Single Molecule Spectroscopy: Single Live Cell

Kankan Bhattacharyya

• 2015: Intl. Yr of Light • 1000 yrs “Optics” by Al-

Haytham

• 200 yrs Fresnel Theory

• 150 yrs of Maxwell

• 100 yrs Light-cosmology

• 1 Nobel, 3 missed

• Raman Effect, Raman-Nath

• JC Bose: Microwave

• SN Bose: BEC

• Sudarshan: Coherent State

• CKN Patel: CO2 Laser

Light & IT

Chemistry Nobel: 2014

Chemistry of Small Things

Single Molecule Spectroscopy

- Every molecule is different

E Betzig S Hell W Moerner

K Bhattacharyya, Resonance (2015)

How to see a Single Molecule?

Focus ~ /2 = 200 nm > molecule (1 nm)

Dilute solution, ONE molecule in focus

Focal Volume = 3 = 10-15 litre = 1 fL

10-9 M= 6 x 1023 x 10-9 mol. per litre

= 6 x 1014 per litre = 0.6 mol. per fL

No of mol. in focal spot = 0.6 WHY??

Molecule in & out of focus: Diffusion

Confocal Microscopy: How to see a single molecule?

W Moerner

Stanford, (1989)

Fluorescence

Confocal

pinhole

Live Cell Imaging: Seeing inside a cell

• Cell: ~20,000 nm ~ 100 times bigger than focus

• Label different parts of a cell with fluorescent dye

• Cancer Cell: How different from a normal cell?

cell Space & time resolution

>100 ns

1.7 ns 1.3 ns

Chattoraj, KB J Phys. Chem C 118 (2014) 22339

• Au25 is characterized by very long >100 ns life time

• Au13 ~ 1 ns life time

• Short life time (1.3 & 1.7 ns) inside cell proves Au25

• Au25 in bulk water becomes Au13 inside cell

Fluorescent Gold Nanocluster Inside a Human Breast Cell

Au25 Au13

Bulk

water

Live

cell

Emission energy = Ef / N1/3

Cancer

N = number of Au atoms in cluster

Normal

Breast Cell

MCF10A Breast

Cancer Cell

MCF 7

• Why higher uptake in cancer cell?

• Cancer cell contains 7-fold higher Thiol (glutathione)

• Thiol (Glutathione) Stabilizes gold nano-clusters (Au-NC) more

• Au-NC with anti-cancer drug for selective killing of cancer cells?

Chattoraj, Bhattacharyya, J. Phys. Chem. C 118 (2014) 22339

Fig. 10 Evaluation and comparition of anticancer activity of doxorubicin loaded Au-NC and

free doxorubicin by MTT assay: (A,B) breast cells, (C,D) lung cells.

(A) (B)

(C) (D)

• Doxorubicin

• Alone kills both

cancer & normal

• On Loading to

Au-NC, normal

Cell survives

Selective killing

of cancer cells

by doxorubicin

loaded Au-NC

Breast cancer Breast normal

Lung cancer

Lung normal

Shyamtanu Chattoraj, KB,

• Warburg Effect (1956) Predicted that Cancer cells contain

more lipid droplets than normal cell

• Cancer causes glycolysis i.e. non-oxidative breakdown of

glucose to pyruvate, instead of respiration (oxidation to CO2)

• Pyruvate: precursor of fatty acid & cytoplasmic lipid droplets

• Can we show that cancer cell has more lipid droplets?

Normal

Cancer Diagnosis

Lung Cancer Cell

Cancer

Normal

Lung Cancer Cell and Lipid Droplets

Rajdeep, Surajit (IICB) KB

Med Chem Comm 5 (2014) 536

2000 downloads in 5 months!!

