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Sudipta Maiti Tata 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. - PowerPoint PPT Presentation
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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
20
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
23
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?!
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?
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
29
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