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Langmuir 1998, 14, 456-462
Effect of Structural Stability on the Characteristics of Adsorbed Layers of T4 Lysozyme Johan C. Fro¨berg,*,† Thomas Arnebrant,‡,§ Joseph McGuire,| and Per M. Claesson† Laboratory for Chemical Surface Science, Department of Chemistry, Physical Chemistry, Royal Institute of Technology, S-100 44 Stockholm, Sweden, and Institute for Surface Chemistry, Box 5607, S-114 86 Stockholm, Sweden, Department of Food Technology, University of Lund, Box 124, S-221 00 Lund, Sweden, and Department of Bioresource Engineering, Oregon State University, Gilmore Hall 116, Corvallis, Oregon 97331-3906 Received June 24, 1997. In Final Form: October 30, 1997 The interferometric surface force technique has been employed to determine how the structural stability of globular proteins affects their adsorption and the interactions between adsorbed protein layers. The system consisted of positively charged bacteriophage T4 lysozyme and negatively charged mica surfaces. The wild type and one synthetic mutant of the protein, Ile3 f Trp, differing in structural stability while the total charge and tertiary structure are the same, were studied. The adsorption leads to a nearly complete neutralization of the negative surface charge of mica, reducing the long-range electrostatic doublelayer interaction acting between mica surfaces. The thickness of the adsorbed layer is for the wild type consistent with the dimensions of the protein, while the Ile3 f Trp mutant gives a layer with a thickness smaller than any of its native dimensions. Another consequence of the difference in structural stability is that the short range attraction between one protein layer and one bare mica surface is an order of magnitude larger for the Ile3 f Trp mutant than for the wild type. The results demonstrate that the less stable mutant loses its tertiary structure upon adsorption, whereas the wild type retains its globular shape. These differences provide an understanding for the differences in adsorbed amount and complements the information about changes in secondary structure upon adsorption observed with other methods.
1. Introduction Conformational changes undergone by globular proteins upon adsorption have been inferred from indirect observations including isotherm shape,1,2 titration data,3 calorimetric measurements,4-6 and more direct observations via antibody binding to epitopes hidden in the native globular structure.7 Norde and co-workers showed that such changes could contribute to the adsorption free energy by increasing the entropy of the polypeptide chain5,8 and thus contribute to the driving force for adsorption. The increase in entropy upon adsorption may at first seem counterintuitive, and it is opposite to the decrease in entropy occurring upon adsorption of synthetic polymers. One should remember, however, the very compact structure of globular proteins, with significantly less free * Corresponding author: e-mail,
[email protected]; fax, +46-8-20 89 98. † Royal Institute of Technology and Institute for Surface Chemistry. ‡ University of Lund. § Present address: Department of Prosthetic Dentistry, Centre for Dental Health Sciences, Carl Gustavs va¨g 34, 214 21 Malmo¨, Sweden. | Oregon State University. (1) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1978, 66, 257. (2) Soderquist, M. E.; Walton, A. G. J. Colloid Interface Sci. 1980, 75, 386. (3) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1978, 66, 266. (4) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1978, 66, 295. (5) Haynes, C. A.; Norde, W. Colloids Surf., B: Biointerfaces 1994, 2, 517. (6) Yan, G.; Li, J.-T.; Huang, S.-C.; Caldwell, K. D. Calorimetric Observations of Protein Conformation at Solid-Liquid Interfaces. In Proteins at Interfaces II; Horbett, T. A., Brash, J., Eds.; American Chemical Society: Washington, DC, 1995. (7) Elwing, H.; Nilsson, B.; Svensson, K.-E.; Askendahl, A.; Nilsson, U. R.; Lundstro¨m, I. J. Colloid Interface Sci. 1988, 125, 139. (8) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267.
