Not just Cheaper. Better.

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Presentation transcript:

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

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

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 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

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

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

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?

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?

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

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)

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 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

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

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

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?

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

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)

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

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

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

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 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

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?!

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

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 – 18-35 residues Folds into a hairpin very similar to the full length Aβ Muralidharan et al., Chem. Phys. (2013), in press

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

But toxicity requires the unstructured part… CTL Aβ40 Aβ10-40 Aβ14-40 Aβ17-40 Aβ22-40 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

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: 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

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

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

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)

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

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 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

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

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)

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>

The process preserves the oligomers After Lyophilization Before Lyophilization