Langmuir 1999, 15, 6829-6836
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Cytochrome c Recognition of Immobilized, Orientational Variants of Cytochrome b5: Direct Force and Equilibrium Binding Measurements C. Yeung,† T. Purves,‡ A. A. Kloss,† T. L. Kuhl,§ S. Sligar,‡ and D. Leckband*,† Department of Chemical Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, and Department of Chemical Engineering, University of California at Santa Barbara, California 93106 Received January 8, 1999. In Final Form: May 14, 1999 Direct force measurements, surface plasmon resonance spectroscopy, and genetic manipulations were used to investigate the impact of the orientation of immobilized cytochrome b5 (cyt b5) on its interactions with cytochrome c (cyt c). In this work, we used two cyt b5 orientational variants that present different regions of the protein surface when immobilized. Direct force measurements demonstrated that the two orientations generate a small difference in the electrostatic surface potential of the protein monolayers, in agreement with the calculated electrostatic potential distribution across the protein surface. This difference did not result in any differences in the electrostatic force between cyt c and the cyt b5 variants, however. The measured equilibrium binding constant for the cyt c interaction with cyt b5 also did not depend on the orientation of the latter. These results suggest that, at large separations, cyt c initially interacts relatively nonspecifically with a large patch of negative charge on the cyt b5. At short separations, it then adopts the optimum relative orientation for electron transfer. The force measurements not only elucidated the molecular basis of the equilibrium binding behavior, but also the possible molecular mechanisms that govern the interactions between these proteins in solution.
Introduction Immobilized proteins and microorganisms are used in bioreactors,1,2 affinity chromatography,3,4 immunoassays,5,6 biosensors,7,8 clinical analysis and diagnostics,7-9 and environmental monitoring.10 One of the main concerns regarding the use of immobilized proteins is the loss of biological activity upon immobilization. This loss is attributed to several causes, which include the immobilization chemistries used, conformational changes in the immobilized proteins, random immobilized orientations, or the influence of the microenvironment. The orientation of bound macromolecules, in particular, affects their apparent biological activity on surfaces.11-14 Selectively oriented proteins are attractive for many * Address correspondence to this author. † Department of Chemical Engineering, University of Illinois at Urbana-Champaign. ‡ Department of Biochemistry, University of Illinois at UrbanaChampaign. § Department of Chemical Engineering, University of California at Santa Barbara. (1) Fodor, S. P. A.; Read, L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 767-773. (2) Faulkner, K. M.; Shet, M. S.; Fisher, C. W.; Estabrook, R. W. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7705-7709. (3) De Ruiter, G. A.; Smid, J. H.; Van, B.; Rombouts, F. M. J. Chromatogr. 1992, 584, 69-75. (4) Hage, D. S.; Kao, P. C. Anal. Chem. 1991, 63, 586-595. (5) Sisson, T. H.; Castor, C. W. J. Immunol. Methods 1990, 127, 215. (6) Suri, C. R.; Jain, P. K.; Mishra, G. C. J. Biotechnol. 1995, 39, 27. (7) Arnold, M. A.; Meyerholf, M. E. CRC Crit. Rev. Anal. Chem. 1988, 20, 149-196. (8) Schultz, J. S. Sci. Am. 1991, 265, 64-69. (9) Guilbault, G. G.; Mascini, M.; Reidel: Boston, 1988. (10) Wiseman, A. J. Chem. Technol. Biotechnol. 1993, 56, 3. (11) Darst, S. A.; Robertson, C. R.; Berzofsky, J. A. Biophys. J. 1988, 53, 533-539. (12) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. Soc. 1991, 113, 18471849. (13) Hamachi, L.; Noda, S.; Kunitake, T. J. Am. Chem. Soc. 1991, 113, 9625-9630.
practical applications because of the high reproducibility of protein film construction, the potential for structural and functional studies, and the retention of biological activity through the controlled presentation of the binding site. The fabrication and characterization of selectively oriented protein films has been the focus of considerable research.15-19 For example, by engineering genetically the location of reactive cysteines on the surface of cytochrome b5, Stayton et al.19 controlled the protein’s orientation when bound to silanized glass slides. Such macromolecular films are also attractive for structural and functional studies of protein-protein interactions. The orientation of immobilized proteins determines not only the accessibility of their binding sites, but also the surface of the protein exposed to the medium. Both can alter the molecular force fields that control the trajectory of soluble ligands to the active site, and thereby affect the apparent biological activity of the bound species. However, because of the technical difficulties involved, there have been few attempts to determine the orientational dependence of protein interactions.20 These studies investigated the effects of the orientation of immobilized cytochrome b5 (cyt b5) on the molecular forces that control its interactions with cytochrome c (cyt (14) Lin, J. N.; Chang, I. N.; Andrade, J. D.; Herron, J. N.; Christensen, D. A. J. Chromatogr. 1991, 542, 41-54. (15) Firestone, M. A.; Shank, M. L.; Sligar, S. G.; Bohn, P. W. J. Am. Chem. Soc. 1996, 118, 9033-9041. (16) Darst, S. A.; Ahlers, M.; Meller, P. H.; Kubalek, E. W.; Blankenburg, R.; Ribi, H. O.; Ringsdorf, H.; Kornberg, R. D. Biophys. J. 1991, 59, 387-396. (17) Rao, S. V.; Anderson, K. W.; Bachas, L. G. Mikrochimica. Acta 1998, 128, 127-143. (18) Song, S.; Clark, R. A.; Bowden, E. F.; Tarlov, M. J. J. Phys. Chem. 1993, 97, 6564-6572. (19) Stayton, P. S.; Olinger, J. M.; Jiang, M.; Bohn, P. W.; Sligar, S. G. J. Am. Chem. Soc. 1992, 114, 9298-9299. (20) McLean, M. A.; Stayton, P. S.; Sligar, S. G. Anal. Chem. 1993, 65, 2676.
