Meir Wilchek, Edward A. Bayer, Sarah Ehrlich-Rogozinski, Tikva Kulik, Talia Miron,
Aharon Rabinikov, Bilhah Schechter and Boris Tchernyshev
Department of Biological Chemistry Tel. (+972)-8-9343808, Fax. (+972)-8-9468256, e-mail: bfwilcek@WICCMail.weizmann.ac.il
Objectives of Research:
The aim of our group is to understand the basis of the molecular biorecognition phenomenon and to apply its principles for different biotechnological applications. Towards this goal, we have mainly used the tenacious affinity interaction between the vitamin biotin and the egg-white glycoprotein avidin (or its bacterial relative, streptavidin, from Streptomyces avidinii). This interaction is the strongest between a protein and a small ligand known to date, boasting an affinity constant in the range of 1015 M-1.
Structure of avidin : We have recently determined the three-dimensional crystal structure of avidin and its complexes with biotin and the dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA) and compared these structures with those of streptavidin (Livnah et al, 1993a,b). The binding of avidin to biotin is extremely strong as mentioned above, but the binding to HABA is relatively weak. These structures enabled us to determine the residues important to the binding of such molecules by avidin (Fig. 1). From the structures, we noticed that in the absence of its ligands, five molecules of water form a defined structure in the binding site of avidin, which is important to the energetics of binding. This observation has important implications for biorecognition in general and enabled us to establish guidelines for the prediction of affinity constants.
Pseudo-enzymatic properties of avidin: In the past, we described the capacity of avidin to cleave certain biotin esters in a manner which seemed to emulate enzymatic hydrolysis of a substrate. In contrast, streptavidin fails to cleave these esters, and even protects them from autohydrolysis. We hoped that additional knowledge gained from such pseudo-enzymatic activity would provide us with new insight into how enzymes work. However, the cleavage was a single event with no turnover, since the product (free biotin) remained in the binding site and prevented additional pseudo-substrate molecules (the biotin ester) from entering the site. Since the binding to HABA is quite weak, we reasoned that its derivatives would be better pseudo-substrates, since the product would be more easily released from the binding site. Indeed, modified HABA derivatives could either be hydrolyzed or acted as an affinity label and bound covalently to a binding site residue (Vetter et al, 1994). Further studies will enable us to design artificial or tailor made protein catalysts.
Reversibility of biotin binding: The importance of a single hydrogen bond: The three-dimensional structure of avidin also showed that the binding site tyrosine, which plays a critical role in biotin binding, has room to accommodate a nitro group. This group would serve to reduce the pKa of the phenol moiety of this tyrosine residue. At low pH, biotin is bound strongly to the nitro-avidin, whereas at high pH biotin is released from the binding site. The binding of biotin thus becomes reversible (Morag et al, in press). The reversibility of biotin binding by nitro-avidin demonstrates the importance of this single hydrogen bond in the binding site. Physicochemical studies (Bayer et al, submitted) also demonstrated that the nitration of avidin may also cause some changes in the quaternary structure of avidin. Nitro-streptavidin behaves in a manner very similar to that of nitro-avidin.
Fig. 1: Schematic representation of amino acid residues in the binding site of avidin which form hydrogen bonds with biotin.
Structural analogs of avidin and streptavidin: Although the binding sites of egg-white avidin and bacterial streptavidin are very similar, there are emphatic differences in the number and character of some of the binding site residues. The possibility that the binding sites may be different in the different types of avidin, led us to search for other types of avidins and streptavidins in different species. Indeed, we found that other strains of produced streptavidin-like proteins (Bayer et al, 1995). Cloning and sequencing the respective genes from two strains of Streptomyces venezuelae revealed that the binding site residues of the streptavidins from different species appear to be identical. This indicated that the binding site of streptavidin is quite impeccable and suggested that the alteration of its residues would severely reduce the affinity for biotin. This information serves to imply that site-directed mutagenesis of the avidins and streptavidins should be performed on non-binding site residues. This system will thus provide an exciting opportunity to examine the contribution of peripheral amino acid residues in the biorecognition phenomenon. In the case of the avidins and streptavidins, this is especially attractive due to the exceptionally high binding constant for biotin. The results of these studies will be particularly applicable to the field of avidin-biotin technology, in which reversible or other specialized forms of avidin may find extensive biotechnological application, e.g., for use in affinity chromatography, cell separation, in-vivo targeting and phage-display libraries (Bayer et al, 1994; Schechter et al, 1995; Tchernychev et al, 1995).
Livnah, O., Bayer, E.A., Wilchek, M. and Sussman, J.L. (1993a) Structure of the complex between avidin and the dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA). FEBS Lett. 328: 165-168.
Livnah, O., Bayer, E.A., Wilchek, M. and Sussman, J.L. (1993b) Three-dimensional structures of avidin and the avidin-biotin complex. Proc. Natl. Acad. Sci. USA 90: 5076-5080.
Bayer, E.A., and Wilchek, M. (1994) Modified avidins for application in avidin-biotin technology: an improvement on nature. In Egg Uses and Processing Technologies. Vol. Sim, J.S., and Nakai, S. (eds). Wallingford, UK: CAB International, pp. 158-176.
