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Sudipta Maiti Tata Institute of Fundamental Research, Mumbai 30 Years of ASET, TIFR, 18Feb13. Not just Cheaper. Better. Biology is not about cutting frogs anymore: Actually, it never was. Robert Hooke’s microscope. Source: Wikipedia. There is a revolution on in microscopy.

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sudipta maiti tata institute of fundamental research mumbai 30 years of aset tifr 18feb13
SudiptaMaiti

Tata Institute of Fundamental Research, Mumbai

30 Years of ASET, TIFR, 18Feb13

Not just Cheaper. Better.

slide2

Biology is not about cutting frogs anymore: Actually, it never was

Robert Hooke’s microscope

Source: Wikipedia

slide3

There is a revolution on in microscopy

Label-free Multi-Photon microscopy of serotonin

Sarkar et al.

Frontiers in Membrane Physiology (2012)

if you cannot see it feel it some other way
If you cannot see it, feel it some other way

Measuring sub-nm size, inside water

“Fluorescence Correlation spectrometer (FCS)”

Magde et al. (1972)

Sengupta et al., Methods (2002)

slide5

Cool Tools

S. Hell (2009)

You are only as good as your microscope

slide6

Combining FCS and Multiphoton

An alignment-free instrument with high sensitivity

Kaushalya et al., US Patent no. 7,705,987 (2010)

slide7

FCS Workshop 2009

Sensitivity > commercially available

Cost < 1/8 th

Teaching colleagues from 10 institutes how to build their own

Why couldn’t they do it before?

slide8

A culture of building instruments

Source: JPK website)

Cutting edge technology is only available in specific labs – until it is marketed

When are our best labs going to put India on the map of cutting edge scientific instruments?

slide9

Perhaps soon!

i2n Technologies, Bangalore

Holmarc, Kochi (Technology from TIFR)

slide10

Not just cheaper. Better.

A dual objective set-up: Half a million photons/sec from a single molecule

(fast)

Auto aligned 4 collection

(Picosecond)

Abhyankar et al. ,

Proc. SPIE (2012)

slide11

Why should we do it?

  • A step forward for someone else to build up the knowledge base
  • Recognition from peers all over the world
  • Promote the culture of instrument building among Indian labs and companies
  • Contribution to the economy (?)
slide12

Who should do it?

With its extra-ordinary legacy of developing scientific instruments,

TIFR MUST TAKE A LEAD

Thanks to all my students and collaborators

Thank you

slide13

Folding intermediates: progress in silico

Folding of villin headpiece

Computational Biophysics Group, UIUC

Experimentalists are far from verifying it

Optical: Fast, low resolution, NMR: Slow, high resolution

slide14

Fragments: concentration can change folding rate

X

Separation

Unfolding

Normal

Amyloid

aggregation

aggregation

Self-complimentarity

Wolynes and coworkers, PNAS (2013)

Amyloids: Aggregation and folding are intertwined

slide15

Concentration affects Amyloid-β aggregation kinetics

Oligomers

150nM

Monomer

15 nM

Nag et al., J. Biol. Chem. (2011)

Why are we interested in Aβ oligomers?

slide16

Amyloid-β intermediates are VERY interesting

<100 nm

Bio-activity

Aggregation Number

(FCS)

Coles et al. Biochemistry (1998)

Crescenzi et al. Eur. J. Biochem (2002)

Misfolding

(FRET)

How do we measure things at a sub-resolution level?

<10nm

Petkova et al. PNAS (2002)

slide17

Photon statistics: Local excitation in a fluorescent solution

Avg. fluorescence

Photon bunching

Anti-bunching

Emitted photons

Emitted photons

Emitted photons

Diffusion time

lifetime

Time ( min)

Time ( µs)

Time ( ns)

slide18

Auto-Correlation: extracting timescales of processes

Fluorescence photon bunching and anti-bunching

Lifetime

(Conformation)

Diffusion

(Size)

Abhyankar et al. , Proc. SPIE (2012)

slide19

Folding: FRET measures conformation change

Monomer

Oligomer

The monomer is “open”, while the oligomer (tetramer or larger) is a “closed” structure

need more detailed more robust information
Need more detailed, more robust information

300K, FCS measures size as a function of time

78K, flash-frozen at appropriate size

240K, lyophilized

ssNMR

(with P. K. Madhu)

slide22

The ssNMR-derived oligomer structure

PDB : 2 BEG, Riek and Coworkers

Structure similar to fibrils found earlier

Mithu et al., Biophys. J., 2011

Tertiary F19-L34 contact is also present

ssNMR shows that the small oligomer has a conformation broadly similar to the fibril

2 origin of toxicity does folding matter

Scale Bar ~ 10 µm

Untreated

150 nM Abeta treated

A mixture of Aβ monomers and oligomers can bind to cell membranes

2) Origin of toxicity: does folding matter?