volume than bulk water, and secondly that their function requires a specific globular conformation. Furthermore, most amino acid residues in the native protein are involved in hydrogen bonding which restricts their rotational freedom. A change in conformation of the protein due to adsorption may therefore lead to a large increase in entropy. This is in agreement with experimental quantification of the loss in secondary structure in desorbed proteins,2,9 as well as upon adsorption to nanoparticles.10,11 The tendency for structural alteration upon adsorption is related to the conformational stability of the protein.5,8,12,13 This has been inferred from a certain degree of correlation between ∆Gdenat and surface tension or foamability and between adiabatic compresssibility and content of structural units as measured by circular dichroism. However, no quantitative correlation has emerged while there is qualitative support that structurally less stable proteins (small ∆Gdenat) lose their structure more easily upon adsorption. Structural changes experienced by proteins upon adsorption are of great importance in areas ranging from biomaterial applications and immunological assays, to emulsification and foam stabilization. Several groups have studied the effect of amino acid substitutions on protein or peptide behavior at interfaces.13-15 Kato and Yutani13 used this approach to show that the structural (9) Chan, B. M. C.; Brash, J. L. J. Colloid Interface Sci. 1981, 84, 263. (10) Kondo, A.; Oku, S.; Higashitani, K. J. Colloid Interface Sci. 1991, 143, 214. (11) Norde, W.; Favier, J. P. Colloids Surf. 1992, 64, 87. (12) Kondo, A.; Higashitani, K. J. Colloid Interface Sci. 1992, 150, 344. (13) Kato, A.; Yutani, K. Prot. Eng. 1988, 2, 153. (14) Elbaum, D.; Harrington, J.; Roth, E. F. J.; Nagel, R. L. Biochim. Biophys. Acta 1976, 427, 57. (15) Arnebrant, T.; Ericsson, B. J. Colloid Interface Sci. 1992, 150, 428.
S0743-7463(97)00674-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/06/1998
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force technique19-21 has been employed. This technique allows a determination of the interaction between two macroscopic surfaces (here negatively charged muscovite mica) as a function of their absolute separation. In this report we demonstrate how the interaction between surfaces bearing adsorbed layers of protein, combined with the known dimensions of the protein can be used to gain information about the orientation and conformation of proteins on the surface and to demonstrate that large scale structural changes occur upon adsorption.21 2. Experimental Section
Figure 1. The backbone of bacteriophage T4 lysozyme (which can be described as an ellipsoid with dimensions 3 × 3 × 5 nm). The site of the Ile3 f Trp substitution is indicated by the arrow. Figure is based on an original provided by Brian W. Matthews.
stabilities of tryptophan synthase R-subunits and mutants thereof strongly influenced their adsorption at air/water and oil/water interfaces. This was evidenced by the ability of the proteins with higher conformational stability to decrease surface tension and stabilize foams and emulsions less efficiently than the less stable wild type. Using single-site mutations, Matsumura et al.16 demonstrated that hydrophobic interactions at the site of Ile3 in bacteriophage T4 lysozyme, cf. Figure 1, contribute directly to its overall thermal stability. This was quantified as the difference in free energy change upon thermal unfolding between each mutant and the wild type, ∆∆G (∆∆G < 0, corresponding to a less stable mutant). The Ile3 mutants are fully functional lysozymes and the threedimensional structures for the crystallized Ile3 f Tyr and Ile3 f Val mutants are similar to that of the wild type.16 With a set of these mutants, remarkable differences in adsorption behavior and interactions with surfactants were recently demonstrated for mutants with relatively low (Ile3 f Trp, ∆∆G ) -2.8 kcal mol-1, pH 6.5) and high (Ile3 f Cys, ∆∆G ) 1.2 kcal mol-1, pH 6.5) structural stability.17,18 On adsorption to silica, lower plateau amounts, i.e., higher area per molecule, and significantly higher and faster loss of R-helix structure implied more pronounced conformational changes for the Ile3 f Trp mutant than for the wild type. In order to obtain information on the interactions between adsorbed layers of bacteriophage T4 lysozyme and to elucidate the difference in interfacial behavior of the wild type T4 lysozyme and the less stable Ile3 f Trp mutant (∆∆G ) -2.8 kcal/mol), the interferometric surface (16) Matsumura, M.; Becktel, W. J.; Matthews, B. W. Nature (London) 1988, 334, 406. (17) McGuire, J.; Wahlgren, M. C.; Arnebrant, T. J. Colloid Interface Sci. 1995, 170, 182. (18) Billsten, P.; Wahlgren, M. C.; Arnebrant, T.; McGuire, J.; Elwing, H. J. Colloid Interface Sci. 1995, 175, 77.
