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J. Phys. Chem. B 2006, 110, 1114-1120
ARTICLES Function of Membrane Protein in Silica Nanopores: Incorporation of Photosynthetic Light-Harvesting Protein LH2 into FSM Ippei Oda,† Kotaro Hirata,† Syoko Watanabe,† Yutaka Shibata,† Tsutomu Kajino,‡ Yoshiaki Fukushima,‡ Satoshi Iwai,§ and Shigeru Itoh*,† DiVision of Material Science, Graduate School of Science, Nagoya UniVersity, Furo-cho, Chikusa-ku Nagoya, Aichi 464-8602 Japan, Toyota Central R&D Laboratories Inc., Yokomichi, Nagakute, Aichi 480-1192 Japan, and Material Engineering DiVision 3, Toyota Motor Corporation, 1200 Mishuku, Susono, Shizuoka 410-1193 Japan ReceiVed: July 25, 2005; In Final Form: NoVember 15, 2005
A high amount of functional membrane protein complex was introduced into a folded-sheet silica mesoporous material (FSM) that has nanometer-size pores of honeycomb-like hexagonal cylindrical structure inside. The photosynthetic light-harvesting complex LH2, which is a typical membrane protein, has a cylindrical structure of 7.3 nm diameter and contains 27 bacteriochlorophyll a and nine carotenoid molecules. The complex captures light energy in the anoxygenic thermophilic purple photosynthetic bacterium Thermochromatium tepidum. The amount of LH2 adsorbed to FSM was determined optically and by the adsorption isotherms of N2. The FSM compounds with internal pore diameters of 7.9 and 2.7 nm adsorbed LH2 at 1.11 and 0.24 mg/mg FSM, respectively, suggesting the high specific affinity of LH2 to the interior of the hydrophobic nanopores with a diameter of 7.9 nm. The LH2 adsorbed to FSM showed almost intact absorption bands of bacteriochlorophylls, and was fully active in the capture and transfer of excitation energy. The LH2 complex inside the FSM showed increased heat stability of the exciton-type absorption band of bacteriochlorophylls (B850), suggesting higher circular symmetry. The environment inside the hydrophobic silica nanopores can be a new matrix for the membrane proteins to reveal their functions. The silica-membrane protein adduct will be useful for the construction of new probes and reaction systems.
Introduction Several reports have demonstrated the introduction of functional organic molecules such as chlorophyll1-3 and soluble small proteins such as horseradish peroxidase (with a molecular weight of 44 kDa),4,5 cytochrome c (12.3 kDa),6,7 and lysozyme (14.4 kDa)8 into mesoporous silica materials. The incorporation seems to increase the stability of the incorporated molecules.1,4 Itoh et al.1 reported the adsorption of large amount of chlorophyll a molecules (0.9 kDa) into a folded silica mesoporous material (FSM). On the other hand, the amount of proteins that had been incorporated into the silica pores was relatively low (e.g., see Table 1), and the incorporation of the large membrane proteins have never been reported, presumably due to the lack of silica materials with large pore sizes. In the present study, we report the high absorption of photosynthetic light-harvesting membrane proteins into FSM. FSM has a honeycomb-like (hexagonal) structure made of silica with ordered hydrophobic pores of several-nanometers diameters inside.9 The interior of the nanometer pores of FSM is expected to provide the environments for proteins similar to that inside * To whom correspondence should be addressed. Phone: +81 (52) 789 2881. Fax +81 (52) 789 2883. E-mail:
[email protected]. † Nagoya University. ‡ Toyota Central R&D Labs Inc. § Toyota Motor Corp.