Normal Lung Cell

• Lipid droplets: ~20 times more in a cancer cell

• Lipid droplets: low polarity of ( ~ 6), slow solvation (1700 ps)

• Cytosol: ~ 24, solvation time ~ 1000 ps

• Cancer cell: Hydrophobics accumulate in lipid droplets &

Favors non-polar reactions

Solvation Dynamics & Intermittent Oscillation of Cell Membrane: Live CHO Cell

CHO Cell:

Cell surface thiols most reactive

Shirsendu, KB,

JPC B 118 (2014) 2949

Labeling of thiols (SH) by coumarin maleimide (CPM dye)

H

S -SH

Cysteine

o

Oxidation

Reduction

S H S H

S S

H S

H

Cytoplasm

Membrane

Cytoplasm

S S S

S

S

Cytoplasm

Membrane

Nucleus Nucleus

• Internalization: rupture of S-CPM bond. Unbound, non-

fluorescent CPM goes inside

• Membrane Potential fluctuates in seconds (due to red-ox)

• Can we see the fluctuations through fluorescence oscillations

• Possible because the ps diode lasers are very stable

(2014 Physics Nobel)

Shirshendu, KB JPC B 118 (2014) 2949

CPM H

CPM

• Origin of Oscillations (10-20%)

a) Red-Ox cycle & environment

b) Unbinding & rebinding of CPM

Shirshendu, KB,

JPC B 118 (2014) 2949

H

S -SH

Non-

fluorescent Fluorescent

Red-Ox Oscillations in Mitochondria of a Live Cell:

Breast Cancer & normal cell

Shyamtanu, KB

J. Phys. Chem. B

119 (2015, in press)

• How different is cancer & normal cell?

• Programmed Cell death: Apoptosis

Human Normal Breast Cell: MCF 10A

Shyamtanu, KB

J. Phys. Chem. B

119 (2015) 8842

• In MCF 10A breast cells : thiols in mitochondria most reactive

• Confirmed by co-localization of CPM with mitotracker green dye

Human Breast Cancer Cell: MCF-7

• CPM labels clustered mitochondria in perinuclear region of cancer cell

Scheme 1. Thiol-disulfide exchange in Mitochondria

Shyamtanu, KB

J. Phys. Chem. B

119 (2015) 8842

Normal Breast Cell

Cancer cell

• Oscillations in Normal Breast Cell

• No oscillation in Breast Cancer cell !

• Red-ox cycle less active in cancer cell

• During red-ox oscillation, cytochrome C

comes out of mitochondria and kills cell

• Normal cell dies, shows oscillation

• Cancer cell never dies, No Oscillation!!

Normal cell

CPM in MCF 10A

Shyamtanu, KB

J. Phys. Chem. B

119 (2015) 8842

Gene Silencing in a Cancer Cell by siRNA

Shyamtanu Chattoraj, S. Saha, SS Jana, KB

J. Phys. Chem. Lett. 5 (2014) 1012−1016

• siRNA= short interfering double-stranded RNA (21-23 nucleotides)

• siRNA: degrades (“silence’) disease related gene in mRNA

• siRNA: binds with ribo-nucleo-proteins to form RNA induced

silencing complex (RISC) & then guides RISC to target gene

• Once siRNA binds target gene, other agents in RISC cleave mRNA

Gene Silencing by siRNA siRNA

Formation of RNA induced Silencing complex (RISC)

Kinetics??

Degradation of mRNA i.e. silencing

Kinetics?? Degraded

mRNA

mRNA

Signaling molecules

mRNA

RISC

Specific binding of siRNA to mRNA

mRNA-siRNA

Confocal image: siRNA labeled with a

fluorescence probe (alexa 488) in MCF-7 cell

• Specific siRNA bound to mRNA:

high fluorescence intensity, many regions

• Free siRNA (in culture medium):

low fluorescence intensity

• Non-specific siRNA does not bind

to mRNA: Low fluorescence intensity

Binding of siRNA to mRNA causes

increase in fluorescence intensity

of Alexa 488

• Normal Breast Cell (MCF 10A)

