Process for characterizing proteins

Details Process for characterizing proteins Process for characterizing proteins

1998-06-19

2002-12-17

Celsa, Bennett (Department: 1627)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S007100, C435S019000, C435S023000, C435S024000, C435S106000, C435S091500, C530S300000, C530S344000, C530S350000

Reexamination Certificate

active

06495314

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The present invention relates to methods for analyzing, altering, and controling the structural basis for protein binding to target molecules. More particularly, the present invention is directed to peptide ladder libraries corresponding to a protein, protein fragment, or other bioactive peptide and to the use of peptide ladder libraries for obtaining a protein signature analysis.
BACKGROUND OF THE INVENTION
One of the major strategies for determining the relationship between the chemical structure of a peptide and its biological activity is to systematically alter the covalent structure and observe the effect on function. Through the use of chemical synthesis, a wide variety of modifications can be made. For example, N-methylation and the use of ester bonds can probe backbone interactions (Arad et al.
Biopolymers
1990, 29, 1633-1649; Bramson et al.
J. Biol. Chem.
1985, 260, 15452-15457; Caporale et al. In:
Peptides: Structure and Function, Proceedings of the Tenth American Peptide Symposium
; Marshall, G. F. Ed. Escom:Leiden: The Netherlands, 1988, pp. 449-451), while sidechain contributions can be probed using D-amino acid or Alanine/Glycine substitutions (Konishi et al. In:
Peptides: Structure and Function, Proceedings of the Tenth American Peptide Symposium
, Marshall, G. F. Ed. Escom, Leiden: The Netherlands, 1988, pp. 479-481; Tam et al. In
Peptides:Proceedings of the Eleventh American Peptide Symposium
; Rivier, J. E.; Marshall, G. R. Ed.; Escom: Leiden, The Netherlands, 1990. pp 75-77). As traditionally practiced, a separate analogue must be prepared and assayed for each position in the peptide sequence that is to be studied.
An alternative, currently popular method of studying peptides is through combinatorial chemistry. This approach has had a major impact on the study of the molecular basis of peptide activity and has contributed to the search for new biologically active peptides (Thompson et al.
Chem. Rev.
1996, 96, 555-600; Gordon et al.
J. Med. Chem.
1994, 37, 1385-1401; Scott et al.
Curr. Op. Biotech
1994, 5, 40-48) ‘Multiple Peptide Synthesis’ has extended the traditional approach by allowing peptides to be synthesized simultaneously (Geysen et al.
J. Proc. Natl. Acad. Sci. USA
1984, 81, 3998-4001; Houghten et al.
Proc Natl. Acad. Sci. USA
1985, 82, 5131-5134). The individual peptide products are spatially separated and can be analyzed either attached to a solid support or in solution. Established ‘split synthesis’ (Furka et al.
Int. J. Pept. Prot. Res.
1991, 37, 487-494; Lam et al.
Nature
1991, 354, 82-84) procedures allow for the rapid generation of huge numbers of peptide sequences through the repetition of a simple divide, couple and recombine process. The compositional diversity made possible by this approach is advantageous for the discovery of new ‘lead’ compounds since, in principle, all possible structural variants can be explored for the desired activity and only the few active oligomers of interest need to be individually identified (Furka et al.
Int. J. Pept. Prot. Res.
1991, 37, 487-494; Lam et al.
Nature
1991, 354, 82-84). However, where information about a complete set of functional and non-functional components is desired over many positions in a peptide sequence, such libraries are too complex to fully characterize and may have limited utility.
