Langmuir 2001, 17, 3719-3726
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P450scc Engineering and Nanostructuring for Cholesterol Sensing Claudio Nicolini,* Victor Erokhin,† Paola Ghisellini, Cristina Paternolli, Manoj Kumar Ram,‡ and Victor Sivozhelezov† Department of Biophysical M&O Sciences and Technologies, University of Genoa, Corso Europa 30, 16132 Genoa, Italy Received October 6, 2000. In Final Form: March 14, 2001 To optimize biodevice assembly and cholesterol sensing, recombinant and wild type P450scc cytochromes are extensively characterized both in solution and in thin solid films, using X-ray scattering, Brewster angle microscopy, quartz crystal nanobalance, ellipsometry, cyclic voltammetry, and circular dichroism. Efficient expression systems are implemented in microbial cells for the production of recombinant P450scc proteins, which are then purified. Modeling of the cholesterol interaction was also studied for the given application. Both types of P450 form monolayers at the air/water interface which can be transferred onto solid substrates, but only in the case of recombinant protein does the engineered monolayer turn out to be more dense and regular, and its thickness corresponds better to the native protein size. By surface pressure and surface potential measurements it is shown that at the air/water interface P450scc molecules orient themselves at the initial stage of the monolayer compression by self-assembly; increasing surface pressure yields high homogeneity, as confirmed by Brewster angle microscopy. CD measurements confirmed a significant increase in the stability of the protein secondary structure in the recombinant monolayers whose regular structure appears by X-ray measurements. The cyclic voltammetric study on LB films of cytochrome P450scc as a function of number of monolayers and of cholesterol concentration pointed to a possible electron transfer from the electrochemical process to cytochrome P450scc that in turn reacted with cholesterol, compatibly with the modeling showing that the nearest cholesterol atoms belonging to the main chain are only 4-5 Å from the heme.
1. Introduction Computational, sensing, and actuating nanotechnology represents a natural evolution of the traditional computational technology for what concerns dimensions, considering that the dimensions of the current smallest electronic silicon-based device (about 200 nm or 2 × 108 atoms) would lead, at the current rate of progress, to the atomic scale in 20 years.1 While such progress with siliconbased devices is just quantitative and is approaching its physical limit, the biopolymers proposed by some authors (for a review see refs 2 and 3) might add a difference in behavior with respect to inorganic semiconductors also in qualitative terms, giving to the words “computer”, ”actuator”, and “sensor” a new and broader sense. Let us indeed remember that the existence of birds was the proof of the possibility of building flying machines before the construction of the first airplane, and birds have been studied in order to understand the principles of flight. In the same way, considering that natural nanocomputers do exist and do work (e.g. DNA, brain, photosynthesis), we should be in principle able to realize artificial nanocomputers and nanosensors based on a biopolymer such as cytochrome P450scc. The performance of this new class of hypothetical devices and systems would then be not only increased but also qualitatively changed. To under* Corresponding author. Phone: +39 010 3538381. Fax: +39 010 3538346. E-mail:
[email protected]. † Fondazione El.B.A. Genova Section. ‡ Polo Nazionale Bioelettronica Genova Section. (1) Toffoli, T. Nonconventional Computers. In Encyclopedia of Electrical and Electronics Engineering; Webster, J., Ed.; Wiley and Sons: 1998. (2) Nicolini, C. Molecular Bioelectronics; World Scientific Publishing: New York, 1996; Chapter 1. (3) Nicolini, C. From Protein Engineering to Bioelectronics. In From Neural Networks and Biomolecular Engineerings to Bioelectronics; Nicolini, C., Ed.; Plenum Publishing Corporation: New York, 1995.
stand if and how the performance will change, we need first to assemble the new biomaterials with the required enhanced physical and chemical properties that we should be able to modify with a “bottom-up” approach by a combination of recombinant DNA, monolayer engineering, and biochemical-biophysical reactions. To outline our progress toward this end is indeed the goal of our communication. Since early 90s the possibility to work at the molecular level appears both physically possible and practically feasible utilizing Langmuir-Blodgett (LB) films in devices.2 Many proteins indeed appear to have such primary function as the transfer and processing of information rather than chemical transformation of metabolic intermediates or the building of cell structures. 3D LB films through an allosteric or local mechanism appear potentially capable of performing computational tasks including amplification and information storage far beyond any expectation.4 Particularly, monolayer studies of the cytochromes are of particular interest because, on the one hand, these proteins display multiple pathways and, on the other hand, “P450scc is unusual among members of this class of enzymes in showing a high degree of substrate specificity”. Its complex with its electron transfer partner protein, adrenodoxin, was found to be easily formed by Langmuir techniques and covalently immobilized on the solid substrates.5,6 The LB technique is found to be a suitable tool for the deposition of thin protein films.7-14 In several cases it is (4) Nicolini, C. Biophysics of Electron Transfer; Plenum Publishing Corporation: New York, 1998; Chapter 1. (5) Gouryev, O.; Erokhin, V.; Usanov; Nicolini, C. Biochem. Mol. Biol. Int. 1996, 39 (1), 205. (6) Guryev, O.; Dubrovsky, T.; Chernogolov, A.; Dubrovskaya, S.; Usanov, S.; Nicolini, C. Langmuir 1997, 13, 299. (7) Langmuir, I.; Schaefer, V. J. Am. Chem. Soc. 1938, 60, 1351.
