Sandeep Verma Department of Chemistry Indian Institute of Technology Kanpur Biomimetic models of protein aggregation 2 nd REACH Symposium March 15-18, 2008
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
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
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: , 2005Reches and Gazit, Science 300: , 2003
Conducting Peptide Fibers silver enhancement gold enhancement Metalated Sup 35 prion fibers PNAS 2003, 100, 4527–4532
Clathrin Mimetic Synthetic Triskelion
Constitution of Clathrin Lattices Nature 432: , 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
Electron Micrographs Of Clathrin Assembly
Bio-inspired Design of Nanocages R = Trp Trp “Triskelion conjugate” MM+ structure
Synthetic Approach
Spontaneous Aggregation of Triskelion a)b)c) d)e)f) Transmission Electron Micrographs (within 5 sec): Scanning Electron Micrographs:
Solvent Dependence Rapid evolution of homogeneously sized vesicles Multilamellar ultrastructure a)b)c) Ghosh et al., Angew. Chem. Int. Ed., 2007, 46, (1 mM, 60% or 90% aq. methanol)
Assembly and Disassembly
5 µm Fluorescent Dye Enclathration Rhodamine B: Fluorescence micrographs 5 µm pH µm pH 2.2
DNA Encapsulation Self-assembled cages for cellular delivery of GFP plasmid Expression in mammalian cells; E.coli unpublished results
Bioinspired Morphological Triggers
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, , 1998
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
Joshi and Verma, Angew. Chem. 2008, in press ( DOI: /anie ) Synthetic Scheme
Self-Assembled Structures
SEM/AFM/Fluorescence microscopy confirmation c a b d e f Denaturing spherical structures (urea)
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
Probing Core Structure: FIB Milling d a b c f e Joshi and Verma, Angew. Chem. 2008, in press ( DOI: /anie )
Inscription on Soft Peptide Structures
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
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