• 60 sec : searching of gene

• 60-125 sec: high intensity, bound

• >125 sec: low intensity after silencing

• Red : Specific siRNA, bound, high intensity & fluctuations

• Black: non-specific siRNA (control): low intensity

High Intensity Period: bound

• At intermediate times (60-160 sec):

high fluorescence intensity & fluctuations

• Several cycles of unbinding & rebinding

Low Intensity Period

• siRNA not bound to target in m-RNA

• Initial period (0 – 60 s): searching

• Long time (>160 s): After degradation

Cancer Cell MCF-7

Fluctuations in Fluorescence Intensity

Shyamtanu, KB

JPC Lett. 5 (2014) 1012

• On-time= residence or dwell time during which

specific siRNA remains bound to mRNA

• On-time (high intensity periods):

• Multiple Values

• 16273 on-time periods detected

• All are odd-integral multiples of 5.5 ms

• Odd-integral multiples:

Evidence of Stochastic Resonance !!

On-time Histogram & Stochastic Resonance:Cancer Cell MCF-7

Shyamtanu, KB

JPC Lett. 5 (2014) 1012

• Random Noise, (t): causes transition from one well to another

• Periodic Noise, Q(): periodically lower & raise two wells, creates bias

• For Q() = sin t, x= positive & highest for t =/2, A-to-B transition

• x = negative & highest for t =3/2, B-to-A transition

• Time in well B (Bound) : 3/2 - /2 = i.e. t=/= T0/2

• If B-to-A does not occur at t =3/2, next one at + 2=3T0 /2

• Time in well B (Bound) : odd-integral multiples of T0 /2

• Stochastic Resonance:

• Noise assisted crossing: Double well

• Langevin Equation:

)()()(

Qtdx

xdUx

Free

Bound

Bound

Free

t = 0-T0/2 t = T0/2-T0

A

B A

B U(x)

x

What did we learn? siRNA

Formation of RNA induced Silencing complex (RISC)

Searching time ~ 60 s Unbinding

& rebinding

Degradation of mRNA i.e. silencing

Silencing time ~ 160 sec

On-time = N x 5.5 ms N=1,3,5,… Stochastic Resonance!

mRNA

Signaling molecules

mRNA

RISC

mRNA-siRNA

Degraded mRNA

Shyamtanu, KB

JPC Lett. 5 (2014) 1012

• Normal Breast Cell

• Faster (~70 s) silencing

• No stochastic resonance

• No unbinding/rebinding

Normal

cell

Cancer

Cell

Normal cell

Cancer Cell • Breast cancer cell

• Silencing difficult (120 sec)

• Stochastic resonance

• Cancer has a signal (sin t)!

Fluctuating Bio-molecules: smFRET & D-A distance

D + A*

A + h

D* + A

A*

D + h D*

FRET

• Forster Theory of FRET

• Rate of FRET 1/ [RDA]6

• Acceptor (Alexa) attached to Microtubules

• Donor (EGFP) bound to end-binding protein Mal3

• Mal3 binds to microtubule: only in Polymerized state

• When microtubule polymerized: FRET occurs, A em high

• When microtubule depolymerized: NO FRET, D em high

Thread Like

microtubule

Cytoskeleton network

= skeleton of

cytoplasm

• Microtubules are dynamic polymers of & tubulins

• Length of microtubule fluctuates: a few nm to 1000 nm

• D (EGFP) – A (Alexa) Distance fluctuates 28-80 Å

Prasenjit, Surajit Ghosh, KB PCCP 17 (2015) 6687

Fluorescence fluctuation of an antigen–antibody complex:

Do they have a rigid structure?

• Antigen: enhanced GFP (EGFP)

DONOR

• Antibody: anti-EGFP VHH-His6

Alexa labeled ACCEPTOR

• smFRET: D-A distance

• D-A distance fluctuates with time

• Structure not RIGID

Surajit Ghosh (IICB) & KB

PCCP 17 (2015) 25250

• Single Molecule: Each Different & organelle specific

• Etching & higher uptake of Au-NC in a live cancer cell

• Lipid droplets: implications in cancer

• Structural oscillations at cell surface

• Oscillations at Mitochondria: Normal vs Cancer Cell

• Stochastic Resonance in a Live Cancer cell

• Viewing Jumping Microtubules by smFRET

• Antigen-Antbody Complex is not Rigid

“Everything which living things do can be understood

in terms of jiggling & wiggling of atoms.”