10.1021/la001418d CCC: $20.00 © 2001 American Chemical Society Published on Web 05/18/2001
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shown that the deposition procedure does not destroy proteins but also gives rise to some new useful properties. Close packing of molecules is shown to be the reason for high thermal stability.14-18 The secondary structure of the proteins in the LB film is slightly affected only till 200 °C, while in solution proteins begin to be denatured even at temperatures of 60-70 °C.14 It is interesting to mention that this property is not possible to observe in chaotically oriented films. In ref 19 the authors developed a technique for the formation of cytochrome P450scc monolayers at the air/ water interface and their transfer to solid substrates. The surface density was estimated with 125I labeled cytochrome and turned out to be 17 pM/cm2. Comparison of the value and the thickness of the monolayer, estimated by interference microscopy, with known sizes of the protein allowed one to conclude a densely packed layer of the protein which was obtained at the substrate. The physical or chemical properties of the formed film were not investigated. In ref 20 atomic force microscopy was applied for studying the cytochrome P450scc structure as well. Practically no works on recombinant cytochrome P450 LB films were done, even if recombinant protein seems to be more adequate for technological reasons, as it represents the homologous population of the molecules with a controlled sequence which can be ad hoc modified by site-specific mutagenesis. Furthermore, it can undergo cheap mass production and has the high level of purity. Escherichia coli is a desirable organism for the heterologous expression of proteins, owing to its ease of manipulation, the availability of a variety of cloning and expression vectors, well-understood genetics, and the low cost of the culture.21 In the case of expression of the metalloproteins, the improvement of the purity level of the final product is possible to carry out not only by using the specific detergents but also by applying the affinic resins (cholatesefarose) to this protein during the chromatography purification.22 The aim of the work is to form LB films of cytochrome P450scc, both wild type and recombinant, to study their structure and properties with several experimental techniques, and to compare their behaviors. 2. Materials and Methods 2.1. Protein and Chemicals. P450scc wild type was obtained from adrenal cortex mitochondria and kindly provided by Dr. P. Pashkevitch (Institute of Biophysics, Genova). The clone of cytochrome P450scc was kindly provided by Prof. M. Eldarov (8) Fromhertz, P. Biochim. Biophys. Acta 1971, 225, 382. (9) Tiede, D. Biochim. Biophys Acta 1985, 811, 357. (10) Okahata, Y.; Tsuruta, T.; Ijiko, K.; Mergura-ku, O. Langmuir 1988, 4, 1373. (11) Kozarac, Z.; Dhathathreyan, A.; Mo¨bius, D. FEBS Lett. 1988, 229, 372. (12) Mecke, W.; Zaba, B.; Mo¨hwald, H. Biochim. Biophys. Acta 1987, 903, 166. (13) Erokhin, V.; Kayushina, R.; Lvov, Yu.; Feigin, L. Stud. Biophys. 1989, 139, 120. (14) Nicolini, C.; Erokhin, V.; Antolini, F.; Catasti, P.; Facci, P. Biochim. Biophys. Acta 1993, 1158, 273. (15) Shen, Yi.; Safinya, C. R.; Liang, K. S.; Ruppert, A. F.; Rothshild, K. J. Nature 1993, 336, 48. (16) Facci, P.; Erokhin, V.; Nicolini, C. Thin Solid Films 1994, 243, 403. (17) Erokhin, V.; Facci, P.; Nicolini, C. Biosens. Bioelectron. 1995, 10, 25. (18) Nicolini, C. Trends Biotechnol. 1997, 15, 395-401. (19) Turko, I. V.; Krivosheev, A. V.; Chaschin, V. L. Biol. Mem. 1992, 9, 529. (20) Kiselyova, O. I.; Guryev, O. L.; Krivosheev, A. V.; Usanov, S. A.; Yaminsky, I. V. Langmuir 1999, 15, 1353. (21) Porter, T. D.; Larson, J. R. Methods Enzymol. 1991, 206, 108. (22) Kastner, M.; Neubert, D. J. Chromatogr. 1991, 587, 43.