10.1021/la990019j CCC: $15.00 © 1999 American Chemical Society Published on Web 08/03/1999
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c). Genetic techniques made it possible to control explicitly the orientation of cyt b5 bound to supported planar bilayers. The affinities of soluble cyt c for immobilized cyt b5 were characterized by surface plasmon resonance. We then used direct force measurements to assess the effects of cyt b5 orientation on the long- and short-range forces that control, respectively, the rates and strengths of the heterologous protein interaction. For the two cyt b5 variants investigated, the similarities in the forces controlling their interaction with cyt c correlate directly with the measured equilibrium binding of the latter protein to cyt b5 monolayers. Our findings provide direct evidence for the molecular basis of the observed protein adsorption behavior, and they provide insights into mechanisms by which these proteins may interact in solution. Materials and Methods Materials. 1,2-Ditridecanoyl-sn-glycero-3-phosphocholine (DTPC) was purchased from Avanti Polar Lipids (Eugene, OR). Iodoacetyl-LC-biotin (LC-biotin), ImmunoPure AffinityPak Immobilized Avidin Columns, and N-chlorobenzenesulfonamide (sodium salt) derivatized nonporous polystyrene beads (IODOBeads) were purchased from Pierce (Rockford, IL). N-((6(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (biotin-X DHPE), was purchased from Molecular Probes. 1-{8-[4-(p-Maleimidophenyl)butaroylamino]-3,6-dioxaloctyl}-2,3-distearyl glycerol-dl-ether (MPB-DSGE) was purchased from Northern Lipids, Inc. (Vancouver, BC). Streptavidin was from Calbiochem. Ethyl alcohol (dehydrated-200 proof) was from McCormick. NaH2PO4, Na2HPO4 (purity > 99.95%), and hexamethyldisilazane (purity >97%) were purchased from Aldrich. dl-dithiothreitol (DL-DTT, purity >99%) was from Sigma. Sodium hydroxide (NaOH) and ethylenediaminetetraacetic acid (EDTA) was from Fisher. Prepacked Sephadex G-25 columns (PD-10) were from Pharmacia Biotech (Piscataway, NJ). Millex-GV syringe filters (0.22 µm) were from Millipore. All aqueous solutions were prepared with water purified with a Milli-Q UV Plus water purification system from Millipore. Water resistivity was greater than 18 MΩ‚cm. Methods. Construction of Oriented Monolayers of Cyt b5 Mutants. To control the immobilized protein orientation, two variants, T8C and T65C, of the soluble fragment of rat liver cyt b5 were used in this study. In these proteins, a threonine was substituted for a single cysteine either at position 8 or at position 65.21 Immobilization via the reactive thiol group on the cysteine determines the orientation of the cyt b5 on the substrates, and therefore the different surface regions of the protein exposed to the medium. The cyt b5 variants were covalently attached to supported lipid monolayers (see below) through the formation of a stable thioether linkage between the engineered sulfhydryl on the protein surface and the maleimide group on the functionalized lipid, MPBDSGE.22 The supported, reactive lipid monolayers were prepared by spreading a chloroform solution of either 2 mol % or 10 mol % MPB-DSGE in a neutral matrix lipid DTPC on the water surface of the Langmuir trough (Nima, type 611). After allowing 5 min for solvent evaporation, we compressed the lipid layer to a target pressure of 30 mN/m (65 Å2/lipid). The monolayer was then transferred by Langmuir-Blodgett deposition at constant pressure onto a hydrophobic solid substrate, by passing the latter vertically through the monolayer into the subphase. For surface force measurements, the hydrophobically modified substrate was a crystalline, Langmuir-Blodgett monolayer of dipalmitoyl phosphatidylethanolamine (DPPE) on mica. Silanized glass slides were used in linear dichroism (LD) measurements, and paraffincoated silver films on glass were used for surface plasmon resonance (SPR) measurements. The mutants were immobilized by incubating reduced cyt b5 for at least 3 h, at room temperature, with MPB-DSGE(21) Stayton, P. S.; Fisher, M. T.; Sligar, S. G. J. Biol. Chem. 1988, 263, 13544-13548. (22) Smyth, D. G.; Blumenfeld, O. O.; Konigsberg, W. Biochem. J. 1964, 91, 589.