Feynman, 1963

Acknowledgements

FUNDS DST (IRHPA)

“Centre for Ultrafast

Spectroscopy & Microscopy”

JC Bose Fellowship

STUDENTS

• Shirshendu Ghosh

• Rajdeep Chowdhury

• Shyamtanu Chattoraj

Collaborator

• Dr. S. S. Jana, IACS

• Dr. Surajit Ghosh, IICB

Thank You Very Much

Gold Nanoclusters: Next Generation Fluorophore

for Cell Imaging

Chattoraj, KB,

JPC C

118 (2014) 22339

Au25 Au13

Em energy

= Ef / N1/3

100 ns ~2 ns

Prasenjit, Surajit Ghosh, KB

PCCP 17 (2015) 6687

• Signal: Fluorescence from bound state (specific siRNA bound to mRNA)

• x = combined distance of ~21 nucleotides (siRNA-to-mRNA)

• Random Noise, (t): Random polymer chain dynamics of siRNA &

mRNA, & fluctuations in concentration of signaling molecules in RISC

• Periodic Noise, Q(): Periodic oscillations in structure & concentration

Structural Oscillation: Co-operative motion of siRNA & mRNA

Concentration oscillations of signaling molecules in RISC

• Stochastic Resonance: Bistable

• Enhancement of signal by noise

)()()(

Qtdx

xdUx

Free

Bound

Bound

Free

t = 0-T0/2 t = T0/2-T0

A

B A

B U (x)

x

Effect of Alcohol on Protein:

Size & Dynamics of Lysozyme

• Fluorescence Correlation Spectroscopy (FCS)

• Diffusion Coefficient, Dt=kT/6r (for a sphere)

• r gives size, changes when a protein unfolds

• (local friction) may vary in heterogeneous medium e.g.

in a vesicle

Dr. Subhadip

Ghosh, NISER

Dr. Dibyendu

Das, USA

Dr. Dibyendu

Sasmal, JNU

Fluorescence Intensity Fluctuation in a microscope

24.0 24.8 25.60

20

40

60

80

Time (s)

Cou

nts

• Conformational Dynamics of Protein

Quenching by NH2 (0.1 nm) OR by FRET (5 nm)

P

NH2 High intensity

Low Intensity P

Variation of size

Variation of

Relaxn time

Lysozyme

Effect of Alcohol on Size & Dynamics of Lysozyme

Shyamtanu, KB

J. Chem. Phys. 140 (2014) 115105

k+

k-

G

Shyamtanu, KB

J. Chem. Phys. 140 (2014) 115105

Oscillatory variation of folding dynamics and size of

a protein on Alcohol concn

Western Blot analysis

Synthesis of protein, NMHC-II-A (translation) by

mRNA in MCF-7 Cells

Specific siRNA stops protein synthesis by 98%

by degrading MYH9 gene in mRNA

Non-specific siRNA does not stop synthesis

Real time PCR 90% mRNA degradation within 48 hours

Indicates transcriptional inhibition

Specific

siRNA

Non-Specific

Solvation Dynamics

• Solvation : stabilization of an ion or dipole by solvent

• How long does it take in water?

Answer: <10-12 sec = 1 ps

• How long does it take in biological system?

Answer: ~100-1000 ps

• Biological water is much slower!!

• KB, Chem Comm Feature (2008), Acc Chem Res

(2003); Bagchi, JPC Feature &Chem Rev (2000)

Real biological water is inside a live cell!!