Nicolini et al. (Center of Bioengineering, Moscow). Reagents for bacterial growth were from Fluka (Buchs, Switzerland). Emulgen 913 was kindly provided by KAO Chemical (Tokyo, Japan). The hydroxyapatite column was from BioRad. Other chemicals were purchased from Sigma Aldrich (USA). 2.2. Clone for Recombinant P450scc Expression. The cDNA gene of the mature form of P450scc was cloned in the pTrc99A vector23 between the NcoI and KpnI sites to obtain bacterial expression of bovin P450scc (the product of CYPA11A genes). The cDNA gene of the mature protein was obtained by deleting the N-terminal mitochondrial targeting sequence coding the first 39 amino acid residues, using a polymerase chain reaction (PCR).24 The correct orientation of the insert was confirmed by restriction mapping. The data of the sequence obtained on the automatic sequencer “Applied Biosystems” Model 373A 1.2.0 version have been confirmed by the correct sequence of the insert. The E. coli strain JM109 was transformed with P450scc-pTrc99a plasmid following the standard protocol.25 The single colony was inoculated in 10 mL of Luria-Bertani broth containing 50 µg/mL of ampicillin and was grown overnight at 37 °C. The 500 mL of Terrific Broth was inoculated with a 1/100 dilution of this overnight culture in the presence of ampicillin. Isopropyl-R-D-thiogalactopyranoside (IPTG) (final concentration 1 mM) and R-aminolevulinic acid (1 mM) were added when the culture achieved the optical density of 0.6-0.8 at 600 nm. The expression was carried out for 24 h at 30 °C by shaking at 120 rpm. 2.3. Extraction and Purification. The purification of recombinant cytochrome P450scc was performed according to ref 26. Cytochrome P450scc integrity was verified by reduced CO difference spectra.27 The concentration was measured according to ref 28 using an extinction coefficient of 91 mM-1 cm-1 for the absorbance difference between 450 and 490 nm. SDSpolyacrylamide gel electrophoresis was carried out according to ref 28. 2.4. LB Technique. LB films were deposited onto solid substrates using an LB trough (MDT, Russia).16 Water purified with a Milli-Q system (18.2 MΩ cm) was used as a subphase. Protein solutions (1 mg/mL) were spread over the water surface with Hamilton syringes. The films were compressed immediately after spreading with a compression speed of 3.5 mm/s (the trough sizes are 300 mm × 100 mm, volume 250 mL; there is a well in the center for the vertical deposition). Films were transferred onto solid substrates by the Langmuir-Shaefer technique (horizontal lift) at 25 mN/m. Excess water transferred with monolayers was removed by nitrogen flux. The substrates, used for deposition, were silicon, for X-ray and ellipsometric measurements; quartz cuvettes, for optical absorbance and CD measurements; and quartz resonators, for gravimetric measurements. 2.5. Brewster Microscopy. Imaging of monolayers of cytochrome P450 at the air/water interface was carried out with a Brewster angle microscope 2 (Nanofilm Technologie GmbH, Germany). Monolayers were spread at the surface of pure water and compressed with a speed of 50 mm2/min. Images were acquired when compression was stopped. 2.6. Gravimetric Measurements.Gravimetric measurements were carried out by means of a homemade gauge with a sensitivity of 0.57 ( 0.18 ng/Hz using quartz oscillators with a frequency of 10 MHz. Calibration of the quartz balance was performed according to ref 29. 2.7. Ellipsometry. Ellipsometric measurements were performed according to the procedure described in ref 30. A PCSA null ellipsometer with a He-Ne laser source (λ ) 632.8 nm) was used for the measurements. The data were processed according to the two-layer model.31 (23) Amann, E.; Ocha, B.; Abel, J. M. Gene 1988, 69, 301. (24) Ortiz de Montellano, P. R. In Cytochrome P450sStructure, Mechanism and Biochemistry, 2nd ed.; Ortiz de Montellano, P. R., Ed.; Plenum Press: 1995; p 201. (25) Sambrook, J.; Fritsch, E.; Maniatis, T. Molecular Cloning; Cold Spring Harbor Laboratory Press: Plainview, New York, 1989. (26) Wada, A.; Waterman, M. R. J. Biol. Chem. 1992, 267, 22877. (27) Omura, T.; Sato, R. J. Biol. Chem. 1984, 239, 2370. (28) Laemmli, U. K. Nature 1970, 227, 680. (29) Facci, P.; Erokhin, V.; Nicolini, C. Thin Solid Films 1993, 230, 86. (30) Tronin, A.; Dubrovsky, T.; Nicolini, C. Langmuir 1995, 11, 385.
P450scc Engineering and Nanostructuring 2.8. Optical Measurements. The UV-visible spectra of cytochrome P450scc LB films deposited on quartz substrates were obtained with a Jasco 7100 spectrophotometer (Japan). The spectra were recorded between 250 and 500 nm under dry conditions. 2.9. X-ray Scattering. X-ray measurements were carried out with small-angle diffractometer with linear position sensitive detector.30 Cu KR radiation (λ) 0.154 nm) was used. The samples were rotated with respect to the incident beam while the intensity was registered by linear position-sensitive detector. Angular resolution of the detector was 0.01°. The curves were acquired in the 2Θ range of 0.3-2.0°. 2.10. Circular Dichroism Measurements. Circular dichroism (CD) measurements were carried out with a Jasco J-710 spectropolarimeter (Japan). Measurements of CD spectra for samples heated till 95 °C were done at the heating temperature using a Peltier cell (model PCT-343, product Jasco, Japan Spectroscopic Co., Ltd., Tokyo, Japan). Heating of samples over 100 °C was done in a usual laboratory oven. Measurements were carried out at room temperature after cooling the samples. Molar ellipticity was calculated according to ref 14. 2.11. Electrochemical Measurements. The electrochemical measurements were made with a Potentiostat/Galvanostat (EG & G PARC, model 263A) with supplied software (M270). A standard three-electrode configuration was used, where LB films of cytochrome P450scc were deposited on either an ITO coated glass plate or platinum, which acted as a working electrode, with platinum as a counter electrode and Ag/AgCl as a reference electrode. The cyclic voltammetries of such LB films were investigated at 10 mM phosphate buffer, pH 6.8-7.4. The cholesterol solution (10 mM) was dissolved in 15% TritonX-100. 2.12. Molecular Modeling. Interaction of cytochrome P450scc with cholesterol was modeled by studying docking cholesterol to P450scc. Since experimental data on the location of the binding site are highly controversial,33-34 a docking study between P450scc and cholesterol was performed in which the entire sixdimensional P450scc/cholesterol orientation space (three Cartesian coordinates plus three Euler angles) was scanned using the program FTDOCK.35 This choice was dictated by the formidability of the computational task, with only the mentioned program allowing us to achieve the result in a reasonable time (within 15 h of CPU time on a SiliconGraphics O2, for the minimum necessary Euler angle variation step, 20°). The P450scc model was based on the model36 optimized by molecular mechanics. The cholesterol structure was also optimized (in both cases, the DISCOVER software package was used).