Yeung et al. containing membranes. The buffer contained 50 mM phosphate and 0.1 mM EDTA at pH 7.2. Before protein coupling to the maleimide, the incubation of the protein with a 50-fold excess of fresh DTT for at least 40 min reduced any disulfide links between cyt b5 molecules. Gel filtration on a PD-10 column that was equilibrated with the coupling buffer (50 mM phosphate with 0.1 mM EDTA at pH 7.2) then removed excess DTT. EDTA was included in the coupling buffer to prevent the oxidation of free sulfhydryls.23 The reduced protein was filtered immediately with a 0.22-µm Millex-GV syringe filter (Millipore), and then added to the reactive supported lipid membrane. The final protein concentration in the incubation solution was 5 µM. Afterward, the samples were rinsed thoroughly with the coupling buffer and mounted either in the SPR cell or in the surface force apparatus (SFA). For the LD and SFA experiments, the immobilization substrate contained 10 mol % MPB-DSGE lipid, whereas a 2 mol % MPB-DSGE lipid monolayer was used in SPR measurements. The surface coverages of the immobilized cyt b5 variants were determined from the amount of 125I-labeled protein bound to the supported bilayers.24 Construction of Oriented Cyt c Monolayers. Yeast cyt c (Sigma) was used directly without further purification. Cytochrome c was first labeled with a biotin at position 102 using IodoacetylLC-Biotin (LC-biotin) according to procedures described previously.24 The biotinylated protein was then immobilized to a supported, oriented monolayer of streptavidin from a 50 mM phosphate solution at pH 6.0. The streptavidin monolayer was prepared according to established procedures.16,25 Previous reports showed that yeast cyt c attached to thiol-reactive monolayers via cysteine 102 forms an oriented array with the heme directed 40° relative to the surface normal.26 The binding site is fully accessible in this configuration. In this study, we attached cyt c to streptavidin, rather than directly to MPB-DSGE membranes, because of both the lowered nonspecific adsorption and the greater selective binding efficiency achieved. The 30 ( 3% cyt c surface coverage was determined by SPR. The cyt c refractive index of 1.62 was determined with an Abbe refractometer. Determination of the Immobilized Cyt b5 Orientation by LD Spectroscopy. The orientation of the heme in the cyt b5 relative to the plane of the substrate was determined by LD spectroscopy.15 The characteristic Soret absorption band of the heme at 410 nm was the spectroscopic probe in these measurements. LD measurements were conducted with a home-built rotation stage mounted in a Hitachi U-3000 spectrometer. Placing two polarizers (Oriel) with the same polarization in the incident light paths of both the sample and reference beams achieved the linear polarization of both the detection and the reference beam. The absorption spectra were then measured from 390 to 450 nm under orthogonal s and parallel p polarized light. Spectra were obtained at a scanning speed of 30 nm/min, and the background signal, obtained with blank samples without protein, was subtracted. The absorption band was integrated using a rectangular integration method to obtain the peak intensity. The error in the measured band intensity was (5%. Monolayers of cyt b5 variants were prepared for these measurements as follows. Glass slabs (8 × 30 mm) were cut from regular glass slides, and cleaned thoroughly with a 1:10 methanol: NaOH solution. The glass substrates were exposed to hexamethyldisilazane vapor for 8 h to render them hydrophobic. The treated glass exhibited advancing and receding water contact angles greater than 100°. Monolayers containing 10 mol % MPBDSGE and 90 mol % DTPC were then deposited onto the latter hydrophobed substrates by Langmuir-Blodgett deposition. The proteins were immobilized to these substrates as described above. The supported protein monolayers were then transferred under water to a quartz cuvette, and the cuvettes were mounted on the rotation stage of the spectrometer. (23) Yoshitake, S. Y., Y.; Ishikawa, E.; Masseyeff, R. Eur. J. Biochem. 1979, 18, 395-399. (24) Yeung, C.; Leckband, D. Langmuir 1997, 13, 6746-6754. (25) Helm, C. A.; Knoll, W.; Israelachvili, J. N. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 8169-8173. (26) Wood, L. L.; Cheng, S. S.; Edmiston, P. L.; Saavedra, S. S. J. Am. Chem. Soc. 1997, 119, 571-576.
