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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 Slide 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 Slide 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 Slide 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 Slide 5 Conducting Peptide Fibers silver enhancement gold enhancement Metalated Sup 35 prion fibers PNAS 2003, 100, 45274532 Slide 6 Clathrin Mimetic Synthetic Triskelion Slide 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 Slide 8 Electron Micrographs Of Clathrin Assembly Slide 9 Bio-inspired Design of Nanocages R = Trp Trp Triskelion conjugate MM+ structure Slide 10 Synthetic Approach Slide 11 Spontaneous Aggregation of Triskelion a)b)c) d)e)f) Transmission Electron Micrographs (within 5 sec): Scanning Electron Micrographs: Slide 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) Slide 13 Assembly and Disassembly Slide 14 5 m Fluorescent Dye Enclathration Rhodamine B: Fluorescence micrographs 5 m pH 5.5 5 m pH 2.2 Slide 15 DNA Encapsulation Self-assembled cages for cellular delivery of GFP plasmid Expression in mammalian cells; E.coli unpublished results Slide 16 Bioinspired Morphological Triggers Slide 17 High Affinity Biotin-Avidin Interaction Most stable biological interaction Role of tryptophan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 Slide 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 Slide 19 Joshi and Verma, Angew. Chem. 2008, in press ( DOI: 10.1002/anie.200705012 ) Synthetic Scheme Slide 20 Self-Assembled Structures Slide 21 SEM/AFM/Fluorescence microscopy confirmation c a b d e f Denaturing spherical structures (urea) Slide 22 NMR studies: Upfield shifts of aromatic protons due to partial face-to-face arrangement of the aromatic side chain, vis--vis biotin moiety. Solution Studies of Self-Organization Slide 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 ) Slide 24 Inscription on Soft Peptide Structures Slide 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 Slide 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