3. Results and Discussion 3.1. Pressure-Area Isotherm. π-A isotherms of wild type and recombinant cytochrome P450 monolayers are presented in Figure 1a. Both isotherms were obtained by spreading 10 µL of protein solutions (1 mg/mL), and compressed with a speed of 3.5 mm/s. The X axis is expressed in barrier coordinate units, as it is impossible to calibrate the axis in area per molecule units due to the impossibility of calculating the actual surface concentration of the protein. This problem is general for protein monolayers, and it results from some partial solubility of proteins in the volume of the subphase.37 As it is possible to see in Figure 1a, the isotherms are practically identical, indicating that the surface-active properties of the proteins are more or less similar. (31) Tronin, A.; Dubrovsky, T.; De Nitti, C.; Gussoni, A.; Erokhin, V.; Nicolini, C. Thin Solid Films 1994, 238, 127. (32) Mogilevski, L. Yu.; Dembo, A. T.; Svergun, D. I.; Feigin, L. A. Crystallography 1984, 587, 587. (33) Pikuleva, I. A.; Mackman, R. L.; Kagawa, N.; Waterman, M. R.; Ortiz de Montellano, P. R. Arch. Biochem. Biophys. 1995, 322, 189. (34) Woods, S. T.; Sadleir, J.; Downs, T.; Triantopolous, T.; Headlam, M. J.; Tuckey, R. C. Arch. Biochem. Biophys. 1998, 353, 109. (35) Gabb, H. A.; Jackson, R. M.; Sternberg, M. J. E. J. Mol. Biol. 1997, 272, 106. (36) Vijayakumar, S.; Salerno, J. C. Biochim. Biophys. Acta 1992, 1160, 281. (37) Lvov, Yu.; Erokhin, V.; Zaitsev, S. Biol. Mem. 1991, 4 (9), 1477.
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Figure 1. (a) π-A isotherm of monolayers of wild type (thin line) and recombinant (bold line) cytochrome P450scc at the water surface. (b) Surface potential dependence on the monolayer area for cytochrome recombinant P450scc.
The monolayer film consisting of cytochrome P450scc molecules exhibits a well-defined surface pressure dependence. This is reflected by a change of the molecular orientation of the proteins at the surface. The dependence of the surface potential of the recombinant protein on the area occupied by the monolayer is presented in Figure 1b (a similar dependence was obtained for wild type protein). The π-A isotherm is presented in the same figure for comparison. As it is possible to see from the figure, a sharp increase of the surface potential takes place when the monolayer is far from its dense state (the surface pressure is practically zero). Then, it begins to increase (in negative values) linearly with the increase of the surface pressure. Such behavior of the surface potential suggests that during compression protein molecules begin to interact even at rather long mutual distances. It results in their reorientation at the water surface, coming to the equilibrium state by self-assembly. Further compression just increases the surface density of the layer, resulting in a practically linear increase (in negative values) of the surface potential. 3.2. Brewster Angle Microscopy. Brewster angle microscopy of the monolayer (Figure 2) revealed a homogeneous distribution of the material in the layer (images are similar for wild type and recombinant protein monolayers). This observation indicates the formation of a rather amorphous structure, as no domains were observed. The presence of domains usually means the existence of 2D crystallites38 having distinguishable boundaries between them.
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Figure 4. Dependence of the thickness of the LB film of cytochrome P450scc, both wild type (solid line) and recombinant (dashed line), upon the number of transferred layers. Table 1. Surface Density and Area Per Molecule of Cytochrome P450scc Wild Type and Recombinant in a LB Film Deposited onto a Solid Substrate
Figure 2. Brewster angle microscope image of the monolayer of cytochrome P450scc. The image size is 600 µm × 400 µm. The monolayer was compressed till 20 mN/m.
Figure 3. Dependence of the surface density of deposited wild type and recombinant cytochrome P450scc LB films upon the number of transferred layers.
3.3. Quartz Crystal Nanobalance. The results of the gravimetric study of deposited monolayers are presented in Figure 3 for wild type and recombinant proteins. The linear dependence of the frequency shift upon the number of deposited layers indicates the reproducibility and homogeneity of the deposition. Knowing the molecular (38) Ho¨nig, D.; Mo¨bius, D. J. Phys. Chem. 1991, 95, 4590.