the hydrophobic membrane matrix. As a membrane protein, we used light-harvesting-2 complex (LH2 with a molecular weight of 90 kDa for protein moiety and 129 kDa together with pigments) that was purified from the membranes of a photosynthetic purple sulfur bacterium, Thermochromatium (Tch.) tepidum. The organism belongs to the γ-subclass of proteobacter and was isolated from a hot spring in Yellowstone National Park.10,11 The organism grows at temperatures known to be the highest for all the purple photosynthetic bacteria: 50 and 58 °C for the optimum and maximum growth temperatures, respectively. The LH2 complex isolated from Tch. tepidum with a suitable detergent, therefore, is heat stable.12 Membrane proteins undergo important biological functions in respiration, sensory response, metabolite transport, photosynthesis, etc. Expressions of their proper functions, however, have been known to be achieved with lipids or detergents that stabilize their structures. Stabilization of the structures and functions of the membrane proteins in silica nanopores, thus, will open new chances for their artificial usages. We report here the stabilization of structure and function of photosynthetic membrane protein LH2 in FSM; it is the first report of function of a membrane protein inside the silica nanopores. LH2 functions to capture sunlight and funnels the excitation energy into the reaction center complex, where the photon energy is used for the charge separation in the photosynthetic
10.1021/jp0540860 CCC: $33.50 © 2006 American Chemical Society Published on Web 01/04/2006
Light Harvesting in Silica Nanopores
J. Phys. Chem. B, Vol. 110, No. 3, 2006 1115
TABLE 1: Adsorption of LH2 to FSM and That of Soluble Proteins to Various Porous Silica Materialsa material
av pore diam, nm
protein (mol wt in kDa)
max amt of adsorbed protein, mg/mg SiO2 materials
ref
Membrane Protein FSM-16
MCM-41 COS CNS MCM-41 MPS-F127 Rod-SBA-15 Con-SBA-15
7.9 2.7 8.9 2.7 6.8 5.5 13.0 2.8 5.0 7.9 8.0
LH2 (90.0) LH2
1.11 0.24
this work this work
Water-Soluble Proteins horseradish peroxidase horseradish peroxidase (44.0) horseradish peroxidase cytochrome c (12.3) cytochrome c cytochrome c cytochrome c lysozyme (14.4) lysozyme
0.18 0.028 0.15 0.047 0.13 0.021 0.084 0.53 0.20
4 4 4 6 6 6 6 8 8
a Maximum amounts of proteins adsorbed per 1 mg of silica nanoporous materials are shown. The amounts of bound LH2 were estimated spectrophotometrically by determining the absorbance decrease at 800 nm of LH2 in the supernatant solution after sedimentation of FSM. Amounts of soluble proteins bound to silica porous materials were obtained from references listed in the table.
structure since its intensity and peak position come from the exciton interactions that are highly sensitive to the symmetry of LH2 ring.20 We here show the high amount of adsorption of LH2 to FSM and the immobilization of LH2 inside the silica nanoporous structure. The LH2 gives the higher B850 band, indicating the maintenance of its ring structure even after its immobilization among solid silica surface inside the FSM pore that has an inner diameter just large enough to accept the LH2 cylinder. Materials and Methods
Figure 1. Molecular structure of LH2 complex (A), arrangements of bacteriochlorophyll a molecules inside a single LH2 molecule (B), schematic view of LH2-FSM7.9 conjugate (C, D), and photographs of vial tubes containing LH2 solution before (E) and after (F) addition of FSM7.9. A value for a in (C) was estimated to be 9.2-11.4 nm upon the binding of 1.11 mg of LH2/mg of FSM7.9 based on the results in this study. See the text for details.
primary reaction.13,14 The LH2 complex exhibits a beautiful ring structure15,16 and is densely scattered on the inner membranes of purple photosynthetic bacteria.17,18 The absorption spectrum of LH2 has two maxima at 800 and 850 nm (named B800 and B850, respectively), both for bacteriochlorophyll a (BChl a) pigments. The LH2 complex is made of nine (or in some case eight) identical subunits. Each subunit consists of two polypeptide chains R and β, with two membrane-spanning helices, and binds three BChl a and one (or two) carotenoids (Car). The complex has a structure like a wheel with a nearly perfect rotational symmetry with nine sets of R- and β-units, i.e., with 27 BChl a molecules (see Figure 1).19 The nine BChl a molecules that bind in the monomer form are responsible for B800, and the other 18 BChl a molecules, which strongly interact with each other, are responsible for B850. The absorption band of B850 can be a sensitive probe of the protein ring
