, A. I.; Castro, M. A.; Perdigon, A. C. Chem. Commun. 2000, 37-38.
10.1021/la0115381 CCC: $22.00 © 2002 American Chemical Society Published on Web 03/19/2002
Notes
Figure 1. 1H MAS spectra recorded at a frequency of 400.1 MHz on a Bruker DSX 400 solid-state NMR spectrometer, with a Bruker 4-mm probe. The dotted line materializes the 7.3 ppm chemical shift, and the arrow indicates the signal already seen at a spinning speed of 8 kHz. Experimental conditions were as follows: 90° pulse of 5 µs, 16 transients separated by a 5-s recycle delay.
Nevertheless, Figure 1 shows that even with a spinning speed of 8 kHz, signals that do not belong to the inorganic framework can be resolved. This is illustrated by the intense signal of water at ca. 4.4 ppm and also by the narrow peak at 6.8 ppm in the spectrum of B, which corresponds to adsorbed NH4+ species. Since M only contains Na+ cations, this signal is not observed. However, upon adsorption of polylysine, a distinct signal at 7.3 ppm is already resolved in the spectrum of M/PL. This peak, which is more clearly seen at the higher spinning speed of 14 kHz, but absent from the spectrum of M, can unambiguously be assigned to the protonated amino group (NH3+) of PL side chains. Similarly, the adsorption of polylysine on B results in a significant reduction of the narrow NH4+ signal at 6.8 ppm, and the appearance of a broader one centered around 7.3 ppm, which therefore can be attributed to NH3+ end groups of PL side chains (Figure 1). It has been shown that unlike the aliphatic groups which experience severely restricted motions, the NH3+ groups of the polylysine side chains adsorbed on silica are still mobile at a temperature as low as 200 K.5 Due to thermal activation, the mobility is necessarily higher at ambient temperature. Therefore, the resolution of the NH3+ peak at 7.3 ppm is explained by the rotational motion around the C3 axis of the NH3+ group, which is shown to be efficient even for adsorbed polylysine.5 The narrower line width of the NH4+ peak is consistent with the high symmetry and a faster isotropic motion of the latter within the interlayer space of B. Internuclear distances that can be probed in a HETCOR experiment strongly depend on the system of spins involved, and in some cases, increasing the contact time does not necessarily allow the screening of greater internuclear distances or weaker dipolar couplings. This is particularly true (i) if the spin-lattice relaxation time (T1F) of the different nuclei involved is of the order of the contact time, which is the case for the quadrupolar 27Al
Langmuir, Vol. 18, No. 8, 2002 3397
Figure 2. 2D 1H spin-diffusion 1H-27Al HETCOR spectrum of the polylysine-adsorbed montmorillonite (M/PL), recorded at frequencies of 400.13 and 100.27 MHz for 1H and 27Al, respectively. Separate 27Al CP-MAS and 1H MAS spectra are plotted along the horizontal and vertical axes, respectively, to account for all the possible correlations. The arrow indicates the correlated signal between PL NH3+ protons and octahedral aluminum. The dotted line corresponds to the indirectly detected 1H spectrum correlated to the octahedral aluminum species, shown in Figure 3 for M/PL with tmix ) 10 ms. Experimental conditions were as follows: The sample was spun at 8 kHz; the proton 90° pulse of 11 µs and the 27Al radio frequency field were optimized on kaolinite for the selective excitation of the central (1/2 transition; the mixing time tmix and the contact time were 10 and 1 ms, respectively; 88 spectra of 192 transients with a 4-s recycle delay were acquired in the t1 dimension.
nuclei (∼500 µs13), and (ii) if the nuclei involved are significantly mobile, as it is the case for protons of either NH4+ or PL NH3+ groups. Since these two points characterize our M/PL and B/PL systems, the 1H-27Al dipolar coupling to be measured is weak, and a modification of the HETCOR experiment,14 which includes 1H spin diffusion during a given mixing time, tmix, prior to the 1Hf27Al polarization transfer, has to be implemented (see Supporting Information). The investigation of the weak 1H-27Al dipolar coupling is made possible by the presence of strong 1H-1H dipolar interactions between different proton species in the sample. This efficient homonuclear dipolar coupling is used to mix magnetization among nearby protons (i.e., protons of the polypeptide, water, and the mineral) during the given period tmix, before it is transferred to 27Al nuclei via the standard shortcontact-time CP-MAS experiment, between proton and aluminum atoms of the mineral. Allowing 1H spin diffusion between the framework hydroxyls and the protons of the interfacial species (Figure 2) clearly reveals three distinct correlations between the octahedral 27Al peak and 1H signals: a broad and dominating one centered at 2.2 ppm, a narrower one at 4.4 ppm, and a well-resolved, though weak, one at 7.3 ppm. The clear observation of the latter involving the protonated amino function of the PL side chains unambiguously establishes their presence within the interlayer space, close to the interface. Figure 3 compares the 1H slices at δ(27Al) ) -1 ppm, drawn from the 2D spin-diffusion 1H-27Al HETCOR spectra of the different samples. For instance, the M/PL spectrum of Figure 3 (with a mixing time of 10 ms) corresponds to the dotted line in Figure 2. These indirectly detected 1H MAS spectra represent the different proton species whose magnetization has been exchanged through spin-diffusion during a mixing time of 10, 20, or 40 ms, (13) Morris, H. D.; Bank, S.; Ellis, P. D. J. Phys. Chem. 1990, 94, 3121-3129. (14) Schmidt-Rohr, K.; Clauss, J.; Spiess, H. W. Macromolecules 1992, 25, 3273-3277.