wild type recombinant
surface density (ng/mm2)
area per molecule (nm2)
2.32 3.16
40.23 29.53
weight and dimensions of the protein, it is possible to compare the area per molecule in the film, calculated from gravimetric measurements, with that for the closely packed system. The comparison of the areas of wild type and recombinant proteins in deposited layers is presented in Table 1. As is clear from the table, we have a denser layer in the case of the recombinant protein. The area per molecule in this case (29.53 nm2) is of the same order of magnitude as that obtained earlier in ref 19 and corresponds well to the calculated area per molecule supposing the close packing of molecules and taking into consideration the molecular sizes from the Protein Data Bank. The protein molecule can be estimated as a block with the following sizes: 5 nm × 6 nm × 4 nm, giving, thus, in one cross section the area of about 30 nm2. The value obtained for the wild type protein monolayer is higher, indicating that the closest possible packing was not reached. 3.4. Ellipsometric Study. The results of the ellipsometric study are summarized in Figure 4. As in the case of gravimetric measurements, we see the linear increase of the film thickness upon increase of the number of deposited layers. The average thickness of one monolayer in a case of wild type protein turned out to be about 4 nm while for the recombinant one it is about 6 nm. This fact indicates once more that in the case of recombinant protein a closely packed monolayer was built up. In fact, as the protein sizes are identical in both cases, the decreased thickness of the monolayer in the case of wild type protein can be explained by the presence of some empty regions in the layer. This decreased thickness results from the fact that ellipsometry provides the averaging through the laser beam spot area.
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Figure 6. CD spectra of an LB film of wild type and recombinant cytochrome P450scc.
Figure 5. X-ray patterns of LB films containing 20 layers of recombinant (bold line) and wild type (thin line) cytochrome P450scc. Table 2. Dependence of the Absorbance at 416 nm on the Number of Deposited Layers of Recombinant and Wild Type P450scc no. of layers
A416nm (recombinant)
A416nm (wild type)
5 7 10 15 20 30
0.0041 0.0062 0.0085 0.0131 0.0151 0.0175
0.0028 0.0044 0.0061 0.0093 0.0107 0.0120
3.5. UV-Visible Study. The cytochrome P450scc after deposition presented the characteristic Soret peak indicating the low spin form.5 The linear dependence of the absorbance at 416 nm on the number of deposited layers (Table 2) confirms the reproducible character of the monolayer transfer. 3.6. X-ray Study. X-ray reflection curves are presented in Figure 5 for wild type and recombinant proteins. The curve obtained from the LB film of the wild type protein shows neither Bragg reflection nor Keissig fringes. Such a result means that the film is not ordered and that there is no uniformity of the thickness along the sample area. In a case of recombinant protein, one can see Keissig fringes, whose angular positions depend on the number of deposited layers. The average monolayer thickness, calculated from these data, is about 6 nm, corresponding well both to the ellipsometric data and to the molecular sizes from the Protein Data Bank. 3.7. Circular Dichroism. CD spectra of LB films of wild type and recombinant proteins are presented in Figure 6. The spectra are similar one to the other and to that in the solution, pointing out that denaturation of the protein during the film deposition cannot be considered as a serious effect. Parts a and b of Figure 7 present the variations of the CD spectra of LB films upon heating for
Figure 7. (a) Dependence of the CD spectrum of a wild type cytochrome P450scc LB film upon temperature. (b) Dependence of the CD spectrum of a recombinant cytochrome P450scc LB film upon temperature. (c) Dependence of the CD spectrum of a wild type cytochrome P450scc solution upon temperature.
wild type and recombinant proteins, respectively. The behavior of the spectra upon temperature change is similar. There is practically no effect of heating on the secondary structure of proteins, both wild type and
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Figure 8. Cyclic votammograms of LB films of cytochrome P450scc on an indium tin oxide glass plate (ITO) in 10 mM phosphate buffer, pH 7.4, containing 0.1 M KCl as a function of layers at a scan rate of 20 mV/s between 0.4 and -0.6 V versus Ag/AgCl.
recombinant, in LB films up to 150 °C, while in the solution (Figure 7c) the protein becomes denatured already at 70 °C. Such behavior is typical for protein LB films, and it was reported for other different types of proteins.14-17 Two factors are responsible for such improved heat stability, namely, molecular close packing and low inner water content, as shown by the atomic resolution study of mesophilic versus thermophilic proteins.39 Previously it was shown also that the molecular close packing plays a dominant role in it.17 3.8. Electrochemistry. Protein studies utilizing electrochemical techniques such as cyclic voltammetry have provided insights into the functional properties of redoxactive centers. This study is conducted by exploiting the redox reaction directly at the electrode surface. To increase the voltammetric signal, studies are conducted on an electrode-containing LB film of cytochrome p450 scc, which is immersed in the electrolyte solution. In this study the electrochemistry of cytochrome p450scc was studied in terms of the number of electrons transferred (n), the kinetic parameters, the standard redox potential (Ec) and the pH dependence. Figure 8 depicts the cyclic voltammetric study on LB films of cytochrome P450scc as a function of number of monolayers. The cyclic voltammetry was performed at 20 mV/sc between 0.4 and -0.6 V versus Ag/AgCl. Various numbers of monolayers displayed a cathodic peak potential (-472 to -480 mV) with other peaks varying from -114 to -120, with a small anodic counter peak varying between -144 and -120 mV and a small peak at -268, indicative of a somewhat irreversible process.40 There is a shift in the peak potential as a function of number of monolayers, as shown in Figure 8. Figure 9 shows the dependence of CV on scan rate for 30 monolayer cytochrome P450scc films on an ITO-coated glass plate in 10 mM phosphate buffer, pH 7.4, containing 0.1 M KCl. It shows the cathodic peak potential at -470 and -134 mV with the feeble anodic peak at -312 at -76 mV. The cathodic peak current at -470 mV was plotted with the scan rate, which also suggests an irreversible (39) Bartolucci, S.; Guagliardi, A.; Pedone, E.; De Pascale, D.; Cannio, R.; Camardella, L.; Carratore, V.; Rossi, M.; Nicastro, G.; de Chiara, C.; Nicolini, C. Biochem. J. 1997, 328, 277. (40) Nicolini, C.; Erokhin, V.; Ram, M. K. Supramolecular layer engineering for industrial nanotechnology. In Nanosurface chemistry; Morton Rosoff, Ed.; New York, in press.