Tch. tepidum cells were grown as reported.11 LH2 protein was extracted from the intracytoplasmic membranes isolated from Tch. tepidum cells by using the detergent lauryldimethylamine oxide (LDAO) and purified by anion-exchange chromatography as reported.21,22 FSM-16 with a pore diameter of 2.7 or 7.9 nm (designated FSM2.7 or FSM7.9) was prepared from kanemite (layered polysilicate) by the use of hexadecyltrimethylammonium chloride.23 Adsorption of LH2 into FSM was performed as follows: 1 mg of dry powder of FSM was added to the 1 mL solution of medium containing 20 mM Tris‚HCl buffer (pH 8.5), 0.011.6 mg/mL LH2 protein, 0.05% LDAO, and 200 mM NaCl. After the mixture was stirred at 25 °C for 3 h, the amount of adsorbed LH2 protein was estimated by measuring the absorbance decrease at 800 nm in the supernatant with a double beam spectrophotometer (UV-3100PC, Shimadzu, Kyoto, Japan) after centrifugation to sediment the FSM-LH2 conjugate. The molar extinction coefficient at 800 nm (800) of LH2 of Tch. tepidum was estimated to be 1350 mM-1 cm-1 according to the value of LH2 of Rhodopseudomonas acidophila.15 We assumed that nine BChl a molecules per LH2 molecule contribute to B800 in LH2 of Tch. tepidum in analogy to LH2 of Rps. acidophila. Fluorescence spectra and lifetimes were measured with a spectrograph system equipped with a fast streak camera as described elsewhere24 (Streak Scope C4334, Hamamatsu Photonics, Hamamatsu, Japan). Samples were excited with a 200 fs excitation laser pulse at 750 nm, fired at 80 MHz (Mai Tai, Ti:sapphire laser, Spectra-Physics, Tokyo, Japan) at room temperature. The suspension of LH2-FSM conjugate was constantly stirred with a magnetic stirrer during the measurements. The amount of adsorbed LH2 inside the pores of FSM was also estimated by the measurement of N2 adsorption isotherms. We measured four samples: FSM7.9 that was just suspended
1116 J. Phys. Chem. B, Vol. 110, No. 3, 2006 in the reaction medium containing 20 mM Tris‚HCl buffer (pH 8.5), FSM7.9 that was immersed in the reaction medium with LDAO at the same concentration as used for the protein adsorption, FSM7.9 that was incubated in the LH2 solution at 0.042 mg/mg FSM, and FSM7.9 that was incubated in the LH2 solution at 0.42 mg/mg FSM. The LH2 solutions contained the same concentration of LDAO, too. The incubated FSM samples were then washed twice with 20 mM Tris‚HCl buffer (pH 8.5) and desiccated. The N2 adsorption isotherms were measured at 77 K using a Quantachrome AS-1 (Quantachrome, Boynton Beach, FL). The pore diameter distribution curves were derived from the N2 adsorption isotherm curves by the BJH method.25 The pore volume was estimated from the amount of adsorbed N2 at the maximum relative pressure. The heat stability of the immobilized LH2 was examined by measuring the spectrum of B-850 absorption band. LH2-FSM conjugate powder was collected by centrifugation at 1500g for 3 min, washed again with a buffer containing 20 mM Tris‚HCl buffer (pH 8.5), and then suspended in the same buffer. Temporal change in the absorbance at 850 nm was measured at each temperature for 2 h. The sample suspensions were constantly stirred by using a magnetic stirrer to avoid precipitation of FSM powder during the measurement. Results Adsorption of LH2 Light-Harvesting Pigment Protein Complex to FSM. Parts A and B of Figure 1 show the molecular structure of LH2 complex and ringlike arrangements of 27 BChl a molecules inside the protein, respectively, of Rps. acidophila determined by X-ray crystallography.16 Parts C and D of Figure 1 show schematic views of a single pore or multiple pores inside FSM containing LH2, respectively. The LH2 complex is made of a 9-mer of units made of two subunit polypeptides, contains 27 BChl a molecules, and has a cylindrical structure with a diameter of 7.3 nm with a 3 nm inner pore and height of 5.7 nm. We can assume a similar complex structure for LH2 of Tch. tepidum used in this study.12,26 The schematic views of LH2 inside pores of FSM7.9 in Figure 1C,D were drawn by assuming that LH2 is adsorbed inside each 7.9 nm pore of FSM7.9 to allow the interaction of its membraneburied hydrophobic surface of cylinder with the inner surface of FSM pore, at the density estimated in this study. The validity of the assumption of the geometry and the packing density will be discussed later. Figure 1 also shows photographs of vials with FSM7.9 powder and LH2 solutions with (Figure 1F) and without (Figure 1E) added FSM7.9 powders. After the addition of FSM7.9 into the solution that contains various amounts of LH2 protein, the white powder of FSM gradually gained a red color parallel to the decrease of concentration of LH2 in solution. After 30 min of gentle stirring at room temperature, almost all the LH2 protein was adsorbed to FSM if LH2 concentration was not extremely high, as seen from Figure 1E,F. The amount of LH2 adsorbed to FSM was measured by the absorption spectrum of LH2 that remained in the supernatant after the FSM-LH2 adducts were precipitated by centrifugation. The amounts of LH2 bound to FSM2.7 and FSM7.9, which have pore diameters of 2.7 and 7.9 nm, respectively, were calculated to be 0.24 and 1.11 mg/ mg of FSM. The amount of LH2 adsorbed to FSM7.9, therefore, was 4.6 times larger than that bound to FSM2.7 (Table 1). The result indicates that LH2 binding to FSM is very sensitive to the pore size inside FSM. If the added amount of LH2 did not exceed 0.7 mg per 1 mg of FSM7.9, free LH2 in solution was almost negligible, as seen in Figure 1. Above this concentration,
Oda et al.