Cyt c Recognition of Variants of Cyt b5 SPR Measurements. SPR measurements characterized the equilibrium binding of soluble horse heart cyt c to immobilized, oriented cyt b5 monolayers. Horse heart cyt c lacks the naturally occurring surface cysteine at position 102 that can bind directly to the maleimide functionalized lipid monolayers. The measurements were conducted with a home-built instrument based on the Kretschmann configuration.27 The instrument uses a miniature GaAs laser (5 mW, 665 nm) as a light source, a precision goniometer (Oriel) driven by a stepper motor (Arrick) for the sample rotation stage, an equilateral triangle prism (Mellis Griot), and a large area Si photodiode detector. The readout of the photodiode detector current and the position of the stepper motor are recorded using a commercial computer program. The detector monitors the intensity of the beam reflected from the sample (Ir) as a function of the external angle of incidence θ. From this, one obtains the resonance curves. The shifts in the resonance angle are then monitored continuously during the adsorption process. A small volume flow cell, which uses regular glass slides as the substrate for the metal coating and sample construction, houses the samples. In this setup, the glass slides are in optical contact with the prism via an index-matching fluid. The shifts in the resonance angles were converted to changes in the optical thickness, and hence the film thickness, with a commercial program purchased from the Institute of Semiconductor Physics (Kiev, Ukraine). Experiments were conducted at room temperature. The buffer solution contained 5 mM or 50 mM phosphate at pH 7.2. The cyt b5 variants were immobilized on 2 mol % MPB-DSGE monolayers according to the procedures described above. To determine the equilibrium binding constants, a series of cyt c solutions with concentrations between 1 and 15 µM were injected into the flow cell housing the immobilized cyt b5 samples, and the equilibrium amount of cyt c adsorbed on the cyt b5 monolayers was determined. Flushing the cell with 4-5 cell volumes of the protein solution fully equilibrated the system. To determine the relative amounts of specific versus nonspecifically adsorbed cyt b5 on the MPB-DSGE lipid monolayers, we first measured the nonspecific adsorption of cyt b5 T8C dimers, which lack free thiols, onto the lipid monolayers. The bulk cyt b5 concentration in these measurements was 5 µM. Surface Force Measurements. We used a Mark II SFA28 to quantify the impact of the cyt b5 orientation on the forces between opposed monolayers of cyt c and cyt b5. With the SFA, one measures the forces between monomolecular films as a function of their separation distance. The forces are measured with a sensitive leaf spring with 10 nN force resolution, and the intersurface distances are determined with a resolution of (1 Å by optical interferometry.29 In these experiments, the samples were bathed in a solution of 5 mM phosphate buffer at pH 6.5 and 25 °C. Because this is near the isoelectric point of streptavidin, the lower pH reduces the background influence of the streptavidin charge.24 Although this differs from the pH used in the SPR measurements, the change only alters the dissociation constant by a factor of 2.30 This will not affect the comparison of the properties of the immobilized, orientational variants because the relative differences or similarities in their properties will not depend on the pH 3sthat is, they will only depend on the protein orientations. All buffer solutions were saturated with DTPC, and filtered through 0.1-µm hydrophilic Durapore filters (Millipore). To minimize protein denaturation during the course of the experiment, all experiments were completed within 12 h of sample preparation. Placing a 500-nm high-pass cutoff filter in the incident light path also prevented the photolysis of the heme due to the strong Soret absorbance band at 410 nm.
Langmuir, Vol. 15, No. 20, 1999 6831 Table 1. Measured Surface Coverage and Orientation of Cyt b5 on Supported 10 mol % MPB-DSGE Lipid Monolayers cytochromes Cyt b5 T8C Cyt b5 T65C
area/binding area/protein fractional heme tilt site (Å2/site) (Å2/cyt) coverage angle 650 650
1650 ( 200 1700 ( 200
46% 46%
78 ( 1° 45 ( 3°