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Figure 9. Cyclic votammograms of LB films of cytochrome P450scc in 10 mM phosphate buffer, pH 7.4, containing 0.1 M KCl as a function of scan rate between 0.4 and -0.6 V versus Ag/AgCl. Table 3. Redox Peak Potential of Cytochrome P450scc at Different pH Values of 10 mM Phosphate Buffer phosphate buffer
anodic peak (mV)
cathodic peak (mV)
pH 6.8 pH 6.8 with 0.1 M KCl pH 7.0 pH 7.0 with 0.1 M KCl pH 7.1 with 0.1 M KCl pH 7.4 with 0.1 M KCl
-315, -159 -316, 78.01 -367.7, 189, 238 -342.0, 74.0 -470, -106 -470, -115.5
-306, -206.7 -224.4, 126.6 -44.5, 65.64 -234, 30.0 42 276.0, -17
redox process (figure not shown). It was interesting to see that two cathodic peaks are visible. The change in the redox peak potential can be observed as the pH of the system varies from 6.8 to 7.4 of 10 mM phosphate buffer. Table 3 shows the redox peak potential of cytochrome P450scc at different pH values of 10 mM phosphate buffer. The dependence of the electron transfer on the scan rate for the irreversible process of the absorbed species is described by the expression
Epc ) E0* - (RT/RnF) ln(RnF/RT)(ν/Ks)
(1)
where E0* ) E0′ - 58.6/Rn pH (at room temperature), E0′ is the surface standard potential and represents the standard potential E0 of the redox reaction in solution if the absorbed coefficients of the oxidized and reduced species are equal, R is the transfer coefficient, ν is the number of electrons exchanged, ν is the potential scan rate, and Ks is the heterogeneous rate constant. Upon fitting the scan rate dependence data to eq 1, the value of Rn ) 1.06 has been determined, and following the relationship, E0* and Ks have been obtained. The values of Ks ) 0.45 s-1 and E0* ) -470 mV have been estimated for the cytochrome redox couple at pH 7.4. The standard conditional potential referred to Ag/AgCl corresponds to -220 mV, from which the standard value of E0 ) -250 ( 15 mV for the cytochrome 450scc. A cholesterol oxidation study has also been performed by the CV technique. Figure 10 shows the redox potential of 40 monolayers of cytochrome P450scc on an ITO glass plate in 10 mM phosphate buffer, pH 7.4, containing 0.1 M KCl. It can be seen that when 50 µL of cholesterol solution was added for each time, the redox peaks were well distinguishable, and the cathodic peak at -90 mV was developed besides the development of the anodic peak at 16 mV. When the potential was scanned from 400 to -400 mV, the data point to a possible reaction with
P450scc Engineering and Nanostructuring
Figure 10. Cyclic votammograms of LB films (40 monolayers) of cytochrome P450scc in 10 mM phosphate buffer at a scan rate of 20 mV/s between 0.4 and -0.4 V versus Ag/AgCl. Cholesterol solution was added in increments of 50 µL: (1) without cholesterol, (2) 50 µL, (3) 100 µL, and (4) 150 µL.
cholesterol. It could have been possible to give electrons from the electrochemical process to cytochrome P450scc that in turn reacted with cholesterol. The kinetics of the adsorption and reduction process could have been the result of an ion diffusion controlled process. 3.9. Determination of Cholesterol Side Interactions on the Cytochrome P450 Surface. The quality of the spatial fit between the two molecules was the main criterion for generating the complexes, so only those parts of the P450scc surface were selected that could geometrically accommodate the cholesterol molecule. This resulted in some 3356 possible configurations of the P450scc/cholesterol complex. Importantly, those configurations are not stochastically distributed over the P450scc sequence. Instead, 12 sequence fragments were identified in P450scc at which the center of the cholesterol molecule is preferentially located, namely 57-63, 73-77, 95-98, 131-139, 147-153, 167-177, 194-198, 235-250, 379-386, 409-419, 426-439, and 468-473. These regions cover about 13% of the P450scc surface. At that stage, two more criteria were applied: (1) The cholesterol-binding site should be essentially hydrophobic because cholesterol itself is almost entirely hydrophobic except for a single OH group. This, however, is necessary but not sufficient to identify the functional binding site because cholesterol is known to bind to P450scc in both a productive and a nonproductive manner.41 Accordingly, one more criterion was introduced: (2) The center of the cholesterol molecule should be as close as possible to the heme because experimental data show that, in the productive complex, the C22 atom of cholesterol located near the center of the molecule should be close to the heme iron.42-45 Application of the first criterion left only 4 out of 12 possible regions for cholesterol contact. Those regions comprised the following residues: (1) Pro131, Leu132, Pro135, and the adjacent Ile130, Leu267, and Val424 (region 1); (2) Ile245, Phe246, Leu250, and the adjacent Leu184 (region 2); (3) Ile409, Leu414, Trp418, and the adjacent Phe390 (region 3); (4) Ile468, Phe469, Leu470, Val471, and the adjacent Leu164 (region 4). (41) Tsubaki, M.; Yoshikawa, S.; Ichikawa, Y.; Yu, N. T. Biochemistry 1992, 31, 8991. (42) Sheets, J. J.; Vickery, L. E. J Biol. Chem. 1983, 258, 11446. (43) Nagahisa, A.; Foo, T.; Gut, M.; Orme-Johnson, W. H. J. Biol. Chem. 1985, 260, 846. (44) Seeley, D.; Schleyer, H.; Kashiwagi, K.; Cooper, D.; Salhanick, H. A. Biochemistry 1987, 26, 1270. (45) Vickery, L. E.; Singh, J. J. Steroid Biochem. 1988, 29, 539.
