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Langmuir 2000, 16, 3414-3421
Effect of the Microenvironment on the Recognition of Immobilized Cytochromes by Soluble Redox Proteins A. A. Kloss, N. Lavrik, C. Yeung, and D. Leckband* Department of Chemical Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 Received July 12, 1999. In Final Form: December 10, 1999 This work examines the effect of the electrostatic properties of an immobilization substrate on the apparent affinity of immobilized receptors for their soluble ligands. In particular, we report measurements of the interaction between cytochrome c or cytochrome b5 bound to an oriented streptavidin monolayer and its soluble reaction partner. Surface plasmon resonance measurements demonstrate that the charge on the underlying streptavidin monolayer has a pronounced impact on the measured equilibrium constants for the heterologous interaction between cyt c and cyt b5. The pH dependence of the equilibrium binding constant is similar qualitatively to that reported for the protein interaction in solution. However, the pH optima of the affinities measured with the immobilized proteins are shifted by up to 1.0 pH units. Whether the shift is to acidic or basic pH depends on which of the two proteins is immobilized. The observed changes are consistent with the long-range repulsion or attraction between the soluble ligand and the supporting streptavidin monolayer.
Introduction The recognition of soluble proteins by immobilized receptors is an important step in many biological and biotechnological processes. It is essential for biomedical applications such as biosensors and immunological testing.1-3 Proteins immobilized on a solid matrix are also widely used in bioreactors,4,5 affinity chromatography,6,7 the production of fine chemicals,1 and environmental monitoring.8 However, the change in the local environment associated with the protein fixation to the surface is known to affect the activity of the immobilized protein. Studies have indicated that the immobilization can (1) induce conformational changes in the protein,9,10 (2) limit the binding site accessibility,11 and (3) alter the orientation of the bound molecules.11-13 The soluble molecules can also adsorb nonspecifically to the underlying support.12 The ability to control the structure and biological activity of immobilized receptor monolayers is therefore a challenge both for practical applications and for fundamental biological research. In addition to the perturbations indicated above, an often underappreciated consequence of immobilization is (1) Norde, W.; Zoungrana, T. Biotechnol. Appl. Biochem. 1998, 28, 133-143. (2) Zhao, S.; Reichert, W. M. American Chemical Society: Washington, DC, 1992. (3) Zhao, S.; Reichert, W. M. Langmuir 1992, 8, 2785-2791. (4) Fodor, S. P. A.; Read, L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 767-773. (5) Faulkner, K. M.; Shet, M. S.; Fisher, C. W.; Estabrook, R. W. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7705-7709. (6) De Ruiter, G. A.; Smid, J. H.; Van, B.; Rombouts, F. M. J. Chromatogr. 1992, 584, 69-75. (7) Hage, D. S.; Kao, P. C. Anal. Chem. 1991, 63, 586-595. (8) Wiseman, A. J. Chem. Technol. Biotechnol. 1993, 56, 3. (9) Tobias, D. J.; Mar, W.; Blasie, J. K.; Klein, M. L. Biophys. J. 1996, 71, 2933-2941. (10) Lee, J. E.; Saavedra, S. S. Langmuir 1996, 12, 4025-4032. (11) Rao, S. V.; Anderson, K. W.; Bachas, L. G. Mikrochim. Acta 1998, 128, 127-143. (12) Wood, L. L.; Cheng, S. S.; Edmiston, P. L.; Saavedra, S. S. J. Am. Chem. Soc. 1997, 119, 571-576. (13) Yeung, C.; Purves, T.; Kloss, A. A.; Kuhl, T. L.; Sligar, S.; Leckband, D. Langmuir 1999, 15, 6829-6836.
the interfering influence of the interfacial force fields on the receptor-ligand potential. The nonspecific interaction energies, which act between the soluble ligand and the underlying substrate, will be superimposed on the intrinsic, lock-and-key potential.12,14,15 This, in turn, may affect the binding free energy and, hence, the apparent affinity between the soluble ligands and the immobilized receptors. For example, the electrostatic properties of the immobilization matrix may enhance the adsorption of oppositely charged ligands to the receptor-functionalized matrix. Conversely, it could impede the binding of ligands with charges similar to that of the substrate. These effects could thereby induce changes in the apparent affinity, even though the intrinsic intermolecular potentials are unaltered under the conditions of the measurements. The electron transfer proteins cytochrome b5 and cytochrome c are convenient proteins for testing the effect of the microenvironment on molecular recognition. The heterologous protein association is electrostatically controlled. Previous studies indicated that complementary charges on the two proteins steer their mutual binding trajectories in solution.16-22 The large number of lysines surrounding the heme pocket of the basic protein cytochrome c (Figure 1a) is believed to facilitate the docking of the acidic cytochrome b5.22-25 The latter protein contains a complement of acidic amino acids surrounding its heme (14) Leckband, D. E.; Kuhl, T.; Wang, H. K.; Herron, J.; Muller, W.; Ringsdorf, H. Biochemistry 1995, 34, 11467-11478. (15) Yeung, C.; Leckband, D. Langmuir 1997, 13, 6746-6754. (16) Vergeres, G.; Waskell, L. Biochimie 1995, 77, 604-620. (17) Rodgers, K. K.; Pochapsky, T. C.; Sligar, S. G. Science 1988, 240, 1657-1659. (18) Rodgers, K. K.; Sligar, S. G. J. Mol. Biol. 1991, 221, 1453-1460. (19) Mauk, M. R.; Barker, P. D.; Mauk, A. G. Biochemistry 1991, 30, 9873-9881. (20) Mauk, M. R.; Mauk, A. G.; Weber, P. C.; Matthew, J. B. Biochemistry 1986, 25, 7085-7091. (21) Matthew, J. B.; Weber, P. C.; Salemme, F. R.; Richards, F. M. Nature 1983, 301, 169-171. (22) Wendoloski, J. J.; Matthew, J. B.; Weber, P. C.; Salemme, F. R. Science 1987, 238, 794-797. (23) Burch, A. M.; Rigby, S. E.; Funk, W. D.; MacGillivray, R. T.; Mauk, M. R.; Mauk, A. G.; Moore, G. R. Science 1990, 247, 831-833. (24) Takano, T.; Dickerson, R. E. J. Mol. Biol. 1981, 153, 79-94. (25) Takano, T.; Dickerson, R. E. J. Mol. Biol. 1981, 153, 95-115.
10.1021/la990909z CCC: $19.00 © 2000 American Chemical Society Published on Web 02/10/2000
Recognition of Immobilized Cytochromes
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Figure 1. NMR minimized average structure of cytochrome c from horse heart, taken from the Protein Databank. Structure a indicates the positively charged lysine and arginine residues shown as dark gray ball-and-stick structures. The heme is shown as a black space-filled structure. Structure b shows the same protein with the histidine residues (protonated at pH