used to immobilize the cyt b5 was reported previously.24 Under identical reaction conditions, and with the same chemical coupling groups, the two cyt b5 variants exhibit similar surface coverage. Table 1 shows that the measured surface densities of the T8C and T65C mutants on 10% MPB-DSGE monolayers are essentially identical. The 46% surface coverage was calculated by comparing the measured average area per protein with that of a close-packed protein monolayer (780 Å2/protein).31 The lateral repulsion between the charged proteins is the likely cause for the less-than-full monolayer coverage achieved. SPR measurements showed similarly that the biotinylated cyt c on streptavidin monolayers occupies 30 ( 2% of the surface area (data not shown). This corresponds roughly to one protein molecule bound to each streptavidin. Cyt b5 Orientation by LD. To investigate the dependence of selective cyt c adsorption on the orientation of the immobilized cyt b5, one requires a homogeneously oriented protein monolayer. Control adsorption measurements indicated that nonspecifically bound cyt b5 accounts for less than 5% of the total protein bound. LD measurements, carried out according to published procedures,32 then verified the different orientations of the specifically bound cyt b5 variants. If the dipole moments µ1 and µ2 in the porphyrin ring are circularly polarized, and the molecular orientation is azimuthally isotropic, then the measured dichroic ratio is33:
F)
∫Asdλ 1 ) 2 R sin2 θ 2 sin ∫Apdλ cos2 R + 2
(1)
2 - sin θ
Results Surface Coverage of Immobilized Cyt b5 and Cyt c. The coupling efficiency of the thiol-maleimide linkage
Here R is the angle between the incident beam and the substrate normal, and θ is the angle between the heme plane normal and the substrate normal. One thus measures the dichroic ratio as a function of the incident angle, R, and determines the angle θ from fits of the data to eq 1. The dichroic ratios for the proteins immobilized on 10 mol % MPB-DSGE lipid monolayers were determined over a range of incident angles (Figure 1). To determine the average heme tilt angle for each cyt b5 variant, we fit the data to eq 1, using a nonlinear curve-fitting algorithm. These results are summarized in Table 1. The average angle of the heme in cyt b5 T8C is 78 ( 3°, with respect to the substrate horizontal. This corresponds to the almost vertical orientation of the porphyrin ring (Figure 2A). The error represents only the uncertainties in the data fitting, and not the actual distribution of the orientation. The latter cannot be determined from LD measurements alone.34,35 Our measured value, however, agrees with the molecular model of immobilized cyt b5 T8C (Figure 2A), and with previous results.19 The orien-
(27) Kretschmann, E.; Raether, H. Z. Naturforsch. 1968, Teil A 23, 2135-2136. (28) Israelachvili, J.; Adams, G. E. J. Chem. Soc., Faraday Trans. I 1978, 74, 975-1001. (29) Israelachvili, J. N. J. Colloid Interface Sci. 1973, 44, 259-272. (30) Mauk, M. R.; Reid, L. S.; Mauk, A. G. Biochemistry 1982, 21, 1843-1846.
(31) Mathews, F. S.; Levine, M.; Argos, P. J. Mol. Biol. 1972, 64, 449-464. (32) Xu, Z.; Lau, S.; Bohn, P. W. Surf. Sci. 1993, 296, 57-66. (33) Yoneyama, M.; Sugi, M.; Saito, M.; Ikegami, K.; Kurod, S.; Iizima, S. Jpn. J. Appl. Phys. 1986, 25, 961-969. (34) Bos, M. A.; Kleijn, J. M. Biophys. J. 1995, 68, 2566-2572. (35) Lee, J. E.; Saavedra, S. S. Langmuir 1996, 12, 4025-4032.
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Figure 1. Linear dichroism measurements with immobilized cyt b5 variants T8C and T65C. The proteins were immobilized on 10 mol % MPB-DSGE monolayers. Solid lines represent the predicted changes in the dichroic ratio as a function of the incident angle, as the normal to the heme plane tilts relative to the surface normal. The filled circles indicate the data obtained with the T8C mutant. Data obtained with the T65C mutant are indicated by the open triangles. The solid lines represent the best nonlinear least-squares fits to the data to eq 3 with the best-fit parameters given in the text.
Yeung et al.
Figure 3. Force vs distance profiles between identical cyt b5 monolayers. Reduced cyt b5 (T8C and T65C) was immobilized on lipid monolayers containing 10 mol % MPB-DSGE. The forces were measured between identical protein monolayers in 5 mM phosphate buffer at pH 7 and 25 °C. The filled circles indicate the data obtained with the cyt b5 (T8C). The solid line is the best visual fit of the data to DLVO theory, and was calculated with the constant surface charge density of -6.0 ( 0.5 mC/m2, and a Debye length of 40 Å (N ) 3). The open circles show the data obtained with the T65C mutant. The dotted line through the open circles was calculated with a constant surface charge density of -7.5 ( 0.5 mC/m2, and a Debye length of 40 Å (N ) 2). The Hamaker constant used in both fits was 10-22 J. Table 2. Measured Electrostatic Properties of Cyt b5 Monolayers as a Function of the Protein Orientationa Cyt b5 variant
salt conc (mM)
surface potential (mV)
charge density (mC/m2)
normalized charge (e/protein)
Cyt b5 T8C Cyt b5 T65C
6.4 6.4
-31 ( 2 -38 ( 2
-6.0 ( 0.5 -7.5 ( 0.5
-0.6 ( 0.1 -0.8 ( 0.1
a The cytochrome b variants T8C and T65C were immobilized 5 on DTPC monolayers containing 10 mol % MPB-DSGE. Force measurements were performed at 25 °C. the solution contained 5 mM phosphate buffer at pH 7, and was saturated with DTPC monomers.