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In view of the second criterion, regions 2 and 4 should be excluded from consideration, as they are too far away from the heme (over 15 Å). The other two regions, 1 and 3, approach the heme iron to about an 8 Å distance and are likely candidates for the cholesterol-binding sites. Region 3 approaches the heme from the side of the B and C porphyrin rings, while region 1 approaches from the A and D rings. Since experimental data suggest that the cholesterol approach is from the A and especially D rings,33 region 1 was the first to be further considered, although region 3 cannot be excluded on this basis only and will have to be considered as well. For further refinement, the cholesterol/P450scc complex among those in region 1 was chosen, in which the cholesterol-heme iron distance was minimal. The complex was subjected to local energy minimization using the program DISCOVER (cholesterol molecule plus P450scc residues within 6 Å, 1000 steepest-descent optimization steps, 400 simplified Newton-Raphson optimization steps, and finally conjugate gradient optimization till convergences3920 steps). Results show that cholesterol is completely and tightly bound to the P450scc surface. Cholesterol penetrates P450scc so that the nearest cholesterol atoms belonging to the main chain are only 4-5 Å from the heme, in the vicinity of the heme’s pyrrole ring D, in agreement with the above-cited experimental data. In the course of optimization, an intermediate structure was identified in which the main chain of cholesterol is even closer to the heme, 3.5-4.5 Å. This shows that the complex obtained at this stage, even if optimal in terms of energy, may not be optimal functionally, so thermal activation is probably needed for full functionality. This kind of activation may in principle be theoretically assessed using molecular dynamics, but only if the conformation-transition rate within the cholesterol/ P450scc complex is high enough (picoseconds to nanoseconds) to allow the use of molecular dynamics (otherwise it is computationally unfeasible). Although the overall turnover number for P450scc/cholesterol is low (about 10 per minute),46 this results from the fact that the reaction is limited by the slow electron transfer from adrenodoxin.47 Generally, although the initial configuration of the P450scc/cholesterol complex was the one with cholesterol contacting mostly with hydrophobic areas of P450scc, the optimized structure lacks this property. Along with the hydrophobic residues Ile126, Ile130, and Val424, cholesterol interacts with the hydrophilic Asn134, Arg421, Gln422, and Cys423 (to which the heme is covalently bound). The only oxygen atom of cholesterol is hydrogenbonded to the terminal nitrogen atoms of Arg421, which no longer interacts with heme propionate D as was the case in the free P450scc. This is in agreement with experimental findings47,48 according to which hydroxylated cholesterol is bound more strongly than nonmodified cholesterol. This, however, does not affect criterion 1 used above, since all those cholesterol derivatives remain strongly hydrophobic. The overall conclusion is that at least one P450scc/ cholesterol complex has so far been identified which may be a precursor of the functionally active complex. Further activities include assessing, by means of molecular mechanics, the other possible cholesterol-binding region (46) Sugano, S.; Morishima, N.; Sone, Y.; Horie, S. Biochem. Mol. Biol. Int. 1995, 35, 31. (47) Tuckey R. C.; Woods, S. T.; Tajbakhsh, M. Eur. J. Biochem. 1997, 244, 835. (48) Shikita, M.; Hall, P. F. J. Biol. Chem. 1973, 248, 5598.
