Sudipta MaitiTata 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

Sarkar et al.Frontiers in Membrane Physiology (2012)

Label-free Multi-Photon microscopy of serotonin

If you cannot see it, feel it some other way

“Fluorescence Correlation spectrometer (FCS)”

Magde et al. (1972)Sengupta et al., Methods (2002)

Measuring sub-nm size, inside water

Cool Tools

You are only as good as your microscope

S. Hell (2009)

Combining FCS and Multiphoton

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

An alignment-free instrument with high sensitivity

FCS Workshop 2009

Teaching colleagues from 10 institutes how to build their own

Sensitivity > commercially availableCost < 1/8 th

Why couldn’t they do it before?

A culture of building instruments

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?

Source: JPK website)

Perhaps soon!

i2n Technologies, Bangalore

Holmarc, Kochi (Technology from TIFR)

Abhyankar et al. , Proc. SPIE (2012)

(Picosecond)

(fast)

Auto aligned 4 collection

Not just cheaper. Better.

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

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 (?)

Thank youThanks to all my students and collaborators

With its extra-ordinary legacy of developing scientific instruments,

TIFR MUST TAKE A LEAD

Who should do it?

Computational Biophysics Group, UIUC

Folding intermediates: progress in silico

Folding of villin headpiece

Experimentalists are far from verifying itOptical: Fast, low resolution, NMR: Slow, high resolution

Fragments: concentration can change folding rate

X

Amyloid

aggregationaggregation

Normal

Wolynes and coworkers, PNAS (2013)

Separation Unfolding

Amyloids: Aggregation and folding are intertwined

Self-complimentarity

Concentration affects Amyloid-β aggregation kinetics

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

Oligomers

Monomer

150nM

15 nM

Why are we interested in Aβ oligomers?

Aggregation Number

Misfo

lding

Bio-

activ

ity

Coles et al. Biochemistry (1998)Crescenzi et al. Eur. J. Biochem (2002)

Petkova et al. PNAS (2002)

<100 nm

<10nm

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

(FCS)

(FRE

T)

Amyloid-β intermediates are VERY interesting

Time ( µs)

Emitt

ed p

hoto

ns

Time ( min)

Emitt

ed p

hoto

ns

Time ( ns)

Emitt

ed p

hoto

ns

Photon bunching Anti-bunchingAvg. fluorescence Diffusion

time

lifetime

Photon statistics: Local excitation in a fluorescent solution

Auto-Correlation: extracting timescales of processes

Fluorescence photon bunching and anti-bunching

DiffusionLifetime

Abhyankar et al. , Proc. SPIE (2012)

(Size)

(Conformation)

Monomer Oligomer

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

Folding: FRET measures conformation change

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The major conformational change is between the monomer and the small oligomer, it remains similar thereafter

1) Is there an intermediate structure?

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)

The ssNMR-derived oligomer structure

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

Tertiary F19-L34 contact is also present Structure similar to fibrils found earlier Mithu et al., Biophys. J., 2011

PDB : 2 BEG, Riek and Coworkers

2) Origin of toxicity: does folding matter?

Untreated 150 nM Abeta treated

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Scale Bar ~ 10 µm

A mixture of Aβ monomers and oligomers can bind to cell membranesNag et al., Biophys. J. (2010)

But everyone has the monomers?!

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Monomers , HEK cells

Oligomers (same concentration as monomers)

0 minute 30 minute

Membrane affinity drastically increases as monomers become oligomers

Do Aβ monomers bind to membranes?

3) Which part of the molecule is the key?

V F F A E D V GS

NK

GAIIM L G

AβS – 18-35 residues

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

Folds into a hairpin very similar to the full length Aβ

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

Truncated peptide also shows cell attachment

A40 AS Control

0 Minutes

30 Minutes

» But toxicity requires the unstructured part…

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

Perc

ent C

ell v

iabi

lity

0

10

20

30

40

50

60

70

80

90

100

Membrane binding may be necessary, but it is not sufficient for toxicityN-terminal part is required for subsequent events

A dominant model for toxicity is the leakage of neurotransmitters from vesiclesAlso, analysis shows neurotransmitter packaging-related genes are affected

» 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

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Thank you

The human parts which made this possible:

(left to right)Christina McLaughlin, Bidyut sarkar, Debanjan Bhowmik, Anand Kant Das, SM, C. Muralidharan, Bappaditya ChandraAlso, Rajiv Abhyankar, and Suman Nag (Now in Stanford)

Acknowledgements:

Venus Singh MithuP. K. MadhuC. MuralidharanS. DandekarV. VaidyaD. KhushalaniG. WalkerElisha Haas Eitan LernerG. KrishnamoorthyM. Kombrabail Lalit Borde

Funding:DIT, DBT, TIFR

National NMR Facility

TIRF measures ms vesicle docking events at the membrane

Experiments with Amyloids are going on….

31A rapid, cell free assay for Aβ bioactivity

Even artificial SUVs show the same effect

Challenge: Excitation is in UV, but UV kills

240 300 360 420 480

0.0

0.2

0.4

0.6

0.8

1.0

Norm

alise

d Unit

s

Wavelength (nm)

270 nm

350 nm

hν/3 hν2

GS

ES

hν

Intensity high enough to cause UV excitation

Localized Excitation

Maiti et al., Science , 1997

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

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

Serotonin

The ssNMR-derived oligomer structure

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

Tertiary F19-L34 contact is also present Structure similar to fibrils found earlier Mithu et al., Biophys. J., 2011

PDB : 2 BEG, Riek and Coworkers

» The Questions:1) Does oligomer formation involve folding?2) Is this structural change linked to function?3) Which part of the peptide is responsible for which property?

The Solutions:4) Size by FCS (Fluorescence Correlation Spectroscopy)5) Conformation by FRET (Forster Resonance Energy Transfer)6) Detailed conformation by solid state NMR (Flash-freezing after 1&2)7) Bio-activity by confocal (membrane attachment) and multiphoton

microscopy (neurotransmitter imaging)

If you cannot see it, feel it some other way

A single molecule level “Fluorescence Correlation spectrometer”

Magde, Elson and Webb, PRL (1972)Review: Maiti, Haupts and Webb, PNAS (1997)

How do you do it experimentally ?

Combined FCS, Antibunching and TCSPC (lifetime):Simultaneously measuring size and conformation

Abhyankar et al. , Proc. SPIE (2012)

(Picosecond)

(fast)

Auto aligned 4 collection

Time ( µs)

Emitt

ed p

hoto

ns

Time ( min)

Emitt

ed p

hoto

ns

Time ( ns)

Emitt

ed p

hoto

ns

Photon bunching Anti-bunchingAvg. fluorescence Diffusion

time

lifetime

Photon statistics: Local excitation in a fluorescent solution

38

DONOR

Acceptor

Forster Resonance Energy Transfer (FRET)

Conformation: Are the oligomers differently folded?

Lifetime measures energy transfer End-to-end distance misfolding

ExcitationkR kNR

kTrDipole-dipole energy transfer efficiency ~ 1/ R6

A nanometric ruler for inter-chromophoric distanceFÖrster (1948); Haugland and Stryer (1976)

|S0>

|S1>

The process preserves the oligomers

Before Lyophilization After Lyophilization