Figure 2. Structural models of the oriented, immobilized cyt b5 variants T8C (A) and T65C (B). The protein backbone is shown as a worm. The heme is shown by solid, gray van der Waals spheres. Residues 8 and 65 are shown as white van der Waals spheres at the base of the protein. The residues shown in black indicate the putative binding site for cyt c.
tation of cyt b5 T8C exposes the binding site to the medium and allows for easy cyt c access (Figure 2A). The fitted tilt angle of 45 ( 1° for T65C on 10 mol % MPB-DSGE membranes (Table 1) was also in good agreement with the model of the bound protein (Figure 2B). In this case, the cyt b5 binding site is also accessible, but it is oriented away from the surface normal. This exposes a different surface to the medium, and it could also generate additional steric impediments for cyt c binding. Electrostatic Properties of Oriented Cyt b5 Monolayers. To characterize the effect of the immobilized cyt b5 orientations on the electrostatic properties of the protein monolayer, we measured the forces between two cyt b5 films.24 Figure 3 shows the force vs distance profile between cyt b5 T8C monolayers on 10 mol % MPB-DSGE lipid membranes in 5 mM phosphate buffer at pH 7. The zero intersurface separation, D ) 0 Å, corresponds to steric contact between the dehydrated lipid headgroups of the supporting bilayers. The thickness of the entire molecular assembly was determined from the change in the distance
of closest intersurface approach after removal of the organic films from the mica surfaces.36,37 We then defined the position of the outer lipid headgroups on the basis of the experimentally determined thicknesses of the crystalline DPPE and fluid DTPC monolayers.37,38 The 30-Å steric thickness of the protein layer, determined by subtraction of the thickness of the lipid bilayer from the overall layer thickness, agrees with the known protein diameter. Upon approach, a long-range, exponentially decaying repulsive force between the cyt b5 T8C monolayers predominates at D > 60 Å (Figure 3). The decay length agrees with the Debye length calculated for the buffer concentration used.39 The steep upturn in the force curve and hard wall repulsion at 55 Å corresponds to the steric force between two slightly overlapping cyt b5 monolayers.31 The electrostatic surface charge density of the film was determined by fitting visually the measured double-layer force to a profile calculated with the nonlinear PoissonBoltzmann equation39,40 at D > 60 Å. The best-fit parameters are summarized in Table 2. With the T8C variant, we obtained a surface potential of -31 ( 2 mV and a surface charge density of -6.0 ( 0.5 mC/m2. The effective charge per protein, determined by normalizing (36) Leckband, D. E.; Schmitt, F. J.; Israelachvili, J. N.; Knoll, W. Biochemistry 1994, 33, 4611-4624. (37) Marra, J.; Israelachvili, J. Biochemistry 1985, 24, 4608-4618. (38) Hauser, H.; Pascher, I.; Pearson, R. H.; Sundell, S. Biochim. Biophys. Acta 1981, 650, 21-51. (39) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: New York, 1992. (40) Hunter, R. J. Foundations of Colloid Science; Clarendon Press: Oxford, UK, 1989.
Cyt c Recognition of Variants of Cyt b5
Langmuir, Vol. 15, No. 20, 1999 6833 Table 3. Measured Attractive Force Between Cyt c and Cyt b5 Variants, T8C and T65C
orientation Cyt b5 T8C Cyt b5 T65C
Figure 4. Force versus distance profile for interactions between cyt B5 and cyt c monolayers. Measurements were obtained with cyt b5 T8C (filled circles, N ) 2) or with cyt b5 T65C (open circles, N ) 2) immobilized on 10 mol % MPB-DSGE lipid monolayers. The biotinylated cyt c was immobilized on a supported, oriented streptavidin monolayer. Measurements were conducted at 25 °C in 5 mM phosphate buffer at pH 6.5. The solid line through the data is merely to guide the eye. The arrow indicates the position at which the bonds yield and the surfaces jump apart.
the surface charge density by the protein coverage, was -0.6 ( 0.1 charge/T8C. Here, the sign of the electrostatic potential and charges were assumed negative because the cyt b5 is negatively charged at pH 7. The force profile between cyt b5 T65C monolayers at similar coverages and under identical conditions was similar to that observed with the T8C variant. The fits of the data gave a slightly larger surface potential of -38 ( 2 mV and a surface charge density of -7.5 ( 0.5 mC/m2 (Table 2). The corresponding charge per protein is -0.8 ( 0.1 (Table 2). The magnitudes of the measured charge densities are lower than one might expect based on the overall charge on the protein. The fits, however, give the effective charge density in a plane tangent to the outer protein surface. Electrostatic screening does attenuate the magnitude of the apparent charge density projected onto this plane. It is also possible that the neighboring charged proteins alter the local pK values of the amino acid side chains. However, other similar measurements of the charge densities of streptavidin monolayers were performed at much higher (∼80%) protein coverage. The values obtained were in very good agreement with the calculated charge in the same plane adjacent to the streptavidin surface.41 Forces Between Oriented Cyt b5 Mutants and Cyt c Monolayers. We used the SFA to quantify the effect of the immobilized cytochrome b5 orientation on the interaction with oriented cyt c. Here the force-distance curves were measured between cyt b5 on an MPB-DSGE lipid membrane and cyt c immobilized on a streptavidin monolayer. Experiments were conducted in 5 mM phosphate buffer at pH 6.5 and 25 °C. The force vs distance profile between cyt b5 T8C and cyt c monolayers is shown in Figure 4. The separation distance D ) 0 Å corresponds to steric contact between the outer streptavidin surface and the 10 mol % MPB-DSGE lipid bilayer. Upon approach, the force profile was purely repulsive. This was attributed to the double-layer force between the negatively charged streptavidin and cyt b5 monolayers. The hard wall repulsion between the two compressed protein films was at 20 Å, and corresponded to fully interdigitated and slightly compressed protein monolayers. Because of the low surface coverage of cyt c (30%) and (41) Sivansankar, S.; Subramanianm, S.; Leckband, D. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12961-12966.
