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(57) ABSTRACT Yan et al., Science, 262:2027-2030 (1993). Holberton et al., J. Mol. Biol., 204:789-795 (1988). Methods for creating and using polypeptide probes with Koski et al., J. Biol. Chem., 267: 12258–12265 (1992). high affinity for any desirable coiled coil region are Ayer et al., Cell, 72:211-222 (1993). described, as well as heterospecific polypeptide probes Gauger and Goldstein, J. Biol. Chem., 268:13657-13666 directed to the coiled coil region of a target polypeptide. (1993). Core residue packing rules are provided for Selective amino Marshal and Holberton, J. Mol. Biol., 231:521-530 (1993). acid pairing and packing in the hydrophobic core of the Zervos et al., Cell, 72:223–232 (1993). interhelical interface. Woolfson and Alber, “Predicting Oligomerization States of Coiled Coils,” Protein Science, 4:1596–1607 (1995). 8 Claims, 6 Drawing Sheets US 6,303.317 B1 Page 2 OTHER PUBLICATIONS Smith et al., Proc. Natl. Acad. Sci. USA, 90:2846-2850 McGregor, Mol. Biotechnol, 6:155–162 (1996). (1993). Janda, Proc. Natl. Acad. Sci. USA,91:10779–10785 (1994). Lu et al., Nature Structural Biology, 2:1075–1082 (1995). Goldman et al., J. Cell Biol. 134:971–983 (1996). Popovic et al., Science, 224:497–500 (1984). Olive et al., J. Biol. Chem., 271:2040-2047 (1996). Wild et al., “A Synthetic Peptide Inhibitor of Human Immu Olive et al., J. Biol. Chem..., 272:18586–18594 (1997). nodeficiency Virus Replication: Correlation Between Solu Sourgen et al., Eur: J. Biochem., 240:765-773 (1996). tion Structure and Viral Inhibition,” Proc. Natl. Acad. Sci. Tripet et al., Prot. Eng., 9:1029-1042 (1996). USA, 89:10537–10541 (1992). U.S. Patent Oct. 16, 2001 Sheet 2 of 6 US 6,303,317 B1 4. O -22OO O -4O 18O 200 22O 24O 26O 28O 3OO wavelength (nm) FIG. 3 O P 5 O APC-55 S o anti-APCp2 as -10 O equimolar s mixture NS - 15 S -2O J$ -25 SNN-30 CD -35 O 2O 40 6O 3O 1OO temperature ('C) FIG. 4 U.S. Patent Oct. 16, 2001 Sheet 3 of 6 US 6,303,317 B1 N SS sysR& s' 3. && s s'sK FIG. s. 97 KDa 66 KDa 46 KDa O KDa FIG. 6C U.S. Patent Oct. 16, 2001 Sheet 4 of 6 US 6,303,317 B1 Human Colon Cell Lysates ac C 9%.2 's incubate with probe Šs2 4. APC antibody t add beads O-Streptavidin O-Streptavidin O-Streptavidin OStreptavidin O-Streptavidin O-Streptavidin isolate beads SDS-PAGE electrophoretic transfer Sprobe with APC antibody v. so -----a- y ézio antibody S Detect phosphorescence on Xray film (Streptavidin/Horse Radish Peroxidase and Peroxide) FIG. 7 U.S. Patent Oct. 16, 2001 Sheet S of 6 US 6,303,317 B1 FIG. 8A FIG 8B S S.9. S N s S$ & S (S&S FIG. 8C FIG 3D U.S. Patent Oct. 16, 2001 Sheet 6 of 6 US 6,303,317 B1 2 3 4 5 6 2 3 4. 293 FIG. 9A FIG 9B -- Anti-APC At Anti-g-catenin Ab FIG. 9C US 6,303,317 B1 1 2 PEPTIDE PROBES AND METHODS FOR interhelical interface adjacent to the core. In a specific MAKING THE SAME embodiment, the probe comprises a Substantially purified polypeptide sequence (SEQ ID NO: 2) directed to the APC This is a continuation-in-part of Provisional Patent protein. In a further embodiment, the invention comprises an Application No. 60/036,219, filed Jan. 28, 1997, in the name isolated polynucleotide Sequence encoding the polypeptide of the inventors listed above. of SEO ID NO: 2. This invention was made with Government support In an alternative embodiment, the present invention com prises a coiled coil Structure formed between a target coiled under Grant No. RO1GM48958 awarded by the National coil region and a probe coiled coil region, wherein the amino Institutes of Health. The Government has certain rights to acid residues located at positions a' and d" in the heptads of this invention. the probe coiled coil region are Selected according to the core residue pairing rules. In a preferred embodiment, a FIELD OF THE INVENTION leucine located at position a in a heptad of the target coiled The present invention relates generally to the field of coil region is paired with a preferred amino acid in the a' biological probes, and more Specifically to polypeptide position in a heptad of the probe coiled coil region, wherein probes directed to coiled coil proteins. 15 the preferred amino acid is Selected from the group com prising: phenylalanine, isoleucine, lysine, methionine, BACKGROUND OF THE INVENTION Serine, threonine and Valine. In a further embodiment, a leucine located at position d in a heptad of the target coiled Interactions among proteins are essential for the con coil region is paired with a preferred amino acid in the d' trolled function of all living cells, and inappropriate or position in a heptad of the probe coiled coil region, wherein adventitous protein-protein interactions are the hallmarks of Said preferred amino acid is Selected from the group com Virtually all diseases. Accordingly, biological probes prising tryptophan, Valine, arginine, glutamine, directed at either the proteins themselves, or alternatively at phenylalanine, asparagine, methionine, isoleucine and ala their nucleic acid precursers, are essential for diagnosis of nine. disease and general human health. 25 In another preferred embodiment, a valine located at Existing biological probes are either nucleic acid-based or position a in a heptad of the target coiled coil region is paired amino acid-based.