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Figure 3. Indirectly detected 1H spectra representing the vertical 1H slice at δ(27Al) ) -1 ppm of the corresponding 2D 1H-spin diffusion 1H-27Al HETCOR spectra. The samples are indicated on the left of the spectra, and the mixing times are on the right. The dotted line indicates a chemical shift of 7.3 ppm. The illustration shows the layer of a 2:1 phyllosilicate with the aluminum atom in the octahedral sheet and one lysine unit along with a water molecule in the interlayer space. The experimental conditions used for the corresponding 2D spindiffusion HETCOR experiments were identical to those indicated in Figure 2.
to finally cross-polarize the octahedral 27Al species via the smectites OH groups. These spectra clearly show that without PL adsorption, no correlated peaks are observed at 7.3 ppm for M, whereas the 6.8 ppm peak confirms that NH4+ species are in the vicinity of the interlayer surface of B. When PL is adsorbed, the side chains approach the interlayer surface for the two clay minerals. For M/PL, this is clearly illustrated by the 7.3 ppm peak, which is already seen for the short mixing time of 10 ms, but which increases for 20 ms. Likewise, with the short mixing time of 10 ms, the spectrum of B/PL displays a correlated signal around 7.3 ppm, which confirms that PL NH3+ side-chain groups have partly replaced NH4+ species at the surface. For the longer mixing time of 40 ms, the maximum of the correlated signal is shifted to lower frequencies in agreement with the presence of remaining NH4+ cations. This selection of the observed correlated signal as a function of the mixing time duration, for B/PL, is in perfect agreement with the fact that NH4+ species are more mobile than PL NH3+ groups and therefore they require longer mixing times to exchange 1H magnetization. The correlated signal around 4.4 ppm in all the spectra of Figure 3 shows that with or without PL adsorption,
Notes
water molecules are present at the interfaces. However, the comparison of the relative intensities of this 4.4 ppm signal between M and M/PL, for a given mixing time, indicates that water interacts more strongly with the clay mineral layer (water dynamics is reduced) when polylysine is adsorbed. The signals between 0 and 4 ppm are due to the smectite OH groups which act as mediators of the proton magnetization. Length scales over which the 1H magnetization is exchanged is determined by the 1H spin-diffusion constant of the material, which in turn depends on the mobilities and densities of the various protons in the sample.15 While a measure of the mobility of protons is provided by the 1D 1 H MAS line widths (800 and 500 Hz for the NH3+ and the water signal in M/PL, respectively), determination of the proton density in a clay mineral is barely feasible owing to the intrinsic heterogeneous nature of this material. Nevertheless, a similar study has reported the length scale l separating the alkyl moieties of the structure-directing surfactant and the octahedrally coordinated Al atoms of as-synthesized MCM-41 mesoporous materials.16 With comparable line widths, an estimated proton density of 0.21 g/cm3 giving a spin diffusion constant of 0.06 nm2/ms and the incipient appearance of the 1H-27Al correlated intensity for tmix ) 20 ms, the authors estimated a length scale l ) 2 nm. Assuming a comparable proton density in our M/PL sample, and given that the correlation between the PL NH3+ protons and the octahedral Al atoms is already seen for a mixing time of 10 ms, an estimated length scale l of less than 1 nm would separate the PL amino function and the octahedral Al atoms. Since the latter are at ca. 0.4 nm from the surface of the layers, this would place the PL NH3+ moiety at less than 0.6 nm from the surface, which is consistent with the basal spacing of the smectites, determined by X-ray diffraction (see Supporting Information). Through the use of an advanced two-dimensional solidstate NMR technique involving both the inorganic host and the organic guest, this study brings a direct observation of the intercalation of a polypeptide side chains between the layers of a clay mineral. Acknowledgment. The Region Champagne-Ardenne is gratefully acknowledged for financial support through the grant for R.D.G. Anne-Catherine Faust is acknowledged for the synthesis of the beidellite sample. Supporting Information Available: Description of the standard and the 1H-spin diffusion 1H-27Al HETCOR experiments used, standard 1H-27Al HETCOR spectra of M and M/PL, showing that no correlation other than the one involving the framework hydroxyl groups can be distinguished without proton spin diffusion, and d001 distances obtained from X-ray diffractograms of the different samples. This material is available free of charge via the Internet at http://pubs.acs.org. LA0115381 (15) VanderHart, D. L.; McFadden, G. B. Solid State Nucl. Magn. Reson. 1996, 7, 45-66. (16) Janicke, M. T.; Landry, C. C.; Christiansen, S. C., Kumar, D.; Stucky, G. D.; Chmelka, B. F. J. Am. Chem. Soc. 1998, 120, 6940-6951.
