Sandeep Verma

Department of ChemistryIndian Institute of Technology Kanpur

sverma@iitk.ac.in

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unpublished results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