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)
Measuring sub-nm size, inside water
“Fluorescence Correlation
spectrometer (FCS)”
Magde et al. (1972)
Sengupta et al., Methods (2002)
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
Teaching colleagues from 10
institutes how to build their own
Sensitivity > commercially available
Cost < 1/8 th
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 (?)
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
Bio-activity
<100 nm
Coles et al. Biochemistry (1998)
Crescenzi et al. Eur. J. Biochem (2002)
<10
nm
Petkova et al. PNAS (2002)
Aggregation Number
(FCS)
How do we measure things at a
sub-resolution level?
Time ( min)
Photon bunching
Diffusion
time
Time ( µs)
Emitted photons
Avg.
fluorescence
Emitted photons
Emitted photons
Photon statistics: Local excitation in a fluorescent solution
Anti-bunching
lifetime
Time ( ns)
Auto-Correlation: extracting timescales of processes
Fluorescence photon bunching and anti-bunching
Lifetime
(Confor
mation)
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
The major conformational change is between the monomer
and the small oligomer, it remains similar thereafter
20
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
Untreated
150 nM Abeta treated
Scale Bar ~ 10 µm
A mixture of Aβ monomers and oligomers can bind
to cell membranes
Nag et al., Biophys. J. (2010)
23
But everyone has the monomers?!
Do Aβ monomers bind to membranes?
Monomers , HEK cells
0 minute
30 minute
Oligomers
(same concentration
as monomers)
24
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
M L G I
AβS – 18-35 residues
I A G
K
N
S
V F F A E D V G
Folds into a hairpin very similar to the full length Aβ
Muralidharan et al., Chem. Phys. (2013), in press
A40
0 Minutes
30 Minutes
AS
Control
» But toxicity requires the unstructured part…
Percent Cell viability
100
90
80
70
60
50
40
30
20
10
0
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
» 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
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
(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)
Lalit Borde
National NMR
Facility
Funding:
DIT, DBT, TIFR
29
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
31
Challenge: Excitation is in UV, but UV kills
Solution: Multiphoton excitation
(here 3-photon excitation with
740nm)
Serotonin
350 nm
1.0
ES
Normalised Units
0.8
0.6
270 nm
hν/3
hν
0.4
hν2
Intensity high enough
to cause UV excitation
0.2
GS
0.0
240
300
360
420
Wavelength (nm)
480
Maiti et al., Science , 1997
Kaushalya et al., J. Neurosci. Res. (2008)
Localized
Excitation
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:
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)
2)
3)
4)
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)
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)
Combined FCS, Antibunching and TCSPC (lifetime):
Simultaneously measuring size and conformation
(fast)
Auto
aligned 4
collection
(Picosecond)
Abhyankar et al. ,
Proc. SPIE (2012)
Time ( min)
Photon bunching
Diffusion
time
Time ( µs)
Emitted photons
Avg.
fluorescence
Emitted photons
Emitted photons
Photon statistics: Local excitation in a fluorescent solution
Anti-bunching
lifetime
Time ( ns)
Conformation: Are the oligomers differently folded?
Forster Resonance Energy Transfer (FRET)
Acceptor
DONOR
Lifetime measures energy transfer
End-to-end distance
|S1>
Dipole-dipole energy transfer
efficiency ~ 1/ R6
kTr
Excitation
kR
kNR
|S0>
38
A nanometric ruler for interchromophoric distance
FÖrster (1948); Haugland and Stryer (1976)
misfolding
The process preserves the oligomers
Before Lyophilization
After Lyophilization