4 m

4

5 6

1 2

3

Z = 10 m above surface

A

• Lipid Vesicles

• Dye C153 only in membrane

• dia ~20 m

• focus ~0.2 m

• 4-6: near water

• 1-3: deep in wall

Supratik, KB,

Langmuir 28 (2012) 10230

• Diffusion coefficient (Dt ) of C153 in water 550 m2 s-1

• Dt of C153 in vesicle 3-21 m2 s-1

• Diffusion in vesicle (membrane) slower

• Friction not uniform : inhomogeneous

• Diffusion controls rate of reaction : varies

0 3 6 9 12 15 18 21 240

3

6

9

12

15

Dt (m2/s)

No o

f O

ccu

rren

ce • Dt in different regions

of a single vesicle

• Dt=kT/6r

• Friction in regions of

200 nm

• Solvation dynamics in live CHO cell

• Rise at long wavelength: in Microscope

0 2 4 6

0.0

0.2

0.4

0.6

0.8

1.0 DCM in cytoplasm

510 nm

570 nm

720 nm

IRF

No

rma

lize

d C

ou

nts

Time (ns)

0.0 0.8 1.6 2.4 3.2 4.00.0

0.2

0.4

0.6

0.8

1.0

Time (ns)

No

rma

lize

d C

ou

nts

420 nm

600 nm

DAPI: nucleus

Sasmal, KB Langmuir 29 (2013) 2289

Solvation Dynamics of C153 in a live CHO cell

Shirsendu, KB Langmuir 29 (2013) 7975

• Solvation time, bulk water ~ 1 ps

• Nucleus ~ 750 ps

• Cytoplasm 1100 ps

• Lipid droplets 3600 ps, ~ 7

• Lipid droplets non-polar, slow

~ 7

40 60 80 100 120 140 160 180100

150

200

250

300

350

Inte

nsi

ty o

f sp

ikes

Time (sec)

• Initial ~60 sec: Low Fluorescence intensity, searching period

• 60-160 sec: High intensity & fluctuations, unbinding-rebinding

• 160-180 sec: Gradual decrease in intensity, silencing

Variation of peak fluorescence intensity against time

Shyamtanu, KB

JPC Lett. 5 (2014) 1012

200 nm

(/2)

Bleached

by em

Absorption

abs

Stimulated

Emission

em

</2

Super Resolution Microscopy: < /2

STED = Stimulated Emission Depletion (S. HELL)

Strategy: two lasers abs & em

1) abs excites to upper state

2) em bleaches outer periphery of ~ 200 nm spot & collect

emission from smaller spot at centre

Effect of GdnHCl in the Hydrodynamic radius of HSA

Sasmal, KB JPC B

115 (2011)13075

Supratik, …Bhattacharyya

J Phys Chem B 116 (2012) 12189

Effect of ionic liq (RTIL) on Cytochrome C

• Two molten globules (MG)

• Denatured>MG>native

• Ionic liq increases size of native

• Ionic liq reduces size of denatured

• Two MG: affected differently

Salt effect on ESPT in Niosome

Addition of NaCl causes

• Decrease in amount of free water

• ESPT: Slower

• Intensity of HA higher

• Decay of HA slower

Tridib KB JPC B 116 (2012) 8105

0 M

NaCl

4M NaCl

HA A-

HA

In what time scale proton transfer

occurs inside a live cell?

Supratik, KB

JCP 138 (2013) 215102

• Ionic dye, HPTS

• No Lipid droplet!

HPTS

CHO cell

R6G

Excited State Proton Transfer (ESPT)

O3S SO3

OHO3S

_

_

_

• Photo-acid: Acidity much higher (107 times) in excited state,

pK*=0.4 and pk=7.4

• Rate of proton transfer (ESPT): ~3 ps, 90 ps in bulk water Monitored by decay of HA emission (460 nm) or rise of A- em (520 nm)

Pyranine, HPTS

HA

A-* HA*

A-

-H+

+H+

Abs HA em

440 nm

A- em

520 nm

HA* = [H+ …. A-* ]= H+ + A-*

A-

HA

• HA emission strong: Normal Lung & CHO cell

• HA emission weak in cancer cell (A549)

• ESPT faster in cancer cell !!

