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

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Presentation on theme: "Sudipta Maiti Tata Institute of Fundamental Research, Mumbai 30 Years of ASET, TIFR, 18Feb13 Not just Cheaper. Better."— Presentation transcript:

1 Sudipta Maiti 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 Sarkar et al. Frontiers in Membrane Physiology (2012) Label-free Multi-Photon microscopy of serotonin

4 “Fluorescence Correlation spectrometer (FCS)” Magde et al. (1972) Sengupta et al., Methods (2002) Measuring sub-nm size, inside water

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

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

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

8 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)

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

10 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

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 Thanks to all my students and collaborators With its extra-ordinary legacy of developing scientific instruments, TIFR MUST TAKE A LEAD Who should do it?

13 Computational Biophysics Group, UIUC Folding intermediates: progress in silico Folding of villin headpiece Experimentalists are far from verifying it Optical: Fast, low resolution, NMR: Slow, high resolution

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

15 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?

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

17 Time ( µs) Emitted photons Time ( min) Emitted photons Time ( ns) Emitted photons Photon bunchingAnti-bunching Avg. fluorescence Diffusion time lifetime Photon statistics: Local excitation in a fluorescent solution

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

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

20 20 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 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

23 Untreated 150 nM Abeta treated 23 Scale Bar ~ 10 µm A mixture of Aβ monomers and oligomers can bind to cell membranes Nag et al., Biophys. J. (2010) But everyone has the monomers?!

24 24 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?

25 3) Which part of the molecule is the key? VFFA E D V G S N K G A II M LG A β S – 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

26 A  40 ASAS Control 0 Minutes 30 Minutes

27 » But toxicity requires the unstructured part… CTL Aβ 40 Aβ Aβ Aβ 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 The human parts which made this possible: 29 (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) 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 Funding: DIT, DBT, TIFR National NMR Facility

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

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

32 Challenge: Excitation is in UV, but UV kills 270 nm 350 nm hν/3hν2hν2 GS ES hνhν 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

33 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

34 » 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: 1)Size by FCS (Fluorescence Correlation Spectroscopy) 2)Conformation by FRET (Forster Resonance Energy Transfer) 3)Detailed conformation by solid state NMR (Flash-freezing after 1&2) 4)Bio-activity by confocal (membrane attachment) and multiphoton microscopy (neurotransmitter imaging)

35 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 ?

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

37 Time ( µs) Emitted photons Time ( min) Emitted photons Time ( ns) Emitted photons Photon bunchingAnti-bunching Avg. fluorescence Diffusion time lifetime Photon statistics: Local excitation in a fluorescent solution

38 38 DONOR Acceptor Forster Resonance Energy Transfer (FRET) Conformation: Are the oligomers differently folded? Lifetime measures energy transferEnd-to-end distance misfolding Excitation kRkR k NR k Tr Dipole-dipole energy transfer efficiency ~ 1/ R 6 A nanometric ruler for inter- chromophoric distance F Ö rster (1948); Haugland and Stryer (1976) |S 0 > |S 1 >

39 The process preserves the oligomers Before Lyophilization After Lyophilization


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