1. Not just Cheaper. Better.
Sudipta Maiti
Tata Institute of Fundamental Research, Mumbai
30 Years of ASET, TIFR, 18Feb13

2. Biology is not about cutting frogs anymore: Actually, it never was
Robert Hooke’s microscope
Source: Wikipedia

3. There is a revolution on in microscopy
Label-free Multi-Photon microscopy of serotonin
Sarkar et al.
Frontiers in Membrane Physiology (2012)

4. If you cannot see it, feel it some other way
Measuring sub-nm size, inside water
If you cannot see it, feel it some other way
Let’s have a look at the optical lay-out of the spectrometer.
Laser light is focused into the sample through a microscope objective lens. This is the region near the focus, the expanded double cone. We know by now that because of the diffusional motion of the molecules, the signal will fluctuate. The fluctuating signal is collected through the same objective lens, separated from the excitation laser light by using a dichroic mirror and collected using an APD detector.
This is how a real data looks… and this is the autocorrelation function calculated from this data. X-axis is the delay time plotted in log scale, y-axis is the autocorrelation function… the decay time of this function is related to the diffusion time of that molecule in the optically defined probe volume.
“Fluorescence Correlation spectrometer (FCS)”
Magde et al. (1972)
Sengupta et al., Methods (2002) 4

5. Cool Tools
S. Hell (2009)
You are only as good as your microscope

6. Combining FCS and Multiphoton
An alignment-free instrument with high sensitivity
Kaushalya et al., US Patent no. 7,705,987 (2010)

7. FCS Workshop 2009 Why couldn’t they do it before?
Sensitivity > commercially available
Cost < 1/8 th
Teaching colleagues from 10 institutes how to build their own
Why couldn’t they do it before?

8. 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?

9. Perhaps soon! i2n Technologies, Bangalore
Holmarc, Kochi (Technology from TIFR)

10. 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)

11. 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 (?)

12. Thank you 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

13. 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

14. Fragments: concentration can change folding rateX
Separation
Unfolding
Normal
Amyloid
aggregation
aggregation
Self-complimentarity
Wolynes and coworkers, PNAS (2013)
Amyloids: Aggregation and folding are intertwined

15. 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?

16. Amyloid-β intermediates are VERY interesting
Aggregation Number
Misfolding
Bio-activity
Coles et al. Biochemistry (1998)
Crescenzi et al. Eur. J. Biochem (2002)
Petkova et al. PNAS (2002)
<100 nm
(FCS)
(FRET)
How do we measure things at a sub-resolution level?
<10nm

17. 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)

18. Auto-Correlation: extracting timescales of processes
Fluorescence photon bunching and anti-bunching
Lifetime
(Conformation)
Diffusion
(Size)
Abhyankar et al. , Proc. SPIE (2012)

19. Folding: FRET measures conformation change
Monomer
Oligomer
The monomer is “open”, while the oligomer (tetramer or larger) is a “closed” structure

20. 1) Is there an intermediate structure?
The major conformational change is between the monomer and the small oligomer, it remains similar thereafter

21. 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)

22. The ssNMR-derived oligomer structure
PDB : 2 BEG, Riek and Coworkers
Tertiary F19-L34 contact is also present
Structure similar to fibrils found earlier
Mithu et al., Biophys. J., 2011
ssNMR shows that the small oligomer has a conformation broadly similar to the fibril

23. 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
Nag et al., Biophys. J. (2010)
But everyone has the monomers?!

24. Do Aβ monomers bind to membranes?
Monomers , HEK cells
0 minute
30 minute
Oligomers
(same concentration as monomers)
Membrane affinity drastically increases as monomers become oligomers

25. 3) Which part of the molecule is the key?
Looking at the core only : the short “S” peptide V F A E D G S N K I M L
AβS – residues
Folds into a hairpin very similar to the full length Aβ
Muralidharan et al., Chem. Phys. (2013), in press

26. Truncated peptide also shows cell attachment
AS
Control
0 Minutes
30 Minutes

27. But toxicity requires the unstructured part…
CTL Aβ Aβ Aβ14-40 Aβ17-40 Aβ S
Percent Cell viability
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

28. 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

29. Thank you The human parts which made this possible: Lalit Borde
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
Lalit Borde
(left to right)
Christina McLaughlin, Bidyut sarkar, Debanjan Bhowmik, Anand Kant Das, SM, C. Muralidharan, Bappaditya Chandra
Also, Rajiv Abhyankar, and Suman Nag (Now in Stanford)
National NMR Facility
Thank you
Funding:
DIT, DBT, TIFR

30. TIRF measures ms vesicle docking events at the membrane
Experiments with Amyloids are going on….

31. Even artificial SUVs show the same effect
A rapid, cell free assay for Aβ bioactivity

32. Challenge: Excitation is in UV, but UV kills
Solution: Multiphoton excitation (here 3-photon excitation with 740nm)
Serotonin
270 nm
350 nm
Intensity high enough
to cause UV excitation
hν/3
hν2 GS ES hν
Localized
Excitation
Dopamine is a small organic molecule which is derived from the amino acid tyrosine and acts mainly as a neurotransmitter. It is packed inside vesicles and released out when a neuron wants to communicate to another neuron. There are two main dopaminergic pathways in our brain.. one to do with locomotion and the other to do with reward and addiction.
Maiti et al., Science , 1997
Kaushalya et al., J. Neurosci. Res. (2008)

33. The ssNMR-derived oligomer structure
PDB : 2 BEG, Riek and Coworkers
Tertiary F19-L34 contact is also present
Structure similar to fibrils found earlier
Mithu et al., Biophys. J., 2011
ssNMR shows that the small oligomer has a conformation broadly similar to the fibril

34. 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)

35. If you cannot see it, feel it some other way
How do you do it experimentally ?
If you cannot see it, feel it some other way
Let’s have a look at the optical lay-out of the spectrometer.
Laser light is focused into the sample through a microscope objective lens. This is the region near the focus, the expanded double cone. We know by now that because of the diffusional motion of the molecules, the signal will fluctuate. The fluctuating signal is collected through the same objective lens, separated from the excitation laser light by using a dichroic mirror and collected using an APD detector.
This is how a real data looks… and this is the autocorrelation function calculated from this data. X-axis is the delay time plotted in log scale, y-axis is the autocorrelation function… the decay time of this function is related to the diffusion time of that molecule in the optically defined probe volume.
A single molecule level
“Fluorescence Correlation spectrometer”
Magde, Elson and Webb, PRL (1972)
Review: Maiti, Haupts and Webb, PNAS (1997) 35

36. Combined FCS, Antibunching and TCSPC (lifetime):
Simultaneously measuring size and conformation
(fast)
Auto aligned 4 collection
(Picosecond)
Abhyankar et al. ,
Proc. SPIE (2012)

37. 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)

38. Conformation: Are the oligomers differently folded?
Forster Resonance Energy Transfer (FRET)
Acceptor
DONOR
Lifetime measures energy transfer
End-to-end distance
misfolding
Excitation kR
kNR
kTr
|S1>
Dipole-dipole energy transfer efficiency ~ 1/ R6
A nanometric ruler for inter-chromophoric distance
FÖrster (1948); Haugland and Stryer (1976)
|S0>

39. The process preserves the oligomers
After Lyophilization
Before Lyophilization