Both the wild type bacteriophage T4 lysozyme and the Ile3 f Trp mutant were produced using individual bacterium strains containing the mutant lysozyme expression vectors kindly provided by Professor Brian Matthews and co-workers.22 The expression and purification followed established procedures.16,17,23-25 The bacteriophage T4 lysozyme has 164 amino acid residues and a molecular weight26 of 18 700 g/mol, and its globular shape can be described as an ellipsoid with dimensions27 (3 × 3 × 5) nm. Its isoelectric point is above pH 9 and it carries a net positive charge of +9 at neutral pH. The ionic strength was adjusted to 1 mM by use of sodium chloride, suprapure grade used as received from Merck, and the pH was that of unbuffered salt solutions, 5.6. The solutions were prepared from deaereated water taken fresh from a two-step Millipore unit: a Milli-RO 10PLUS pretreatment unit with depth filtration, carbon adsorption, and decalcination preceeding reverse osmosis, and a Milli-Q PLUS185 unit consisting of a UV source (185 and 254 nm) treating the feed water to the Q-pack, an active carbon unit followed by a mixed bed ion exchanger and an Organex cartridge. The water is then passed through a final 0.22 µm Millipak 40 filter. The surface force measurements were performed using a MkIV SFA20 utilizing interferometric detection of the surface separation, D, via fringes of equal chromatic order (FECO).28 The force is calculated according to Hooke’s law, using the force constant of the spring onto which the lower surface is mounted and the spring deflection. This latter quantity is calculated as the difference between the observed change in D and the expansion of the piezoelectric tube which holds the upper surface. The piezo expansion is determined prior to each force run, while the surfaces are kept far apart for no interaction to be present. The two silica disks, onto which the silvered side of the mica sheets (A ) 1 cm2) are glued using an Epoxy resin (Epon 1004), are mounted in a crossed-cylinder configuration. A droplet of approximately 0.1 mL protein solution of concentration 0.02 mg/mL in 1 mM NaCl was then placed between the two surfaces. After allowing the adsorption to take place for 2 h or more, when the adsorption was found to have reached a plateau, the force-distance relationship was monitored. The box was then filled with protein-free 1 mM sodium chloride solution, an effective dilution by a factor of 3000, and left for at least 12 h to allow for any possible desorption. The force-distance relationship was then determined in this diluted system before and after one of the surfaces was replaced with a clean mica surface. (19) Israelachvili, J. N.; Adams, G. E. J. Chem. Soc., Faraday Trans. 1 1978, 74, 975. (20) Parker, J. L.; Christensson, H. K.; Ninham, B. W. Rev. Sci. Instrum. 1989, 60, 3135. (21) Claesson, P. M.; Blomberg, E.; Fro¨berg, J. C.; Nylander, T.; Arnebrant, T. Adv. Colloid Interface Sci. 1995, 57, 161. (22) Institute of Molecular Biology, University of Oregon, Eugene, OR. (23) Alber, T.; Matthews, B. W. Methods Enzymol. 1987, 154, 511. (24) Muchmore, D. C.; McIntosh, L. P.; Russell, C. B.; Andersson, D. E.; Dahlquist, F. W. Methods Enzymol. 1989, 177, 44. (25) Poteete, A. R.; Dao-Pin, S.; Nicholson, H.; Matthews, B. W. Biochemistry 1991, 30, 1425. (26) Matthews, B. W.; Dahlquist, F. W.; Maynard, A. Y. J. Mol. Biol. 1973, 78, 575. (27) Matthews, B. W.; Remington, S. J. Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 4178. (28) Israelachvili, J. N. J. Colloid Interface Sci. 1973, 44, 259.
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The results, based on several measurements at each condition and position, are presented with the force normalized by the local geometric mean radius of the interacting surfaces, F/R, as a function of surface separation, D. This in turn allows the force to be converted to the free energy per unit area of interacting flat surfaces, Gf(D), according to the Derjaguin approximation29
F(D)/R ) 2π Gf(D)
(1)
and therefore makes a comparison between experiments and theory possible. This relationship is valid as long as D , R and insofar as R does not vary with D; both requirements were fulfilled for the results presented here with the exception of the highly attractive forces between bare mica and mica covered with the Ile3 f Trp mutant. The resolution of the interferometric surface force technique, and thus the numbers for the distance given, are within 0.2 nm, implying a detection limit of (0.01 mN‚m-1 for the normalized force. A mechanical instability occurs whenever the gradient of the measured force exceeds the spring constant,30 which is observed as a jump to the closest separation where the system again is mechanically stable. To facilitate further analysis of the data, a comparison of the observed forces with those calculated using DLVO theory, i.e., additive contributions from the nonretarded van der Waals force and the electrostatic double-layer force, was carried out. The electrostatic contribution was calculated assuming interaction at constant charge and constant potential in the nonlinear Poisson-Boltzmann approximation, using the algorithm of Chan et al.31 for the symmetrical case of two protein-covered surfaces. In the asymmetric case, i.e., with one bare mica surface and one protein-covered surface, the electrostatic contribution was calculated using the algorithm of Bell and Peterson32 for solving the nonlinear Poisson-Boltzmann equation in the case of constant charge boundary conditions, while the approach of Deveraux and deBruyn33 was used to solve the same equation for the case of constant potential.