jump-off adhesive repulsive total attractive position force at force at force at Dj (Å) Dj (mN/m) Dj (mN/m) Dj (mN/m) 52 ( 5 55 ( 5
-0.3 ( 0.1 -0.5 ( 0.2
0.8 ( 0.1 0.8 ( 0.1
-1.1 ( 0.2 -1.3 ( 0.2
that of cyt b5 (46%), the interdigitation of the two protein films at modest compression was expected. During separation, the proteins first moved apart to 50 Å. At the latter distance, the interprotein bonds yielded, and the surfaces began to slowly jump apart. When the gradient of the applied force exceeds the spring constant, the materials jump out of contact.37 The final detachment was from D ∼ 70 Å (Table 3). The point at which the attractive forces first lock in corresponds to partially overlapping cyt c and T8C monolayers. We attribute the sluggish outward movement to 70 Å to the stretching of the anchoring spacers. The adhesive force, which is the tensile force required to separate the two protein monolayers, was -0.3 ( 0.1 mN/m (Table 3). The total attractive force of -1.1 ( 0.2 mN/m (Table 3), which is the difference between the net repulsive and adhesive force at the yield point (Figure 4), gives the strength of the short-range cyt b5-cyt c attraction. The forces between cyt b5 T65C and cyt c were then measured under identical conditions (Figure 4). Within experimental error, the magnitude of the long-range electrostatic repulsion was the same as measured with the T8C variant. The final compressed protein layer thickness was also ∼20 Å, and the detachment behavior was similar to that observed with T8C variant. The measured adhesive force was -0.5 ( 0.2 mN/m, and the total attractive force was -1.3 ( 0.2 mN/m (Table 3). Within experimental error, the tensile strengths of the cyt b5-cyt c complexes are the same. In none of these cases did the adhesive junction fail by pulling the lipid anchors out of the supporting membranes.42 This is not surprising because the equilibrium binding constants are sufficiently low30 that the estimated bond rupture force should be smaller than the lipid pullout force.42,43 Thus, the attractive forces were attributed to the rupture of the protein complex. Adsorption of Horse Heart Cyt c onto Monolayers of Oriented Cyt b5 Mutants. SPR measurements were used to determine whether the similarity in the measured interprotein attractive forces in fact generate similar equilibrium binding between soluble horse heart cyt c and the immobilized cyt b5 variants. For a first-order reaction between the soluble cyt c and immobilized cyt b5, the rate equation can be expressed in terms of the % surface coverage of cyt c, R,
dR/dt ) ka[c](Rmax - R) - kdR
(2)
Here [C] is the concentration of the injected solution of cyt c, ka, and kd are the association and dissociation rate constants, respectively, and Rmax is the maximum % coverage at saturated cyt c binding. At equilibrium, dR/dt ) 0, and eq 2 can be rearranged to
K)
R [c](Rmax - R)
(3)
(42) Leckband, D.; Muller, W.; Schmitt, F. J.; Ringsdorf, H. Biophys. J. 1995, 69, 1162-1169. (43) Bell, I. G. Science 1978, 200, 618-627.
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Yeung et al.
Figure 5. Double-reciprocal plot horse heart cyt c binding to immobilized cyt b5 monolayers. Data obtained with cyt b5 T65C (filled circles) and T8C (open circles) were measured at pH 7.2 in 50 mM phosphate buffer at 25 °C. The solid lines through the data are the linear least-squares fits of the data to eq 4. The affinity constants, obtained from the slopes, for the immobilized cyt b5 T8C and cyt b5 T65C are given in Table 4.
