sandeep verma department of chemistry indian institute of technology kanpur [email protected]
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Biomimetic models of protein aggregation. Sandeep Verma Department of Chemistry Indian Institute of Technology Kanpur [email protected]. 2 nd REACH Symposium March 15-18, 2008. Objectives. Ordered peptide assemblies following rules of self-organization in natural systems; morphologies - PowerPoint PPT PresentationTRANSCRIPT
Sandeep Verma
Department of ChemistryIndian Institute of Technology Kanpur
Biomimetic models of protein aggregation
2nd REACH SymposiumMarch 15-18, 2008
Objectives
Ordered peptide assemblies following rules of self-organization in natural systems; morphologies
Stimuli-responsive systems following biologically relevant principles
BiomimeticsBiomimetics
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 incomplexity
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 435:773-778, 2005
H2N CN
O
H
CO2H
Reches and Gazit, Science 300:625-627, 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 432:573-579, 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
ElectronMicrographsOf ClathrinAssembly
Bio-inspired Design of Nanocages
R = Trp Trp
“Triskelion conjugate”
MM+ structure
N
NN
N
C
C
C
O R
O
R
R
O
H
H
H
Synthetic Approach
HN
N
NH
NH
O
NH
NH
OHN NH
O
NH
NH
OHN
HN
O
HN
HN
O
NH
NH
HN
NHN
NH
O
NH
NH2O
HN
NH
O
HNNH2
ONHHN
O
NH
H2N
O NH
NH
OO
OO
O
O
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, 2002-2004
(1 mM, 60% or 90% aq. methanol)
Assembly and Disassembly
5 µm
5 µm
Fluorescent Dye Enclathration
Rhodamine B: Fluorescence micrographs
5 µm
pH 5.5
5 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
AvidinB
B
B
B
J. Mol. Biol. 279, 211-221, 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
NHHN
S
O
2OH
O
1. EDC.HCl, DMAP, DMF
2. Trp-Trp-OMe. HCl N2, 6-8h
NHHN
S
O
2 NH
OHN
O
O
O
R
HN
NH
HH
HH
1. EDC.HCl, DMAP, DMF
2. Phe-Phe-OMe.TFA N2, 6h
NHHN
S
O
2 NH
OHN
O
O
O
R
HH
R= CH3 ,
R= H, 1a
1
2
1N NaOH :MeOH (1:1)RT, 4h
R= CH3 ,
R= H, 2a
1N NaOH :MeOH (1:1)RT, 4h
NHHN
S
O
2OCH3
OHH
Joshi and Verma, Angew. Chem. 2008, in press (DOI: 10.1002/anie.200705012)
Synthetic Scheme
Self-Assembled Structures
SEM/AFM/Fluorescence
microscopy confirmation
c
a b
d
e f
Denaturing spherical
structures (urea)
NHHN
S
O
2 NH
OHN
O
O
O
R
HN
NH
HH
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
fe
Joshi and Verma, Angew. Chem. 2008, in press (DOI: 10.1002/anie.200705012)
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