A) Understanding the Genome: We will develop combinatorial methodologies that provide precise temporal control over the structure and activity of any protein of choice, thus providing an otherwise unattainable chemical-genetic strategy for dissecting cellular pathways. We will structurally dissect individual proteins that can be functionally reconstituted in vivo and be subsequently regulated by cell-permeable peptides or small molecules. We will demonstrate the feasibility of our approach on the green fluorescent protein and subsequently target specific members of the protein phosphatase family of cellular regulators involved in multiple signal transduction cascades that are widely implicated in cellular growth, differentiation and proliferation.

Figure 1. Conceptual strategy for designed protein reassembly. The covalent complex is dissected into A and B fragments, which can subsequently reassemble only upon attachment of oligomerization domains to afford a functionally equivalent non-covalent protein complex. The active protein complex can be subsequently inhibited by either small molecules or peptides.

B) Cancer Therapeutics: We will develop novel cancer therapeutics as we answer questions regarding determinants of protein-protein recognition and b-sheet self-assembly processes. We will use phage-display methodology for the selection of thermostable variants of the B1 domain of the streptococcal Protein G (GB1) that serve as rigid 400 Ų b-sheet receptors for the cytokine, IL-6 and growth factor, VEGF, and impair their in vitro and possibly in vivo efficacy. Residues selected from phage-display will subsequently be grafted onto designed b-sheet stabilizing molecular scaffolds utilizing peptide nucleic acids.

Figure 2. a) Ribbon representation of IL-6, with residues implicated in IL-6 receptor binding shown in light blue (inset shows IL-6 with respect to GB1). b) Ribbon representation of the VEGF dimer with residues implicated in receptor binding shown in dark blue (inset shows VEGF with respect to GB1).

C) Biosensors: We will develop small molecule allosteric regulators of protein folding and therefore function. In this proposal we will use both de novo design and combinatorial approaches to redesign GB1 in order to bind designed small molecules that result in the concomitant folding of GB1. In related studies we will screen targeted libraries of the green fluorescent protein (GFP) mutants that will be fluoresce only in the presence of environmental pollutants, such as dioxin, thus serving as folding dependent molecular biosensors.

Figure 3. Strategy for the identification of dioxin dependent GFP biosensors. Mutations are introduced in the GFP sequence to create a cavity. Residues adjacent to the cavity are randomized (yellow). Colonies that show a change in fluorescence upon addition of ligand (dioxin) can be easily identified by replica plating.
 

2. Ghosh, I; Hamilton, A. D.; Regan,L. "Leucine Zipper Assisted Protein Reassembly: Application to the Green Fluorescent Protein" J. Am. Chem. Soc., 2000, 122, 5658-5659.

3. Ghosh, I, Bishop, P.,Chmielewski, J. " DNA binding properties of basic-helix-loop-helix fusion proteins of Tal and E47" J. Pept. Res. Accepted., 2000

4. Ma, Y. S.; Cunningham, M. E.; Ghosh, I; Regan, L.; Longley, B. J. "Inhibition of spontaneous receptor phosphorylation by residues in a putative alpha-helix in the KIT intracellular juxtamembrane region," J. Biol. Chem. 1999, 274, 13399-13402.

5. Ghosh, I; Issac, R.; Chmielewski, J. "Structure-function relationship in a beta-sheet peptide Inhibitor of E47 Dimerization and DNA Binding", Bioorg. Med. Chem. 1999, 7, 61-66.

6. Ghosh, I.; Chiemelewski, J., "A b- Sheet Inhibitor of IEB E47-Dimerization and DNA Binding," Chem. Biol. 1998, 5, 439-445.

7. Yao, S.; Ghosh, I.; Zutshi, R.; Chmielewski, J.; "Selective Amplification via Auto- and Cross-catalysis in a Replicating Peptide System", Nature, 1998, 396, 447-450.

8. Yao, S.; Ghosh, I.; Chmielewski, J.; "Natural Selection in Self-Replicating Peptides", Peptides: Chemistry, Structure and Biology, 1998, 15, 0000.

9. Yao, S.; Ghosh, I.; Zutshi, R.; Chmielewski, J.; "Self-replicating Peptide under Ionic Control", Angew. Chem. Int. Ed. Eng., 1998, 37, 478-481.

10. Ghosh, I.; Yao, S.; Chmielewski, J., " DNA Binding Peptides," Comprehensive Natural Products Chemistry, Vol. 7, 1998, 477-490.

11. Yao, S.; Ghosh I.; Zutshi, R.; Chmielewski, J.; "A pH-Modulated Self-Replicating Peptide", J. Am. Chem. Soc., 1997, 119, 10559-10560.

12. Bishop, P.; Ghosh, I.; Jones, C.; Chmielewski, J., "The Basic-Helix-Loop-Helix Region of Tal: Evaluation of Structure and DNA Affinity," J. Am. Chem. Soc. 1995, 117, 8283