Figure 2. (A) N2 adsorption isotherm curves: a, FSM7.9 treated with 20 mM Tris‚HCl buffer solution at pH 8.5; b, FSM7.9 treated with buffer solution containing 0.05% (v/v) LDAO (FSM7.9-LDAO); c, FSM7.9 treated with buffer solution containing LH2 at 0.042 mg/mg FSM (FSM7.9-0.042 mg of LH2) and 0.05% (v/v) LDAO; d, FSM7.9 treated with buffer solution containing LH2 at 0.42 mg/mg FSM (FSM7.9-42% LH2) and 0.05% (v/v) LDAO. The ordinate indicates the adsorbed volume of N2, and the abscissa indicates the ratio between the adsorption equilibrium pressure and the saturated vapor pressure (expressed as relative pressure). (B) Pore diameter distribution curves calculated according to the BJH method25 applied to the N2 adsorption curves in (A). Symbols a-d are the same as in (A). See Materials and Methods for details.
free LH2 molecules increased as the increase of bound LH2 (not shown). FSM2.7 and FSM7.9 have different pore sizes but have similar physicochemical properties and particle sizes (2-5 µm).23 The pore-size dependence of the adsorption efficiency, therefore, suggests the binding of LH2 into the inner pores of FSM7.9. LH2, which has a 7.3 nm diameter of its cylindrical structure, seems to bind only to the outer surface of FSM2.7, and to bind to both the outer surface and the inner pores of FSM7.9. It was also evident from the optical measurements that LH2 was not decomposed into smaller subunits that are small enough to be adsorbed into the 2.7 nm pores inside FSM2.7, as shown in the following section. The hydrophobic moiety of LH2 around the cylindrical surface, which had been buried within hydrophobic carbohydrate tails of lipid inside the intact membranes or covered by the detergent in the solution, seems to have high affinity to the inner surface of the silicate pores. Effects of LH2 Binding to the Adsorption Isotherms of N2 to FSM. Figure 2A shows the adsorption isotherms of N2 to FSM. We used four samples: (a) untreated FSM7.9, (b) FSM7.9 immersed in a 0.05% (v/v) LDAO solution (FSM7.9LDAO), (c) FSM7.9 in a solution containing LH2 at 0.042 mg/ mg FSM (FSM7.9-0.042 mg of LH2) and 0.05% (v/v) LDAO, and (d) FSM7.9 in a solution containing LH2 at 0.42 mg/mg FSM (FSM7.9-0.42 mg of LH2) and 0.05% (v/v) LDAO. The ordinate indicates the volume of N2 adsorption, and the abscissa indicates the ratio between the adsorption equilibrium pressure and the saturated vapor pressure (expressed as relative pressure). The total pore volume and BET surface area calculated for each sample were as follows: FSM2.7 (0.84 cm3 g-1, 927 m2 g-1), FSM7.9 (1.75 cm3 g-1, 973 m2 g-1), FSM7.9-LDAO (0.7 cm3 g-1, 390 m2 g-1), FSM7.9-0.042 mg of LH2 (0.66 cm3 g-1, 362 m2 g-1), and FSM7.9-0.42 mg of LH2 (0.39 cm3 g-1, 228 m2 g-1). The volume of adsorbed N2 decreased drastically in
Light Harvesting in Silica Nanopores FSM7.9-LDAO (curve b) compared to that of the untreated FSM7.9 (curve a), suggesting that the detergent molecules are adsorbed to FSM7.9. The volume of adsorbed N2 further decreased upon the adsorption of LH2 at 0.042 mg (curve c) and significantly at 0.42 mg (curve d), even at the same LDAO concentrations. The result strongly suggests that LH2 is adsorbed mainly into the internal pores of FSM. Figure 2B shows the effective pore-size distribution estimated for each sample. The distribution curves were calculated according to the BJH method25 applied to the N2 adsorption curves in Figure 2A. The method calculates the inner surface area by assuming N2 to be adsorbed to the intrapore surfaces of the silica materials and estimates the distribution of pore diameters with the assumption of sufficiently long pore lengths.25 The peak indicated the pore diameter centered at 7.9 nm (curve a) in untreated FSM7.9. The peak shifted to the