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(region 3 above) and studies of others present in the Brookhaven Databank. 4. Conclusions Metalloproteins can be considered perspective material for bioelectronics.49 The first step toward device applications is the formation of regularly ordered structures from these recombinant materials utilizing monolayer engineering, namely LB and self-assembly techniques. Cytochromes, an essential characteristic of which is the presence of porphyrin rings with a metal inside, are objects of several studies using the LB technique. Cytochrome c, for example, was inserted into LB films by electrostatic interaction with a preformed fatty acid monolayer.11 LB films with cytochrome c were also deposited using reversed micelles as a spreading solution.50 Multiheme cytochromes were also deposited by the LB technique in a complex with photosynthetic reaction centers from Rhodobacter sphaeroides,9,13,14,50 Rhodopseudomonas viridis,50,51 and Chromatium minutissimum.52,53 Cytochrome P450s are a large superfamily of heme/ thiolate enzymes involved in the metabolism of many different toxic compounds and seem to be best among various candidates for bioelectronic reasons. In particular, P450scc (side chain cleavage)2 is a monooxygenase heme protein54 that catalyzes the conversion of cholesterol to pregnenolone by cholesterol side chain cleavage. The P450scc/cholesterol complex has been identified from molecular docking, where the P450scc residues Pro131, Leu132, and Pro135 and the adjacent Ile130, Leu267, and Val424 are involved in the contact with cholesterol. They belong to the proximal side of the P450scc heme, which is the side from which adrenodoxin, the electron-transfer protein, approaches. This is in agreement with the experimental indication54 of synergism between cholesterol binding and adrenodoxin binding to P450scc. Minimization results in penetration of cholesterol molecule into P450scc in such a way as to approach the D ring of the heme, which is in agreement with experiment.33 The structure used in the above cholesterol-binding studies of P450scc was compared to the 3D structures available for other P450s, P450 BM3, P450 2B4, P450 eryF, and P450cam in the distal heme region in which substrate is typically bound. It was found that the volume available for accommodating the cholesterol molecule in that region is at least 70 A3 larger for P450scc than for any of the above-listed cytochrome P450s. This suggests that binding of cholesterol to non-scc P450s is thermodynamically disadvantageous; very high kinetic barriers exist for productive cholesterol binding to those proteins, most likely preventing this binding. Work is currently in progress to estimate the heights of those barriers with theoretical calculations. At this stage, however, even the qualitative results obtained allow us to assume that, among P450s, P450scc is the best choice for analyzing processes related to cholesterol binding and detection. The comparative study, utilizing recombinant protein and wild type P450scc from adrenal cortex mitochondria, revealed similar surface-active properties and secondary structure. The improved thermal stability is also similar (49) Nicolini, C. Thin Solid Films 1996, 284-285, 1. (50) Alegria, G.; Dutton, P. L. Biochim. Biophys. Acta 1991, 1057, 239. (51) Yasuda, Y.; Sugino, H.; Toyotama, H.; Hirata, Y.; Hara, M.; Miyake, J. Bioelectrochem. Bioenerg. 1994, 34, 135. (52) Kayushina, R.; Erokhin, V.; Dembo, A.; Sabo, J.; Knox, P.; Kononenko, A. Biol. Mem. 1991, 4 (11), 1827. (53) Erokhin, V.; Kayushina, R.; Dembo, A.; Sabo, J.; Knox, P.; Kononenko, A. Mol. Cryst. Liq. Cryst. 1992, 221, 1. (54) Hanukoglu, I.; Hanukoglu, Z. Eur. J. Biochem. 1986, 157, 27.
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for LB films built up from these proteins. Marked differences for LB films of wild type and recombinant protein were observed in surface density and thickness of the deposited layer. These differences can be explained by the improved quality of the recombinant sample, attributable to the lack of endogenous cholesterol and other contaminants. In fact, the expression system in E. Coli gives the possibility to obtain the purification product more easily than via the extraction from the adreno-cortex. Moreover, strong treatments are omitted, preserving the enzymatic activity and the structure of the protein. On the other hand, the decreased quality of the wild type sample can disturb the layer formation, preventing closest packing and diminishing the surface density and the average monolayer thickness, and can also be the reason for the poor homogeneity of the deposited layers in the case of wild type protein. The similarity of the behavior of the monolayers of wild type and recombinant proteins is not so easy to understand taking into account the significant differences in the structure of layers, deposited onto solid substrates. However, in the case of recombinant protein all the experimental techniques had provided data consistent with those from the Protein Data Bank. This correspondence together with the film uniformity (X-ray data) allows us to suggest that the layer is composed of protein molecules in a closely packed form. Instead, in the case of wild type protein we observed decreased values of both surface density and thickness. This fact together with the low uniformity of the layer allows us to suggest that the sample can contain some impurities, which have similar surface active properties but change the structure of the resultant layers. An additional reason might be the different biological nature (in vivo and in vitro) and the different synthetic pathways. The cathodic peak current of cytochrome P450scc was found to be pH dependent over 6.8 to 7.4, with the exchange of two protons. The kinetics of the adsorption and reduction process of cytochrome P450scc could have been those of the ion diffusion-controlled process. At present we are using polypyrroles or polyanilines as electron-transfer carriers for the hydroxylation of cholesterol by P450scc for our future work. In conclusion, the quality and performance of the engineered and nanostructured P450scc’s warrant achieving the goals set for optimal biodevice assembly. For sensing, the voltametric measurements of cytochrome P450scc Langmuir-Blodgett films revealed the possibility of cholesterol monitoring by registering variations in cyclic voltamograms. Indeed, the cyclic voltammetric study on LB films of cytochrome P450scc as a function of number of monolayers and of cholesterol concentration points to a possible electron transfer from the electrochemical process to cytochrome P450scc that in turn reacted with cholesterol compatibly with the modeling, showing that the nearest cholesterol atoms belonging to the main chain are only 4-5 Å from the heme. Acknowledgment. This work was supported by the Fondazione El.B.A. and by Polo Nazionale Bioelettronica within the framework of the Programma Nazionale di Ricerche “Biotecnologie Avanzate 2” contract number “Tema 7” sponsored by the Minestry of University and Scientific Technological Research. LA001418D
