Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-07-16
2001-12-18
Celsa, Bennett (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S023000, C435S024000, C436S506000, C436S536000, C436S057000, C436S086000, C436S089000, C436S161000, C436S173000, C436S174000, C436S175000
Reexamination Certificate
active
06331400
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the characterization of the binding site involved in binding between a binding protein and a binding partner.
2. Background Art
Biochemical Binding, Generally
Many biological processes are mediated by noncovalent binding interactions between a protein and another molecule, its binding partner. The identification of the structural features of the two binding molecules which immediately contribute to those interactions would be useful in designing drugs which alter these processes.
The molecules which preferentially bind each other may be referred to as members of a “specific binding pair”. Such pairs include an antibody and its antigen, a lectin and a carbohydrate which it binds, an enzyme and its substrate, and a hormone and its cellular receptor. In some texts, the terms “receptor” and “ligand” are used to identify a pair of binding molecules. Usually, the term “receptor” is assigned to a member of a specific binding pair which is of a class of molecules known for its binding activity, e.g., antibodies. The term “receptor” is also preferentially conferred on the member of the pair which is larger in size, e.g., on avidin in the case of the avidin-biotin pair. However, the identification of receptor and ligand is ultimately arbitrary, and the term “ligand” may be used to refer to a molecule which others would call a “receptor”. The term “anti-ligand” is sometimes used in place of “receptor”.
While binding interactions may occur between any pair of molecules, e.g., two strands of DNA, the present specification is primarily concerned with interactions in which at least one of the molecules is a protein. Hence, it is convenient to speak of a “binding protein” and its “binding partner”. The term “protein” is used herein in a broad sense which includes, mutatis mutandis, polypeptides and oligopeptides, and derivatives thereof, such as glycoproteins, lipoproteins, and phosphoproteins. The essential requirement is that the “binding protein” feature one or more peptide (—NHCO—) bonds, as the amide hydrogen of the peptide bond (as well as in the side chains of certain amino acids) has certain properties which lends itself to analysis by proton exchange.
A “binding site” is a point of contact between a binding surface (“paratope”) of the binding protein and a complementary surface (“epitope”) of the binding partner. (When the binding partner is a protein, the designation of “paratope” and “epitope” is essentially arbitrary. However, in the case of antibody-antigen interactions, it is conventional to refer to the antigen binding site of the antibody as the “paratope” and the target site on the antigen as the “epitope”.) A specific binding pair may have more than one binding site, and the term “pair” is used loosely, as the binding protein may bind two or more binding partners (as in the case of a divalent antibody). Moreover, other molecules, e.g., allosteric effectors, may alter the conformation of a member of the “pair” and thereby modulate the binding. The term “pair” is intended to encompass these more complex interactions.
Limitations of Current Methods of Characterizing Protein Binding Sites
Considerable experimental work and time are required to precisely characterize a binding site. In general, the techniques which are the easiest to use and which give the quickest answers, result in an inexact and only approximate idea of the nature of the critical structural features. Techniques in this category include the study of proteolytically generated fragments of the protein which retain binding function; recombinant DNA techniques, in which proteins are constructed with altered amino acid sequence (site directed mutagenesis); epitope scanning peptide studies (construction of a large number of small peptides representing subregions of the intact protein followed by study of the ability of the peptides to inhibit binding of the ligand to receptor); covalent crosslinking of the protein to its binding partner in the area of the binding site, followed by fragmentation of the protein and identification of crosslinked fragments; and affinity labeling of regions of the receptor which are located near the ligand binding site of the receptor, followed by characterization of such “nearest neighbor” peptides. (Reviewed in 1, 2).
These techniques work best for the determination of the structure of binding subregions which are simple in nature, as when a single short contiguous stretch of polypeptide within a protein is responsible for most of the binding activity. However, for many protein-binding partner systems of current interest, the structures responsible for binding on both receptor and ligand or antibody are created by the complex interaction of multiple non-contiguous peptide sequences. The complexities of these interactions may confound conventional analytical techniques, as binding function is often lost as soon as one of the 3-dimensional conformations of the several contributing polypeptide sequences is directly or indirectly perturbed.
The most definitive techniques for the characterization of the structure of receptor binding sites have been NMR spectroscopy and X-ray crystallography. While these techniques can ideally provide a precise characterization of the relevant structural features, they have major limitations, including inordinate amounts of time required for study, inability to study large proteins, and, for X-ray analysis, the need for protein-binding partner crystals (Ref. 3).
Applicant's technology overcomes these limitations and allows the rapid identification of each of the specific polypeptides and amino acids within a protein which constitute its protein ligand binding site or antibody binding subregion in virtually any protein-ligand system or protein antigen-antibody system, regardless of the complexity of the binding sites present or the size of the proteins involved. This technology is superior in speed and resolution to currently employed biochemical techniques.
Tritium Exchange
When a protein in its native folded state is incubated in buffers containing tritiated water, tritium in the buffer reversibly exchanges with hydrogen present in the protein at acidic positions (for example, O—H, S—H, and N—H groups) with rates of exchange which are dependent on each exchangeable proton's chemical environment, temperature, and most importantly, its accessibility to the tritiated water in the buffer. (Refs. 4, 5) Accessibility is determined in turn by both the surface (solvent-exposed) disposition of the proton, and the degree to which it is hydrogen-bonded to other regions of the folded protein. Simply stated, acidic protons present on amino acid residues which are on the outside (buffer-exposed) surface of the protein and which are hydrogen-bonded to solvent water will exchange more rapidly with tritium in the buffer than will similar acidic protons which are buried and hydrogen-bonded within the folded protein.
Proton exchange reactions can be greatly accelerated by both acid and base-mediated catalysis, and the rate of exchange observed at any particular pH is the sum of both acid and base mediated mechanisms. For many acidic protons, a pH in the range of 2.7 results in an overall minimum rate of exchange (Ref. 6, pg.238,
FIG. 3
, refs. 7-11). While hydrogens in protein hydroxyl and amino groups exchange with tritium in buffer at millisecond rates, the exchange rate of one particular acidic proton, the peptide amide bond proton, is considerably slower, having a half life of exchange (when freely hydrogen bonded to solvent water) of approximately 0.5 seconds at 0° C., pH 7, which is greatly slowed to a half life of exchange of 70 minutes at 0° C. pH 2.7.
When peptide amide protons are buried within a folded protein, or are hydrogen bonded to other parts of the protein, exchange half lives with solvent protons are often considerably lengthened, at times being measured in hours to days. Proton exchange at peptide amides is a fully reversible reaction, and rates of on-exchange
Carta Proteomics, Inc.
Celsa Bennett
Pennie & Edmonds LLP
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