smaller side at 6.7 nm upon the adsorption of LDAO (curve b). FSM7.9-0.042 mg of LH2 (curve c) showed a pore-size distribution at 6.2 nm (curve b), suggesting the adsorption of a small amount of LH2 into the pore. The peak of FSM7.9-0.42 mg of LH2 (curve d), on the other hand, was found at 5.1 nm and was significantly shifted to the smaller side from that of untreated FSM7.9. The smaller pore-size distribution, as well as the significantly lower extent of N2 adsorption seen in Figure 2A in the case of FSM7.9-0.42 mg of LH2 (curve d), thus suggests the high adsorption of LH2 into the pores with diameters large enough to accept the LH2 ring structure. The adsorption isotherms of N2 suggest that LH2 is adsorbed into the pores inside FSM7.9. This is consistent with the fact that the diameter of the LH2 ring is approximately 7.3 nm and is slightly smaller than the average 7.9 nm pore diameter of FSM7.9. The result also implies that LH2 is adsorbed into the pores inside FSM7.9 mainly in a geometry with its ring axis parallel to the pore axis because the LH2 cylinder does not fit into the 7.9 nm pores in the other geometries. The major driving force for the high adsorption, therefore, can be assumed to be the hydrophobic interaction between the inner surface of pores in FSM and the outer surface of LH2. The binding of LDAO molecules to the FSM pores also seems to exist. The binding of LH2 to the outer surface also seems to occur, as can be seen in the binding of 0.24 mg of LH2/mg of FSM2.7 (see Table 1). We observed that the amount of LH2 remaining in the supernatant was almost negligible below 0.7 mg of LH2/mg of FSM7.9 and increased significantly above this amount. The binding to the outer surface, therefore, seemed to be a little weaker and became more significant at the higher concentrations of LH2. Optical Absorption Spectra of LH2 in Solution and in LH2-FSM7.9 Conjugate. The extent and shape of the B850 absorption band are known to vary depending on temperature or medium conditions, and to be lost as the degradation of the ring structure.14 Figure 3 shows the absorption spectra of LH2 in a buffer solution (curve a) and in the LH2-FSM7.9 conjugate (curve b) suspended in the same medium. The spectra were measured by using a spectrophotometer with optical fibers to minimize the effect of strong light scattering by FSM. The LH2-FSM conjugate showed the B800 absorption band essentially identical to that of LH2 in solution. The spectrum of the conjugate, however, revealed the peak of B850 at 853 nm, which is red-shifted by 2 nm from that at 851 nm in solution. Furthermore, the conjugate had a B850/B800 peak ratio a little higher than that in solution. The results indicate that the ring structure of LH2 that is essential for the B850 absorption band is well maintained inside FSM7.9. The B800 band is known to
J. Phys. Chem. B, Vol. 110, No. 3, 2006 1117
Figure 3. Absorption spectra of LH2 in the reaction medium and LH2-FSM conjugate: a, LH2 in a 20 mM Tris‚HCl buffer at pH 8.5; b, 0.57 mg of LH2/mg of FSM7.9 conjugate dispersed in the same medium. Each spectrum was normalized at the 800 nm peak of B800 band.
Figure 4. (A) Time courses of absorption decrease of LH2 at 850 nm during heat treatment. Curves a and b, LH2 in 20 mM Tris‚HCl buffer at pH 8.5 at 60 and 70 °C, respectively. Curves c and d, LH2-FSM conjugate (0.57 mg of LH2/mg of FSM7.9) in the same medium at 60 and 70 °C, respectively. Absorbance at 850 nm was monitored at each temperature. Abscissa indicates the time of heat treatment. (B) Arrhenius plot of the degradation rate of B850. Curves a and b, LH2 in a 20 mM Tris‚HCl buffer at pH 8.5 and LH2-FSM conjugate in the same medium, respectively. The rate constants were calculated from the measurements as in (A).
