1. Sandeep Verma Department of Chemistry Indian Institute of Technology Kanpur sverma@iitk.ac.in Biomimetic models of protein aggregation 2 nd 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 Non-covalent interactions  Hydrogen bonding  Aromatic interactions Spatially defined or random Recruitment of Building Blocks Constituents: 162 capsomers Herpes Simplex Virus Capsid Increase in complexity Assembly

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, 2005Reches and Gazit, Science 300:625-627, 2003

5. Conducting Peptide Fibers silver enhancement gold enhancement Metalated Sup 35 prion fibers PNAS 2003, 100, 4527–4532

6. Clathrin Mimetic Synthetic Triskelion

7. Constitution of Clathrin Lattices Nature 432:573-579, 2004  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

8. Electron Micrographs Of Clathrin Assembly

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

10. Synthetic Approach

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

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

13. Assembly and Disassembly

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

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 DNA Encapsulation  Self-assembled cages for cellular delivery of GFP plasmid  Expression in mammalian cells; E.coli unpublished results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  Most stable biological interaction  Role of tryptophan residues in recognition and binding Avidin B B B B J. Mol. Biol. 279, 211-221, 1998

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

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

20. Self-Assembled Structures

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

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

23. Probing Core Structure: FIB Milling d a b c 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