Sandeep Verma Department of Chemistry Indian Institute of Technology Kanpur sverma@iitk.ac

1. Biomimetic models of protein aggregation Sandeep Verma Department of Chemistry Indian Institute of Technology Kanpur sverma@iitk.ac.in 2nd REACH Symposium March 15-18, 2008

2. Objectives • Ordered peptide assemblies following rules of self-organization in natural systems; morphologies • Stimuli-responsive systems following biologically relevant principles Biomimetics • Mimicry of vesicle formation: clathrin pits • Morphological triggers: biotin-avidin interaction

3. Protein/Peptide Self-Assembly Increase in complexity Assembly Recruitment of Building Blocks Herpes Simplex Virus Capsid • Constituents: 162 capsomers • Non-covalent interactions • Hydrogen bonding • Aromatic interactions • Spatially defined or random

4. Importance Peptide Self-Assembly • Importance in modeling protein aggregation in neurodegeneration • Recent advances pertaining to designed fibers and filaments for advanced applications Nelson et al. Nature 435:773-778, 2005 Reches and Gazit, Science 300:625-627, 2003

5. Conducting Peptide Fibers Metalated Sup 35 prion fibers silver enhancement gold enhancement PNAS2003, 100, 4527–4532

6. Clathrin Mimetic Synthetic Triskelion

7. Required component of vesicular transport • Clathrin building blocks are constituted of six polypeptide chains (~6000 amino acids) forming a three-legged structure - "triskelion“ • Triskelions self- assemble into spherical structures which look like a hexagonal barrel Nature 432:573-579, 2004 Constitution of Clathrin Lattices

8. Electron Micrographs Of Clathrin Assembly

9. Bio-inspired Design of Nanocages MM+ structure R = Trp Trp “Triskelion conjugate”

10. Synthetic Approach

11. Spontaneous Aggregation of Triskelion Transmission Electron Micrographs (within 5 sec): a) b) c) Scanning Electron Micrographs: d) e) f)

12. Solvent Dependence a) b) c) (1 mM, 60% or 90% aq. methanol) • Rapid evolution of homogeneously sized vesicles • Multilamellar ultrastructure Ghosh et al., Angew. Chem. Int. Ed.,2007, 46, 2002-2004

13. Assembly and Disassembly

14. Fluorescent Dye Enclathration pH 5.5 5 µm 5 µm pH 2.2 5 µm 5 µm Rhodamine B: Fluorescence micrographs

15. DNA Encapsulation • Self-assembled cages for cellular delivery of GFP plasmid • Expression in mammalian cells; E.coli unpublished results

16. Bioinspired Morphological Triggers

17. High Affinity Biotin-Avidin Interaction Avidin B B B B J. Mol. Biol.279, 211-221, 1998 • Most stable biological interaction • Role of tryptophan residues in recognition and binding

18. Mutational Analysis of Binding Site • Trp-120 to Phe-120 mutation reduces biotin binding affinity • Tryptophan contacts are crucial for recognition and binding; role of hydrophobic interactions

19. Synthetic Scheme Joshi and Verma, Angew. Chem.2008, in press (DOI: 10.1002/anie.200705012)

20. Self-Assembled Structures

21. a b d c f e • SEM/AFM/Fluorescence microscopy confirmation • Denaturing spherical structures (urea)

22. Solution Studies of Self-Organization • NMR studies: Upfield shifts of aromatic protons due to partial face-to-face arrangement of the aromatic side chain, vis-à-vis biotin moiety.

23. Probing Core Structure: FIB Milling a b c d f e Joshi and Verma, Angew. Chem.2008, in press (DOI: 10.1002/anie.200705012)

24. Inscription on Soft Peptide Structures

25. Summary • Formation of clathrin-like vesicular morphologies • Stimuli-responsive soft structures • Cellular delivery of plasmid DNA • Morphological triggers for structural control • Biotin-avidin interaction (Trp requirement) • Processing of soft biomaterials

26. Acknowledgments • Mr. K.B. Joshi, Mr. Surajit Ghosh • Chandra, Ashutosh, Nidhi, Sudipta, Jitendra, Vijay Krishna, Apurba, Prabhpreet, Rajni • IIT Kanpur Swarnajayanti Fellowship, DST Special Bioinorganic Initiative, DST