be sensitive to the change of pigment-protein interaction between the monomer-like BChl a molecule and the surrounding amino acid residues.20 The band shape of B800 in the LH2FSM conjugate suggests that the interaction between each BChl a and its protein environment is almost unaffected. On the other hand, the red shifts of B850 absorption and fluorescence peaks, as well as the slight increase of the B850 absorption band, inside FSM may indicate the higher circular symmetry of BChl a molecules to stabilize or to populate the energy levels of the B850 exciton band. Stabilization of LH2 Structure inside FSM. Heat stability of LH2 either in solution or adsorbed to FSM7.9 was tested by monitoring the B850 absorption band. The peak intensity was only slightly decreased at 60 °C, indicating the high stability of LH2 structure (Figure 4A, curve a). The rate of decrease became significantly higher at 70 °C (curve b). The rate became lower if LH2 was adsorbed to FSM7.9 (curves c and d) at these temperatures, indicating that LH2 inside FSM is more stable against the heat treatment. The degradation rate was calculated from the time courses in Figure 4A and plotted against temperature in Figure 4B. The
1118 J. Phys. Chem. B, Vol. 110, No. 3, 2006
Figure 5. (A) Fluorescence spectra of LH2 excited by 750 nm laser pulse that is mainly absorbed by B800. Curves a and b, LH2 in 20 mM Tris‚HCl buffer at pH 8.5 and LH2-FSM conjugate (at 0.57 mg of LH2/mg of FSM7.8) dispersed in the same reaction medium. (B) Fluorescence kinetics of LH2 measured at 870 nm (a, b) and 800 nm (c) after 0.2 ps laser excitation. Curve a, LH2 in 20 mM Tris‚HCl buffer at pH 8.5. Curves b and c, LH2-FSM conjugate (0.57 mg of LH2/mg of FSM) dispersed in the same reaction medium. Curve d, profile of Ti:sapphire laser pulse measured by the same experimental conditions at 750 nm. Solid lines represent simulated curves with amplitude and time constants as indicated in text.
difference between the degradation rate in FSM (Figure 4B, curve b) and that in solution (Figure 4B, curve a) became larger especially at the higher temperatures. The Arrhenius plots in Figure 4B gave a lower activation energy and lower frequency factor for the LH2 adsorbed to FSM7.9. The results indicate that the degradation rate of LH2 is slower in FSM than that in solution because of the significantly lower frequency factor even with the lower activation energy value. The stabilizing effect was more marked at the higher temperatures because of the lower apparent activation energy. Furthermore, it was also noted that the heat-induced decrease of B850 band in FSM, but not in solution, was highly reversible if cooled to the lower temperature (not shown). Excitation Energy Transfer from B800 to B850 in FSM. Fluorescence of LH2-FSM7.9 conjugate was excited by the illumination at the 750 nm short-wavelength edge of B800 absorption band at room temperature (Figure 5A). The emission spectrum showed a peak at 885 nm of B850 fluorescence and showed very low fluorescence from B800 below 840 nm. Approximately 80% of photons in the 750 nm laser beam was estimated to be absorbed by the B800 absorption band so that the fluorescence from B850 is mainly excited through the energy transfer from B800 to B850. The result in Figure 5A indicates the efficient energy transfer from B800 to B850 in the LH2FSM conjugate as well as in solution. The extent of the 20 nm red shift of the peak wavelength of B850 fluorescence compared to that in solution was larger than the 2 nm red shift of the absorption band. It therefore seems to occur due to the selfabsorption of fluorescence by the B850 absorption bands by the densely packed nearby LH2 molecules, as assumed in Figure 1D. We also investigated the dynamic function of LH2 by the measurement of fluorescence lifetimes. Figure 5B shows the time course of B850 fluorescence at 870 nm upon the excitation of B800 band at 750 nm with repetitive 0.2 ps laser flashes.