CHO Cell

Suprotik, KB,

JCP (2013)

A-

HA

A-

HPTS in Live cell: Emission spectrum under microscope

Rajdeep, KB, JPC B (PPT Spl issue)

Lung Cell

WI38, Normal

A549, Cancer

Water

Rise time

• Water ~90 ps

• Normal lung cell 300 ps

• Cancer cell 200 ps

Rajdeep, KB JPC B (PPT Spl issue, 2014)

Rise & Decay of 430 nm (HA) & 510 nm (A-) emission):

In live cell under a microscope

HA

A-

Rajdeep, KB JPC B (PPT Spl issue)

AHA

HA

kk

AHA

HA

Z

Y

X

dt

d

diss

PT

rec

0

0

0k

System PT(ps) rec (ps) diss (ps)

_____________________________________

Water 5 7 50

Normal Lung Cell 40 25 120

Lung Cancer Cell 40 30 80

CHO cell 50 25 120

Niosome (no NaCl) 40 30 75

• Initial proton transfer 8 times slower in lysosome than water

• Recombination ~4 times slower in cell than water

• Dissociation ~ 2 fold slower in cell

• Dissociation in normal cell 1.5 times slower than cancer cell

• Dissociation of ion pair faster in the cancer cell: cause of faster ESPT!!

Rajdeep, KB JPC B (PPT Spl issue)

Single Molecule: Why Important?

Student X

100/100 Student Y

00/100

Average 50/100

Average does not give correct picture!

Chemistry Nobel 2013 & A Missed Indian Hero

Aneesur Rahman

Born: 1927 (Hyderabad)

Died: 1987 (Minnesota)

M. Karplus M. Levitt A. Warshel

“for the development of Computer Simulation

for complex chemical systems”

What is Fluorescence Correlation

Spectroscopy (FCS)?

0 5 10 15 20 25 30 3518.8

19.0

19.2

19.4

19.6

19.8

Time (s)

De

tect

ed

Inte

nsi

ty (

kcp

s)

t1

t2

t3

t4

t5

Fluorescence observed: t2, t3, t4

No Fluorescence detected: t1, t5

< F(0)F()> G() =

<F(0)>2

F(0)

Fluorescence Intensity Fluctuation in a microscope

• Binding/unbinding of Probe

to a Protein

24.0 24.8 25.60

20

40

60

80

Time (s)

Cou

nts

• Diffusion : in & out of focus

Picosecond Dynamics under a microscope

A few molecules: How to record ?

• Emission Spectrum

• Fluorescence Decay

• Each time collect one photon: Repeat many times

• Spectrum & decay correspond to focus ~200 nm

R6G in

Membrane

DCM in

Cytoplasm

DAPI in

Nucleus

Image of Chinese Hamster Ovary (CHO) Cell

Sasmal, KB Langmuir 29 (2013) 2289

Shirshendu, KB Langmuir 29 (2013) 7995

DAPI in Nucleus and

DCM in Cytoplasm

540 600 660 720

0

2k

4k

6k

8k

Wavelength (nm)

Em

. In

ten

sit

y

400 450 500 550 600

0.0

0.2

0.4

0.6

0.8

1.0

Wavelength (nm)

Em

. In

ten

sit

y

Emission Spectra of DCM & DAPI in CHO Cell under a Microscope

Ethanol

2-Propanol

1:1

CH3OH-

water

Cytoplasm DAPI

Water

• Polarity of Cytoplasm is ~ 2-propanol, ~ 15

• Polarity of Nucleus is ~ 1:1 CH3OH-H2O mixture, ~ 65

DCM Nucleus

Sasmal, KB Langmuir 29 (2013) 2289

Conformational Dynamics in Protein

• HSA covalently labeled at cys-34 by the dye, CPM

• During conformational dynamics quenching of CPM

emission by NH2 group (in tryptophan & other residues)

H

S -SH

Cysteine

Sasmal, KB JPC B 115 (2011) 13075

Suprotik, KB JPC B 116 (2012) 12189

0 1k 2k 3k 4k 5k

0.0

0.2

0.4

0.6

0.8

1.0

Wavenumber (cm-1)

C(t

)

Nucleus

Cytoplasm

Av. Solvation time

• Cytoplasm: 1300 ps

• Nucleus: 750 ps

• Bulk water: 1 ps

Solvation Dynamics in a live CHO cell under a Microscope

Sasmal, KB Langmuir 29 (2013) 2289

Conclusion

Gene Silencing involves

• Initial search for target gene (~ 60 s)

• does not occur in one shot

• Several cycles of unbinding & rebinding

• On-time: odd-integral multiple of 5.5 ms

• Silencing occurs in ~160 s

• Stochastic Resonance

IACS

• Cancer Cell: How Different ??