A more systematic investigation of the molecular basis of peptide function requires a different type of molecular diversity. Instead of a peptide mixture of high compositional diversity, it would be useful to construct an array of peptides which differ from each other in a precise and defined manner. In principle, one way to access this population would be as a minor fraction of a large, fully combinatorial library. For example, such an array of analogues could consist of all peptides which differ from a target sequence by a single amino acid substitution at each position in a peptide sequence (cf. ‘Ala scans’). By removing this defined subset of analogues from the context of a complex, fully combinatorial mixture of peptides, handling and analysis would be greatly simplified and a more useful profile of the effects of substituting the amino acid throughout the peptide chain would be obtained. Current split resin methods do not allow for this type of control over the composition of a peptide library. (Furka et al.
Int. J. Pept. Prot. Res.
1991, 37, 487-494; Lam et al.
Nature
1991, 354, 82-84).
Typically, to investigate the molecular basis of protein function systematic modifications are made to the protein structure and the effects of those modifications on the properties of the protein are evaluated. Site-directed mutagenesis (Smith et al.
Angew. Chem. Int. Ed. Engl.
1994, 33, 1214-1220) has been the principle tool used to implement this approach and has given many insights into the contribution of individual sidechains to protein function. In particular, ‘alanine scanning’ (Wells et al.
Methods in Enzymology
1991, 202, 390-411) has been used to identify specific amino acid sidechains involved in ligand binding interactions. This technique involves the sequential substitution of native amino acids by individual alanine residues which are regarded as functionally and structurally neutral. To extend the repertoire of modifications beyond the twenty genetically encoded amino acids, methods have been developed to substitute non-natural groups into proteins (Noren et al.
Science.
1989. 244, 182-185). Although a variety of both novel sidechain and backbone modified proteins have been generated, there are apparent limits to the modifications possible using the methods of molecular biology and ribosomal synthesis (Ellman et al.
Science
1991, 255, 197-200; Cornish et al.
Angew Chem Int. Ed. Engl.
1995, 34, 621-633).
Recent advances in the total synthesis of polypeptides have opened the world of proteins to direct application of the tools of organic chemistry (Schnölzer et al.
Science
1992, 256, 221-225; Jackson et al.
Science
1994, 266, 243-247; Dawson et al
Science
1994, 266, 776-779; Canne et al.
J. Am. Chem. Soc.
1995, 117, 2998-3007; Liu et al
J. Am. Chem. Soc.
1995. 118, 307-312; Englebretsen et al.
Tet. Lett.
1995, 36, 8871-8874). Using total chemical synthesis, a variety of protein analogues has been synthesized. Of particular note have been proteins containing &bgr;-turn mimics (Baca et al.
Prot. Sci.
1993, 2, 1085-1091), N-methylated amino acids (Rajarathnam et al.
Science
1994, 264, 90-92), modified backbone atoms (Baca et al
J. Am. Chem. Soc.
1995, 117, 1881-1887), and mirror image proteins composed entirely of D-amino acids (Zawadzke et al.
J. Am. Chem. Soc.
1992, 114, 4002-4003; Milton et al.
Science
1992, 256, 1445-1448; Fitzgerald et al.
J. Am. Chem. Soc.
1995, 117, 11075-11080; Schumaacher et al.
Science
1996, 271, 1854-1857). In addition, important insights into the mechanism of action of enzymes have been attained through the total chemical synthesis of unique analogues (Baca et al.
Proc. Natl. Acad. Sci. U.S.A.
1993, 90, 11638-11642).
Although structure-function relationships in proteins can be studied using individual analogues prepared by either recombinant or chemical techniques, development of a profile of effects across the whole protein molecule is hindered by the time and effort required to generate and analyze multiple protein analogues (Matthews et al.
Ann. Rev. Biochem.
1993, 62, 139-160). The use of combinatorial oligonucleotide synthesis in conjunction with protein expression in bacteria (Reidhaar-Olsen et al.
Science
1988, 241, 53-57; Gregoret et al.
Proc. Natl. Acad. Sci. USA.
1993. 90. 4246-4250) or on phage (Scott et al.
Science
1990, 249, 386-390; Lowman, H. B. Bass, S. H.; Simpson, N.; Wells, J. A.
Biochemistry
1991. 30 10832-10838) has provided a powerful method for studying large numbers of analogue proteins. These techniques allow pools of expressed proteins to be probed for a desired function. With appropriate screening procedures, a statistical sampling of numer

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