Nag et al., Biophys. J. (2010)

But everyone has the monomers?!

slide24

Do Aβ monomers bind to membranes?

Monomers , HEK cells

30 minute

0 minute

Oligomers

(same concentration as monomers)

Membrane affinity drastically increases as monomers become oligomers

slide25

3) Which part of the molecule is the key?

Looking at the core only : the short “S” peptide

AβS – 18-35 residues

A

M

L

G

I

I

G

K

N

S

V

F

F

A

D

G

E

V

Folds into a hairpin very similar to the full length Aβ

Muralidharan et al., Chem. Phys. (2013), in press

slide27

But toxicity requires the unstructured part…

Percent Cell viability

CTL Aβ40 Aβ10-40 Aβ14-40 Aβ17-40 Aβ22-40 S

Membrane binding may be necessary, but it is not sufficient for toxicity

N-terminal part is required for subsequent events

A dominant model for toxicity is the leakage of neurotransmitters from vesicles

Also, analysis shows neurotransmitter packaging-related genes are affected

slide28

The Questions and the answers

1) At what stage of aggregation does the molecule fold?

As early as tetramer , perhaps earlier

2) Is there an intermediate structure?

None detected

3) Does folding determine bioactivity?

Yes, it seems to be required for membrane attachment

4) Which part of the molecule is the key?

The core (18-35) determines folding and membrane attachment, but unstructured N-terminus required for toxicity

thank you

The human parts which made this possible:

Acknowledgements:

Venus Singh Mithu

P. K. Madhu

C. Muralidharan

S. Dandekar

V. Vaidya

D. Khushalani

G. Walker

Elisha Haas

Eitan Lerner

G. Krishnamoorthy

M. Kombrabail

LalitBorde

(left to right)

Christina McLaughlin, Bidyutsarkar, DebanjanBhowmik, Anand Kant Das, SM, C. Muralidharan, Bappaditya Chandra

Also, Rajiv Abhyankar, and Suman Nag (Now in Stanford)

Thank you

National NMR Facility

Funding:

DIT, DBT, TIFR

slide30

TIRF measures ms vesicle docking events at the membrane

Experiments with Amyloids are going on….

slide31

Even artificial SUVs show the same effect

A rapid, cell free assay for Aβ bioactivity

slide32

ES

hν/3

hν2

GS

Intensity high enough

to cause UV excitation

Challenge: Excitation is in UV, but UV kills

Solution: Multiphoton excitation (here 3-photon excitation with 740nm)

350 nm

Serotonin

270 nm

Maiti et al., Science , 1997

Kaushalya et al., J. Neurosci. Res. (2008)

Localized

Excitation

slide33

The ssNMR-derived oligomer structure

PDB : 2 BEG, Riek and Coworkers

Structure similar to fibrils found earlier

Mithu et al., Biophys. J., 2011

Tertiary F19-L34 contact is also present

ssNMR shows that the small oligomer has a conformation broadly similar to the fibril

slide34

The Questions:

  • Does oligomer formation involve folding?
  • Is this structural change linked to function?
  • Which part of the peptide is responsible for which property?

The Solutions:

  • Size by FCS (Fluorescence Correlation Spectroscopy)
  • Conformation by FRET (Forster Resonance Energy Transfer)
  • Detailed conformation by solid state NMR (Flash-freezing after 1&2)
  • Bio-activity by confocal (membrane attachment) and multiphoton microscopy (neurotransmitter imaging)
if you cannot see it feel it some other way1
If you cannot see it, feel it some other way
  • How do you do it experimentally ?

A single molecule level

“Fluorescence Correlation spectrometer”

Magde, Elson and Webb, PRL (1972)

Review: Maiti, Haupts and Webb, PNAS (1997)

slide36

Combined FCS, Antibunching and TCSPC (lifetime):

Simultaneouslymeasuring sizeand conformation

(fast)

Auto aligned 4 collection

(Picosecond)

Abhyankar et al. ,

Proc. SPIE (2012)

slide37

Photon statistics: Local excitation in a fluorescent solution

Avg. fluorescence

Photon bunching

Anti-bunching

Emitted photons

Emitted photons

Emitted photons

Diffusion time

lifetime

Time ( min)

Time ( µs)

Time ( ns)

slide38

Conformation: Are the oligomers differently folded?

Forster Resonance Energy Transfer (FRET)

kTr

Acceptor

kNR

kR

Excitation

DONOR

misfolding

Lifetime measures energy transfer

End-to-end distance

|S1>

Dipole-dipole energy transfer efficiency ~ 1/ R6

A nanometric ruler for inter-chromophoric distance

FÖrster (1948); Haugland and Stryer (1976)

|S0>

slide39

The process preserves the oligomers

After Lyophilization

Before Lyophilization