Oda et al. The intensity of fluorescence at 870 nm of LH2 in solution rose within 2 ps and decayed slowly. The decay can be simulated with a single-exponential component with a relative initial amplitude and decay time constant of 111 and 997 ps, respectively. The kinetics of B850 fluorescence indicates that the laser energy absorbed by B800 is rapidly transferred to the B850 exciton band (within a few picoseconds27) and then emitted as the fluorescence from B850 as reported previously.28,29 LH2 adsorbed to FSM7.9 showed similar but somewhat different fluorescence kinetics. The fluorescence at 870 nm rose rapidly, indicating the fast energy transfer from B800 to B850 within the time of response function of the apparatus as was the case in solution. The decay of fluorescence at 875 nm in FSM was significantly faster than that in the LH2 solution. The curve can be simulated by a fast rise time constant of less than 2 ps and the three exponential decay components with relative amplitudes (and decay time constants) of 399 (13 ps), 84.6 (52 ps), and 3.69 (207 ps), respectively. Although the fluorescence from B800 band was not distinguished as a distinct single component in the present photon-counting measurement, the kinetics at 800 nm measured in LH2-FSM7.9 conjugate (Figure 5B, curve c) revealed the higher contribution of the fast decay component indicating the fast energy transfer from B800 to B850. It is also noted that almost no free pigment that give long lifetimes were detected. The result of fluorescence study indicates the fast energy transfer from B800 to B850 in LH2 in FSM and somewhat faster energy dissipation from B850 in FSM. It is, thus, suggested that LH2 inside FSM still keeps the symmetrical arrangement of BChl a molecules that enables the fast excitation energy transfer from B800 to B850. Discussion Adsorption of Membrane Protein into Silica Nanopores. Membrane proteins are essential in the energy conversion as well as regulation of many cell activities. They are essentially insoluble in aqueous media without suitable detergents or lipids. The incorporation of the functional membrane proteins into the silica mesoscopic materials done in this study, therefore, opened a new method for their use in vitro or for artificial purposes. We indicated the high adsorption of membrane protein LH2 into FSM. The LH2 pigment-protein complex is a typical membrane protein that is well-known for its elegant ring structure made of 18 membrane-spanning R-helices and 27 BChl a molecules.15,16 The protein structure and the pigment arrangements are highly optimized for the efficient energy transfer required for solar energy capture. The extent of LH2 bound to FSM amounted to 1.11 mg/mg FSM, and is the highest among the proteins thus far tested for the conjugation with silica nanoporous materials (Table 1). The molecular size of LH2 is the largest among the proteins tested thus far. A small water-soluble protein, lysozyme, with a molecular weight of 14.4 kDa was shown to be adsorbed to SBA-15,30 which is a silica nanoporous material synthesized by the use of amphiphilic block copolymers as structure-directing organic agents in aqueous acidic conditions. SBA-15 with a pore diameter of 7.9 nm bound lysozyme at a comparable efficiency of 0.53 mg/mg SBA.8 The high adsorption efficiency seems to come from the strong Coulomb interaction between the positive charges of lysozyme and the negative internal silicate surface that is rich in -Si-OH or -Si-O- group in SBA. Different morphologies of SBA also may contribute to the different extents of adsorption since the adsorption was larger with Rod-
Light Harvesting in Silica Nanopores SBA-15, which possesses a discrete rodlike morphology with uniform rod lengths (1-2 µm), than with Con-SBA-15, which has a fibrous macrostructure extending tens of micrometers.8 Except for the combination of lysozyme and Rod-SBA-15, water-soluble proteins seem to be rather poorly adsorbed, in general. Mammalian cytochrome c (12.3 kDa) and horseradish peroxidase (HRP; 44.0 kDa) were adsorbed only at 0.021-0.18 mg/mg silica materials even when the pore sizes were large enough to accept them. The structural integrity or arrangements of these water-soluble proteins inside silica nanopores have not been known yet. The N2 adsorption isotherm measurements in the present study indicated the binding of LH2 into the interior of porous honeycomb-like cavities inside FSM. This conclusion is also supported by the high and low adsorptions of LH2 to FSM7.9 and FSM2.7, respectively. The extent of binding of LH2 was highly dependent on the pore size of FSM. The rather hydrophobic nature of the inner pore surface of FSM, as revealed by the binding of high amounts of chlorophylls shown previously by Itoh et al.1,2 or of the detergent LDAO to FSM shown in this study, also seems to be suitable for the binding of the hydrophobic membrane proteins. The extent of LH2 bound to FSM was also highly dependent on the experimental conditions such as pH, detergent, and ionic conditions (not shown). Arrangement of LH2 in FSM Nanopores. We estimated the bindings of LH2 inside and outside FSM according to the following two approaches by using the amounts of bound LH2 and the total pore volumes of 0.84 and 1.75 cm3/g for FSM2.7 and FSM7.9, respectively, obtained by the N2 isotherm adsorption measurements. (1) The amount of LH2 bound to the outer surface of FSM7.9 was assumed to be the same as that to FSM2.7 per unit weight (0.24 mg/mg FSM), so the amount of LH2 bound to the