Here we introduce the affinity constant K ) ka/kd (M-1). The affinity constant can therefore be obtained by measuring the equilibrium coverage R as a function of the cyt c concentration [c]. For this purpose it is convenient to write eq 3 as a reciprocal dependence44:
1 1 1 ) + R Rmax KRmax[c]
(4)
By plotting 1/[R] vs 1/[c] (double-reciprocal graph) one obtains the maximum coverage Rmax from the intercept of the linear dependence. Then, the affinity constant K is calculated from the slope. This analysis assumes that the binding between cyt c and cyt b5 follows pseudo first-order kinetics. We verified this by monitoring the adsorption kinetics of horse heart cyt c binding to immobilized cyt b5. In these experiments, we used 2 mol % MPB-DSGE to immobilize the cyt b5 to minimize the amount of excess maleimide, which can hydrolyze to maleic acid. The resulting negative charges will promote the nonspecific adsorption of positively charged cyt c and cause deviations from pseudo first-order behavior.45 SPR measurements demonstrated that the affinities of cyt c for the two cyt b5 variants are the same in 5 mM buffer, but differ by a factor of 1.5 in 50 mM buffer. To demonstrate this, the equilibrium amount of horse heart cyt c adsorbed to immobilized cyt b5 monolayers was measured over the specified range of bulk cyt c concentrations. We then constructed double-reciprocal plots as described above for cyt c adsorption both in 5 mM and in 50 mM buffer. The results of the measurements at the higher ionic strength and pH 7.2 are shown in Figure 5. The affinity constants, determined by linear regression analysis of the data shown in the figure, gave values of (44) Surface Analytical Techniques for Probing Biomaterial Pricesses; Davies, J., Ed.; CRC Press: Boca Raton, 1996. (45) Jiang, M., University of Illinois at Urbana-Champaign, 1995.
Table 4. Measured Affinity Constants of Horse Heart Cytochrome c Binding to Immobilized Cytochrome B5 Monolayers
Cyt b5 variant Cyt b5 T8C Cyt b5 T8C Cyt b5 T65C Cyt b5 T65C
measured measured phosphate affinity Cyt b5 binding free coverage concentration constant K energy -∆G (%) (mM) (105 M-1) (kT) 24.1 20.1 35.3 29.0
50 5 50 5
2.2 ( 0.3 5.8 ( 0.4 1.5 ( 0.2 5.3 ( 0.8
12.3 ( 0.1 13.3 ( 0.07 11.9 ( 0.1 13.2 ( 0.1
K ) (2.2 ( 0.3) × 105 M-1 and K ) (1.5 ( 0.2) × 105 M-1 for T8C and T65C, respectively. The results are summarized in Table 4. By contrast, the determined affinity constants for T8C and T65C measured in 5 mM phosphate buffer are K ) (5.8 ( 0.4)× 105 M-1 and K ) (5.3 ( 0.8)× 105 M-1 for T8C and T65C, respectively. Within experimental error, these latter values are the same. The overall lower affinity at the higher buffer concentration is as expected, because the electrostatic interactions are screened. Discussion These studies investigated the molecular forces that control both the equilibrium binding of cyt c and cyt b5 and of cyt c interactions with different faces of cyt b5. To achieve this we used the cyt b5 mutants, T8C and T65C, which formed selectively oriented protein monolayers on mercaptol-silane-derivatized glass substrates.21 We then measured directly the molecular forces that control the binding of soluble cyt c to immobilized cyt b5 monolayers. These investigations show the attractiveness of using supported lipid membranes as supports. The excellent agreement between the model-predicted cyt b5 orientation and the measured average tilt angles of the hemes, as well as the stability of the LD signal, indicate that the protein activity is retained on these supports. By contrast,
Cyt c Recognition of Variants of Cyt b5
Figure 6. Calculated electrostatic potential contours of the cyt b5 variants, T8C and T65C. Calculations were based on the crystal structure of bovine heart cytochrome b5 obtained from the Brookhaven protein data bank.32 The protein backbone is shown as a worm. The heme, residue 8, and residue 65 are shown as filled van der Waals spheres. The electrostatic potentials were calculated using the linearized PoissonBoltzmann equation. Full charges were assigned to the amino acid side chains of the protein according to the Full.crg charge file provided with the program. We used dielectric constants of 2 and 80 for the protein and the solvent, respectively, and a salt concentration of 5 mM. The grids surrounding the proteins indicate the (3 kT electrostatic potential surfaces.
on silane-derivatized substrates, the average tilt angle of T65C was not determined because of the low signal-tonoise ratio.45 This indicates the low efficiency of the immobilization method. In addition, denaturation of the immobilized protein on silanized glass was also evident in IR spectra, which were similar to those measured after thermal treatment of the protein.45 LD measurements verified that the heme orientations are consistent with the molecular models of the immobilized proteins. Although the 45° heme tilt angle of the T65C variant is close to that predicted for random protein orientations, the latter possibility is unlikely. A similar site-selective method used to immobilize yeast cyt c on reactive monolayers gave highly oriented protein films with a very narrow orientation distribution.26 Nonspecific adsorption is the main source of randomly oriented protein, but the plasmon resonance measurements showed negligible (
