Life Sciences Open Day 2002

Objectives of Research:
Our group consists of experimentalists who employ encoded combinatorial peptide libraries expressed on the surface of phage in order to determine peptide-specific binding by selectors, as well as theoreticians involved in the elucidation of the basic principles determining biological specificity.

Recent Findings:
Mapping of [[alpha]]-bungarotoxin binding site within the nicotinic acetylcholine receptor: A 15-mer phage-peptide library was used to select an epitope reacting specifically with [[alpha]]-bungarotoxin (BTX). A peptide with sequence similarity to a region containing the two tandem cysteines 192 and 193 of the, [[alpha]]-subunits of the muscle nicotinic acetylcholine receptor and, more strikingly, to BTX-sensitive neuronal nAChRs was identified. This peptide, as well as synthetic peptides corresponding to residues 187-199 of Torpedo nAChR and to the respective residues in the neuronal nAChRs, inhibited the binding of BTX to Torpedo nAChR.

NMR analysis of the [[alpha]] -bungarotoxin-library derived peptide complex: NMR study of the structure of the library peptide-BTX complex suggests that only four residues of the peptide participate in direct interactions with the toxin. Other two residues of the peptide interact within the peptide to form its horseshoe-like structure appearing in the complex.

Identification of receptor epitope on cultured cells:

A peptide that binds specifically to the muscarinic acetylcholine receptor type 1 (m1AChR) was selected by applying 15-mer phage peptide library on CHO cells transfected with the receptor. A single peptide was isolated by elution of the bound phage with a high affinity antagonist.

Mapping functional epitopes for acidic Fibroblast Growth Factor and generating a neutralizing single chain Fv': A 15-mer phage display peptide library was used to identify the binding site of a neutralizing mAb to aFGF. A restricted epitope, homologous to a peptide sequence within the putative receptor binding site, was localized to the C-terminal region of aFGF. A single chain (Fv') antibody targeted against this region was constructed and expressed in bacteria. Its use in vitro and in vivo as a neutralizing agent of aFGF is being tested.

Selection of phage bound in high affinity by the nitro-streptavidin: A novel approach was developed for selecting high-affinity phage from phage-peptide library using a chemically modified streptavidin (nitro-streptavidin developed in M. Wichek lab.), which shows reversible attraction for biotin. Phage which were not released by acid elution may be dissociated from a nitro-strepavidin by biotin. Using this approach, phage which bind to BTX with high affinity were isolated.

Sub-fractionation of polyclonal antibodies by phage-peptide library: Sub-fractionation of polyclonal antibodies generated against Fibroblast Growth Factor receptors was achieved by the use of phage-peptide library,which enabled the characterization of mono-specific antibody fractions from polyclonal antisera to well- defined epitopes.

Modelling supramolecular helices: An extension of the geometric fit algorithm to supramolecular helices was recently carried out. First we identified a large number of good binary matches between the constituents of a helical aggregate, then built helices using the geometrical characteristics of these matches, and estimated their quality. This approach was successful in building the helical protein coat of tobacco mosaic virus from the structure of one monomer. Moreover, our results show that the unique shape of the protein monomer allows good lateral interaction for a range of axial translations. This explains how the 34-mer double layered disk, which is formed at the initial step of virus assembly, can transform to a helical fragment without dissociation into subunits.

Normal mode of biopolymers: It is possible to characterize the long-time dynamics and internal flexibilities of proteins by computing the normal mode (NM) of these systems. An algorithm that permits direct NM analysis of crystal coordinates, without initial energy minimizations, was developed. The algorithm reproduces earlier results, and extends the use of NM analysis in structural biology.

Fig. 1: Schematic illustration of peptide libraries on filamentous phage and a selector protein

Recent Publications:

Katchalski-Katzir, E. (1993) Recollections - Poly-amino Acids as the Simplest Protein Models: Recollections of a Retired President. Protein Sci. 2: 476-482.

Katchalski-Katzir, E. (1993) Immobilized Ensymes - Larning from Past Successes and failures. Trends in Biotechnol. 11: 471-478.

Balass, M., Heldman Y., Cabilly S., Givol, D., Katchalski-Katzir E., Fuchs S. (1993) Identification of a hexapeptide that mimics a conformation-dependent binding site of acetylcholine receptor by use of a phage-epitope library. Proc. Natl. Acad. Sci USA, 90: 10638-10642.

Yayon, A., Aviezer, D., Safran, M., Janet L. Gross, Y., Heldman, S., Cabilly, D., Givol, D. and Katchalski-Katzir, E. (1993) Isolation of peptides that inhibit binding of basic fibroblast growth factor to its receptor from a random phage-epitope library. Proc. Natl. Acad. Sci. USA 90: 10643-10647.

Lancet, D., Horovitz, A. and Katchalski-Katzir, E. (1994) Molecular Recognition in Biology: Models for Analysis of Protein-Ligand Interaction. In Lock and Key Principle, Edited by J.-P. Behr, John Wiley & Sons Ltd.

Barchan, D., Balass, M., Souroujon, M.C.,Katchalski-Katzir, E. and Fuchs, S. (1995) Identification of Epitopes Within a Highly Immunogenic Region of Acetylcholine Receptor by a Phage Epitope Library. J. Immunol. 4264-69.

Natarajan Venkatesh, Sin-Heyong Im, Moshe Balass, Sara Fuchs and Ephraim Katchalski-Katzir (2000) Prevention of passively transferred experimental autoimmune myasthenia gravis by a phage library-derived cyclic peptide. Proc. Natl. Acad. Sci. USA 97/2 p. 761-766

S. Moshitch-Moshkovitz, Y. Heldman, A. Yayon, E. Katchalski-Katzir (2000) Sorting polyclonal antibodies into functionally distinct fractions using peptide phage display. J. of Immunological Methods, 242 183-191

Ephraim Katchalski-Katzir, Dieter M. Kraemer (2000) Eupergit C, a carrier for immobilization of enzymes of industrial potential. J. of Mol. Catalysis B: Enzymatic 10, 157-176

M. Balass, E. Kalef, S. Fuchs, E. Katchalski-Katzir (2001) A cyclic peptide with high affinity to a-bungarotoxin protects mice from the lethal effect of the toxin. Toxicon 39(7):1045-1051

Roni Kasher, Moshe Balass, Tali Scherf, Mati Fridkin, Sara Fuchs & Ephraim Katchalski-Katzir (2001) Design and Synthesis of Peptides that bind a-bungarotoxin with high affinity" Chemistry & Biology 8(2):147-155

Scherf T., Kasher R., Balass M., Fridkin M., Fuchs S & Katchalski-Katzir E. (2001) A b-hairpin structure in a 13-mer peptide that binds a- Bungarotoxin with high affinity and neutralizes its toxicity. Proc. of Natl. Acad. Sci. (USA) June 5, 98, No. 12, pp 6629-6634

Maya R., Balass M., Kim ST., Buschmann T., Martinex Leal JF., Shifman O., Shkedy D., Shiloh Y., Ronai Z., Kastan MB., Katchalski-Katzir E., and Oren M. ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage. Gene and Development (2001) 15 (9) pp 1067-1077