Sudipta Maiti Tata Institute of Fundamental Research, Mumbai 30 Years of ASET, TIFR, 18Feb13

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

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 “Fluorescence Correlation spectrometer (FCS)” Magde et al. (1972) Sengupta et al., Methods (2002)

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 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. 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 rate X 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 <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)

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

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 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

23. 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?!

24. 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

25. 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

26. Control AS A40 0 Minutes 30 Minutes Truncated peptide also shows cell attachment

27. 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

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. 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

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. 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 hν Maiti et al., Science , 1997 Kaushalya et al., J. Neurosci. Res. (2008) Localized Excitation

33. 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

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 ? A single molecule level “Fluorescence Correlation spectrometer” Magde, Elson and Webb, PRL (1972) Review: Maiti, Haupts and Webb, PNAS (1997)

36. Combined FCS, Antibunching and TCSPC (lifetime): Simultaneouslymeasuring sizeand 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) 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>

39. The process preserves the oligomers After Lyophilization Before Lyophilization