internal pores of FSM7.9 was calculated simply as the difference between the bindings to FSM7.9 and to FSM2.7 per unit weight (1.11 mg - 0.24 mg ) 0.87 mg/mg FSM7.9). The calculation is correct if the external surface areas of FSM7.9 and FSM2.7 are equal per unit weight, i.e., if the average particle size of FSM2.7 is smaller than that of FSM7.9. (2) In the other estimation, we assumed equal amounts of LH2 binding per unit areas of the outer surface of FSM7.9 and FSM2.7. Simply calculating with average particle sizes of 2 µm and thickness of silica walls of 0.3 nm for both types of FSM, we can calculate that the surface area of FSM 7.8 is 1.71 times larger than that of FSM2.7 per unit weight. The amount of LH2 inside the pores was, then, calculated to be 0.7 mg/mg FSM7.9. The amount of LH2 at 0.87 or 0.7 mg/mg FSM7.9 inside the 7.9 nm silica nanopores estimated above gives the mutual spacing between LH2 molecules as 3.5 or 4.6 nm, respectively, or the average value of a in Figure 1 as 9.2-11.4 nm. Although the distribution and orientation of LH2 inside the silica pores are not fully clear yet, we estimated the arrangement of LH2 inside nanopores of FSM7.9 as illustrated in Figure 1 to fit an LH2 molecule with 7.3 nm diameter and 5.7 nm height into the 7.9 nm diameter pores inside FSM without gross changes of the LH2 structure. The distribution of detergent LDAO that may also exist around LH2 is not clear yet. The estimated distribution of LH2 inside FSM explains the almost intact but slightly modified absorption/fluorescence properties of LH2 and suggests some interaction of LH2 with FSM in a way to stabilize the LH2 ring structure. The dense and linear packing estimated in Figure 1C,D seems to be the most unique feature of the LH2FSM conjugate. The packing density of LH2 inside FSM may
J. Phys. Chem. B, Vol. 110, No. 3, 2006 1119 be a little higher than that in the stacked membranes inside bacterial cells. The accelerated decay of B850 fluorescence in LH2-FSM conjugate might also be related to the dense packing of LH2 inside FSM because the dense packing will increase the chance of reabsorption of excitation energy by the nearby LH2 molecules or by some quenching molecules such as oxidized or denatured pigments even when they are in other complexes. This is interesting if the high-density packing accelerates the decay of excited state through the mutual interaction of excited states, as seen in the laser resonance emission, along long distances. This interesting phenomenon is now being studied. Function of Membrane Proteins inside Silica Nanopores. The absorption bands of B800 and B850 of LH2 have been known to be sensitive markers of the ring structure of LH2. The molecule of bacteriochlorophyll a gives the absorption band at 800 nm in its monomeric form and the exciton-type absorption band at 850 nm in its multiple interacting forms with a highly circular symmetry expanded over half of a ring. Destruction of the ring structure by the detergent treatment12,19 or thermal perturbation31 seems to result in the decrease and blue shift of the B850 band. The LH2 conjugated to FSM showed an almost intact but a little stronger and red-shifted B850 absorption band. The result indicates that LH2 adsorbed to FSM has an intact or even more symmetrical cylindrical structure. The adsorption to mesoscopic silica materials has been reported to increase the heat stability of the water-soluble proteins4 and chlorophylls.1-3 The adsorption of the heat-stable LH2 complex of Tch. tepidum into FSM further elevated the heat stability by about 10 degrees. The stabilization was achieved by the decreases in both the apparent activation energy value and the collisional factor for the degradation process as shown in Figure 4B. Although protein degradation is known to be a complex series of reactions, it seems that the mode of fluctuation of LH2 is modified and restricted inside the FSM7.9 to stabilize the ring structure. The restriction that comes from the interaction with the silica walls seems to prevent the irreversible breakage of the ring structure and the irreversible loss of pigments, and might interpret the lower apparent activation energy value for the degradation process inside FSM. The incorporation of the membrane protein LH2 into mesoscopic porous silica has opened a new chance for the stabilization/fabrication of membrane proteins. By the use of nontoxic silica nanoporous materials as the matrix for the membrane proteins, we will be able to realize their functions without membranes, liposomes, and detergent micelles. Proper combination of the pore size, material structure, and proteins will produce a wide variety of new functional systems. Acknowledgment. We are grateful to Drs. Tsunenori Nozawa and Masayuki Kobayashi, Department of Biochemistry and Engineering, Faculty of Engineering, Tohoku University, for their kind gift of Tch. tepidum and valuable advice on the preparation of LH2 complex. We also thank Dr. Govindjee at the University of Illinois for his critical reading and revision of manuscript. The work was supported by Grants-in-Aids (No. 15370067 and 17370055) to S.I. and by the 21st COE program for “the origin of the universe and matter” from the Japanese Ministry of Education, Science, Sports and Culture. References and Notes (1) Itoh, T.; Yano, K.; Inada, Y.; Fukushima, Y. J. Am. Chem. Soc. 2002, 124, 13437-13441. (2) Itoh, T.; Yano, K.; Inada, Y.; Fukushima, Y. J. Mater. Chem. 2002, 12, 3275-3277.
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