Figure 2. Interaction between mica surfaces after adsorption (squares) from a solution of 0.02 mg/mL bacteriophage T4 lysozyme wild type in 1 × 10-3 M NaCl solution at pH 5.6 and T ) 20 °C. The triangles show the interaction after a dilution with pure buffer by a factor of 3000. Filled and unfilled symbols represent the interaction on approach and separation, respectively.
3. Results The observed interactions between the two negatively charged mica surfaces in a solution of 0.02 mg/mL of the wild type bacteriophage T4 lysozyme in 1 mM NaCl at pH 5.6 are presented in Figure 2. A very weak repulsive force is observed on approach, which contrasts to the situation in the electrolyte in absence of protein where the longrange interaction is dominated by a strong electrostatic double-layer repulsion decaying in agreement with Poisson-Boltzmann theory (decay length, κ-1 ) 9.6 nm). With the wild type protein in solution a hard wall is present at a separation of D ) 9.5 nm, originating from contact between the two adsorbed layers. We note that unlike the previously studied hen egg white lysozyme, we do not see any evidence of surface dimerization of the T4 wild type. Also evident from Figure 2 is that a dilution by a factor of 3000 does not alter the interaction on approach. There is however a small increase in the force needed to separate the two surfaces from the adhesive minimum at 9.5 nm, leading to a sudden jump apart to separations where no force acts between the surfaces. The magnitude of this pull-off force increases from 0.3 before to 1 mN‚m-1 after dilution. Figure 3 shows the force curves measured when the single-site Ile3 f Trp mutant of bacteriophage T4 lysozyme is present in 1 mM NaCl to a concentration of (29) Derjaguin, B. Kolloid Z. 1934, 69, 155. (30) Horn, R. G.; Israelachvili, J. N. J. Chem. Phys. 1981, 75, 140011. (31) Chan, D. Y. C.; Pashley, R. M.; White, L. R. J. Colloid Interface Sci. 1980, 77, 283. (32) Bell, G. M.; Peterson, G. C. J. Colloid Interface Sci. 1972, 41, 542. (33) Devereux, O. F.; de Bruyn, P. L. Interaction of Plane-Parallel Double Layers; MIT Press: Cambridge, MA, 1963.
Figure 3. Interaction between mica surfaces after adsorption (squares) of the Ile3 f Trp mutant of bacteriophage T4 lysozyme from a solution of 0.02 mg/mL in 1 × 10-3 M NaCl solution at pH 5.6 and T ) 20 °C. The triangles show the interaction after a dilution with pure buffer by a factor of 3000: triangles pointing upward are the first approach while those pointing downward represent subsequent ones. Filled and unfilled symbols represent the interaction on approach and separation, respectively. The solid line is the nonretarded van der Waals force calculated according to the triple layer film model using A232 ) 2.34 × 10-21 J as the Hamaker constant for the protein-solutionprotein interface.
0.02 mg/mL in the droplet between the surfaces. The forces measured on approach with protein present in the bulk solution display no repulsive double-layer interaction showing that the surfaces are uncharged. At 12 nm separation an attraction sets in, leading to a sudden jump into contact between the adsorbed layers at a separation of 3.5 nm. After dilution with 1 mM NaCl by a factor of 3000 a weak double-layer interaction is observed during the first approach, and as a consequence no jump into contact occurs. The steric wall at 3.5 nm remains. During subsequent approaches the double-layer force has nearly disappeared and the attraction giving rise to an inward sudden jump, now from 8.5 nm, has reappeared. Another
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Table 1. Parameters (K-1, Debye Length; Ψ∞, Apparent Interfacial Potential; σ, Surface Charge; A, Hamaker Constant) Obtained When Fitting DLVO Theory at 1 mM NaCl/ to the Experimental Results As Described in the Text, Together with Measured Layer Thicknesses, L, and Pull-Off forces, F0/R system
κ-1 (nm)
Ψ1,∞ (mV)
σ1 × 103 (C/m2)
Ψ2,∞ (mV)
σ2 × 103 (C/m2)
L (nm)
A × 1020 (J)
F0/R (mM/m)
buffera wild type symmetric wild type asymmetric mutant symmetric mutant asymmetric
9.6 9.6 9.6 9.6 9.6
-120 -10 -120 -11 -120
-43.5 -0.423 -43.5 -0.450 -43.5
-120 -10 -25 -11 -55
-43.5 -0.423 -0.924 -0.450 -3.25
0 4.5 4.5 1.5 1.5
2.2 2.2 2.2 1.4 2.2