Vetter, S., Bayer, E.A., and Wilchek, M. (1994) Avidin can be forced to adopt catalytic activity. J. Am. Chem. Soc. 116: 9369-9370.
Bayer, E.A., Kulik, T., Adar, R., and Wilchek, M. (1995) Close similarity among streptavidin-like, biotin-binding proteins from Streptomyces. Biochim. Biophys. Acta 1263: 60-66.
Schechter, B., Arnon, R., Colas, C., Burakova, T., and Wilchek, M. (1995) Kidney International, 47: 1327-1335, Renal accumulation of streptavidin: Potential use for targeted therapy to the kidney.
Tchernychev, B., Rabinkov, A., Mirelman, D. and Wilchek, M. (1995) Immunol. Lett., 47: 53-57. Natural antibodies to dietary proteins: the existence of natural antibodies to alliinase (alliin lyase) and mannose-specific lectin from garlic (Allium sativum) in human serum.
Morag, E., Bayer, E.A., and Wilchek, M. (1996) Reversibility of biotin-binding by selective modification of tyrosine in avidin (In press).
Bayer, E.A., Ehrlich-Rogozinski, S., and Wilchek, M. (1996) Sodium dodecyl sulfate-polyacrylamide gel electrophoretic method for assessing the quaternary state and comparative thermostability of avidin and streptavidin (Submitted).
Wilchek, M. and Chaiken, I. (2000). An overview of Affinity Chromatography. In Methods in Molecular Biology. Affinity Chromatography: Methods and Protocols (P. Bailon, G.K. Ehrlich, W.-J Fung, W. Berthold, eds.) Humana Press Inc., USA, Vol. 147, 1-6.
Miron, T., Rabinkov, A., Mirelman, D., Wilchek, M. and Weiner, L. (2000) The mode of action of allicin: its ready permeability through phospholipid membranes may contribute to its biological activity. Biochim. Biophys. Acta 1463, 20-30.
Marttila, A.T., Laitinen, O.H., Airenne, K.J., Kulik, T., Bayer, E, Wilchek, M. and Kulomaa, M.S. (2000) Recombinant neutraLite avidin: a non-glycosylated, acidic mutant of chicken avidin that exhibits high affinity for biotin and low non-specific binding properties. FEBS Lett. 467, 31-36.
Tchernychev, B., Rabinkov, A., Miron, T. and Wilchek, M. (2000) Natural antibodies against alliinase in human serum and polyclonal antibodies elicited in rabbit share the same immunogenic determinants. Immunol. Lett. 71, 43-47.
Hofstetter, H., Morpurgo, M., Hofstetter, O., Bayer, E. and Wilchek, M. (2000) A labeling, detection, and purification system based on 4-hydroxyazobenzene-2-carboxylic acid: An extension of the avidin-biotin system. Anal. Biochem. 284, 354-366.
Hofstetter, O., Hofstetter, H. Wilchek, M., Schurig, V. and Green, B.S. (2000) An immunochemical approach for the determination of trace amounts of enantiomeric impurities. Chem. Comm. 1581-1582.
Hofstetter, O., Hofstetter, H. Wilchek, M., Schurig, V. and Green, B.S. (2000) Production and applications of antibodies directed against the chiral center of a-amino acids. Int. J. Biochromatography (in press).
Wilchek, M., Hofstetter, H and Hofstetter, O. (2000) The application of biorecognition. In: Novel Approaches in Biosensor and Rapid Diagnostic Assays. (Z. Liron, ed.) Kluwer Academic/Plenum (publ.). (in press).
Chen, L., Schechter, B., Arnon, R. and Wilchek, M. (2000) Tissue selective affinity targeting using the avidin-biotin system. Drug Develop. Res. 50, 258-271.
Laitinen, O.H., Marttila, A.R., Airenne, K.J., Kulik, T., Livnah, O., Bayer, E.A., Wilchek, M. and Kulomaa, M.S. (2000) Biotin induces tetramerization of a recombinant monomeric avidin: A model for protein-protein interactions. J. Biol. Chem., in press.
Rabinkov, A., Miron, T., Mirelman, D., Wilchek, M., Glozman, S., Yavin, E. and Weiner, L. (2000) S-allylmercaptoglutathione: The reaction product of allicin with glutathione possesses SH-modifying and antioxidant properties. Biochem.Biophys. Acta (in press).
Elkayam, A, Mirelman, E., Peleg, E., Wilchek, M., Miron, T., Rabinkov, A., Sadetzki, S. and Rosenthal, T. (2000) Comparison of the effects of allicin and enalapril on blood pressure, insulin and triglycerides levels in fructose-induced hyperinsulinemic-hyperlipidemic hypertensive rats. Am. J. Hypertension (in press).
Kirsch, K., Danilenko, M., Giat, Y., Miron, T., Rabinkov, A., Wilchek, M., Mirelman, D., Levy, J. and Sharoni, Y. (2000) The effect of purified allicin, the major ingredient of a freshly crushed garlic, on cancer cell proliferation. Nutrition and Cancer (in press).
Outline of Program