• Cancer cell: Increased glycolysis,

pyruvate, lipid

• Lipid droplet (LD): ~10-fold higher

in number in a Cancer cell, A549

450 500 550 600 6500.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4 Lipid Droplet

C153 (CHO)

C153 (A549)

Inte

nsit

y

Wavelength (nm)

4 m

4

5 6

1 2

3

Z = 10 m above surface

A

• Dye C153 only

in membrane

• 4-6: near water

• 1-3: deep in wall

4 m

4

5 6

1 2

3

Z = 10 m above surface

A

• Dye C153 only in membrane

• 4-6: near water

• 1-3: deep in wall

Average solvation times

• 1200 ps in buried region 1 ( = 2000 cm-1)

• 750 ps in 6 near water pool ( = 1300 cm-1)

Solvation Dynamics under a Microscope:

Single Giant Lipid Vesicle

10 m

A few Molecule: How to record ?

• Emission Spectrum

• Fluorescence Decay

• Each time collect one photon

& then repeat many times

• C153 in DLPC (Fluid phase)

Supratik, KB, Langmuir 28 (2012) 10230

Size of a Protein in Native & Non-native States

• Cytochrome C

• Molten globules (MG): one with SDS; another with urea & SDS

• Effect of Ionic liquid on size of Native, denatured & MG

Conformational Dynamics in Protein

• HSA covalently labeled at cys-34 by the dye, CPM

• During conformational dynamics quenching of CPM

emission by NH2 group (in tryptophan & other residues)

H

S -SH

Cysteine

Sasmal, KB JPC B 115 (2011) 13075

Suprotik, KB JPC B 116 (2012) 12189

I║ ┴

Basic Design of Confocal Microscope

TSUNAMI

Pratik

IIT Kanpur

Prof GS

Agarwal

Kalyan

IIT Guwahati

Saptarshi

IISER-Bhopal Durba

BITS/

Presidency

Sudip, Visva-Bharati

University

(A)

N N

Br

(B) (C)

O

N

O

N

O

CH3

N

CH3

CH3

CPM

CPM-HSA

Ionic liquid

Rh (Ǻ)

native 38

1.5 M Ionic liquid 61

6 M GdnHCl 61

1.5 M RTIL + 6M GdHCl 41

• Ionic liquid unfolds native HSA: size increase

• Ionic liquid reduces size of HSA denatured

by GdnHCl

• Hydrophobic Collapse in presence of two

Denaturing agents

Sasmal, KB J Phys. Chem. B 115 (2011)13075

Effect of Ionic liquid

on Size of HSA

Lysosomes

• waste disposal system of the cell, digests biomolecules

• spherical vesicles containing > 50 hydrolytic enzymes

• acidic environment of about pH 5

• discovered by Christian de Duve, Nobel in 1974.

LYOSOSOME Cytoplasm Nucleus

Cargo

Building

Blocks

Lytic

Enzymes

Lung Cancer cell (A 549)

• HPTS and lysotracker dye localizes in same place

• HPTS located in lysosome

• Dt (in bulk water) = kT/6r =350 m2 s-1

• Normal Lung cell, Dt = 12 m2 s-1 ~ 30 cP

• Lung Cancer cell, Dt = 8.5 m2 s-1 ~ 40 cP

• CHO (cytoplasm) Dt = 15 m2 s-1 =~25 cP

• Viscosity of Lysosome: First measurement

HPTS

Bulk

water

CHO Cell

Cytoplasm

Fluorescence Correlation Spectroscopy

HPTS

Rajdeep, KB

JPC B (PPT Spl issue)

• Rotational Relaxn. in water (200 ps)

• Rot. Relaxn. in Cancer cell (1350 ps)

• Rotational Motion ~7 times slower

• FCS: transl. diffusion 40 times slower

• FCS & anisotropy decay probe

different motions & length scale

HPTS in live cell

under microscope

Rajdeep, KB

JPC B (PPT Spl issue)

Anisotropy Decay in a live Cell under a microscope

• No Power Law !!

Rajdeep, KB JPC B (PPT Spl issue, 2014)

Solvation Dynamics in a Live Cell: Under a Microscope

• Pure water: ~ 1 ps

• Lung Cells: Med Chem Comm (2014)

Cytoplasm: Normal (800 ps); Lung cancer cell (1000 ps)

Lipid droplets: Normal (1700 ps); Lung Cancer cell (3700 ps)

Nucleus: Normal (450 ps); Lung Cancer cell (300 ps)

• Similar slow solvation in Mitochondria, Membrane of other cells

Langmuir (2013 & 2013), JPC B (2014 & 2014)

Lung Cancer Cell

Normal Lung Cell

Factors controlling Proton Transfer

• Solvation Dynamics: slow inside a cell

• Number of “free” water molecules: Few inside a Cell

• Slow ESPT inside lysosome (compared to bulk water):

caused by slow solvation & lack of “free” water

Chemistry Nobel 2013 & A Missed Indian Hero

Aneesur Rahman Born: 1927 (Hyderabad) Died: 1987 (Minnesota)

M. Karplus M. Levitt A. Warshel

“for the development of multiscale models (Computer

Simulation) for complex chemical systems”

• Tripos, Cambridge: Math (1948), Phys („49)

• DSc: 1953, Leuven, Belgium

• Osmania Univ: 1953-57, Lecturer

• TIFR: 1957-60, Fellow

• Argonne: 1960-1985

• Father of Molecular Dynamics Simulation (PR A, 1964)

“Motion of atoms in liquid Ar” 864 Ar atoms on a CDC 3600

• Collaborators: Parinello, Klein, ….

• Micro-canonical ensemble approach to lattice gauge theory

• 1985: shifted to Minnesota for better treatment of cancer

“Accurate simulation of protein dynamics in solution,”

LEVITT* & SHARON PNAS 85 (1988) 7557-7561

“The first simulation of crystals of bovine pancreatic trypsin inhibitor (BPTI)

lasted for only a few ns(7)”

7. Hermans, J. & Rahman, A. (1976) in Models for Protein Dynamics, ed.

Berendsen, H. J. C. (CECAM, France), pp. 153-158.

Scientists who missed Nobel for Single Molecule

• Rotman (1961): hydrolysis of sugar by single enzyme

under microscope by fluorescence of dye liberated

• Magde, Elson & Webb (1972): FCS

• Keller (1983) : LIF to detect single molecule

• Bjorklund (1980): Frequency-Modulation Spectroscopy

used by Moerner (1989) for single molecule absorption

• Oritt (1990): Single molecule by fluorescence

PULSED LASER Powerful, TSUNAMI!!

Peak Power

= Pulse energy/duration

= 10-9 J/10-15 sec

= 106 (Mega) Watt !

CW LASER

Time Time

I

MODE LOCKING

RAMAN-NATH 1935

Amplified,

x 106 1012

(Tera watt)

Indian Pioneer in Laser

• invented carbon dioxide laser in 1963

• used for cutting & welding metal

• as a laser scalpel in surgery

• laser skin resurfacing

• military range finding C. Kumar N. Patel

(1938-)

TSUNAMI

Prof GS

Agarwal

2014 Physics Nobel

“Invention of efficient blue LEDs (for) white light sources”

“Incandescent light lit 20th century; 21st century will be lit by LED”

Akasaki, Amano (Nagoya), Nakamura (Nichia-Santa Barbara)

• Red & green LEDs (1960), but NO white light without blue LED

• Amano’s 1500 failures to grow GaN crystals

• Thin layer (30 nm) of AlN nucleated on sapphire at 500 C & then

heated up to 1000 C (growth temp of GaN, APL, 1986)

Prasenjit, Surajit Ghosh, KB PCCP 17 (2015) 6687

220 ms 430 ms