Interface Physics Group

Padua 15 April 2010 1

Interface Physics Group

Biophysics

The FluRedox Principle: The FluRedox Principle: Biosensors and Sensing Biosensors and Sensing

Single EnzymesSingle Enzymes

LeidenUniversity

Nijmegen U.R. NolteA. RowanH. EngelkampN. Hatzakis

A. Patil

Oxford U.J. J. DavisG. Mizzon

T.LION, Biophysics

J. AartsmaM. ElmalkJ. SalverdaN. Akkilic

Lorentz/EdRox, 1 Nov 2010

S.

L. Tabares

Zauner

LIC, METPROT

G. W. CantersG.

KuznetsovaA. Tepper

D. HeeringM. Strianese

Newcastle U.C. DennisonD. Kostrzc

2

Förster Resonant Energy Transfer

FRET


3

Fluorescence detection of redox state

No FRET FRET

+e-

-e-

Energy

Reduced Oxidized


4

+ ++

Proof of principle

Anal. Biochem. 350 (2006) 52

Now for:


5

Single Molecules


6Proc. Natl. Acad. Sci. (1961), 47. 1981Lorentz/EdRox, 1 Nov 2010

7

Oil

O

HO

HOOH

OH

O

O

O

O

O

HO

HOOH

OH

OH

OO

COOH

β-D-Galactosidase

B. Rotman, P.N.A.S. 1961, 47, 1981.

OilH2O

The first single enzyme experiment (1961)

+


8

H. P. Lu, L. Xun, X. S. Xie,H. P. Lu, L. Xun, X. S. Xie,Science, Science, 1998,1998, 282 282, 1877, 1877..

OH O

H2O2 O2

Fluorescent

CholesterolCholesteroloxidaseoxidase

Cox in oxidized form


9

FRET & Electrochemistry:

Fluorescent CVThe quest for single molecules


10

Fluorescence detection with Potentiostatic control

Potentiostat

Protein with attached dye

CCD camera

Fluorescencemicroscope

Reference electrode

Work electrode

Counter electrode

Gold with C8 monolayer and wt-azurin


11

Fluorescence image (32x32 μm) of WT azurin

200 mV/s

Cyclic Voltammetry


12

-0.2

-0.1

0

0.1

0.2

0.3

0 10 20 30 40 50

Time (s)

Po

ten

tial

vs.

SC

E (

V)

37 μm16 μm

0

500

1000

1500

2000

0 10 20 30 40 50Time (s)

Flu

ore

sc

en

ce

(a

.u.)

-2000

-1000

0

1000

2000

0 20 40 60 80 100

Time (s)

Flu

ore

sc

en

ce

(a

.u.)

Fluorescence traces show cyclic redox switching

Widefield (Leiden): near-monolayer w. variable brightness

TIRF (Oxford): very low coverage in clusters

Results – fluorescence switching


13

0

0.4

0.8

1.2

-0.2 -0.1 0 0.1 0.2 0.3

Potential vs. SCE (V)

Flu

ore

scen

ce (

a.u

.)

0

0.4

0.8

1.2

-0.2 -0.1 0 0.1 0.2 0.3


Flu

ore

scen

ce (

a.u

.)

0

0.4

0.8

1.2

-0.2 -0.1 0 0.1 0.2 0.3


Flu

ore

sce

nc

e (a

.u.)

10 mV/s

100 mV/s

1 V/s

FCV and CV: increase of separation to ~40 mV at 1V/s(widefield (Leiden) data example, FCVs from full images)

Scan rate dependence

150

100-100

-50

0

50

100

0.01 0.1 1 10

Scan rate (V/s)

Po

ten

tial

(m

V, v

s. S

CE

)


14

0

1

2

3

4

5

6

7

8

-50

-30

-10 10 30 50 70

E 0 vs. SCE (mV)Fr

eque

ncy

E0 dispersion much larger in more dilute TIRF sample!

TIRF (N42C) (Oxford)

Widefield (wt azurin) (Leiden)

Thermodynamic (E0) dispersion

0

10

20

30

40

50-5

0

-30

-10

10 30 50 70

E0 vs. SCE (mV)

Fre

qu

enc

y


15

0

5

10

15

20

0.2

5 1 4

16

64

25

6

k 0 (s−1)

0

5

10

15

20

0.25 0.

5 1 2 4 816 32 64

128

256

512

Fre

qu

ency

0

5

10

15

20

0.2

5 1 4

16

64

25

6

k 0 (s−1)

* Large k0 dispersion in both datasets!

* Factor 100 difference within 10 micron on surface possible

Kinetic (k0) dispersion

TIRF (N42C) (Oxford)

Widefield (wt azurin) (Leiden)

Angew. Chemie 2010, in press

0

5

10

15

20

Fre

qu

ency

0

5

10

15

20

0.2

5 1 4

16

64

25

6

k 0 (s−1)


16

Dispersion E0

Protein-protein complexes

Effect of charges

Dielectric between partners

Protein-surface interaction

El. Fields of 3-30mV/Å

ΔE0: 0-100 mV

Batie & Kamin, JBC 256(1981)7756

Knaff cs BBA 635(1981)405

Davidson cs JBC 263(1988)13987

Haehnel cs Biochem 35(1996)1282

Murgida & Hildebrandt Chem S Rev 37(2008)937


17

S

S

n

Dispersion k0

S S S S S S SS S S S S S S S S S S S S S S S S S S S S S S S S

S S S S S S S S S S S S S S

k0

Feng et al. J.Chem.Soc.

Far. Trans. 1997 93, 1367


18

Nitrite Reductase

NiR


19

Cu-containing Nitrite Reductase - NiR

NO2-

e-

e-

Xox

NO

Xred


20

What will happen during turnover?

e-

NO2-NO

Ex Em Ex ExEm Em


21NO2- + e- + 2H+ NO + H2O

Nitrite Reductase

J. Biol. Chem. 281 (2006) 16340Lorentz/EdRox, 1 Nov 2010

22

Confocal Fluorescence Spectroscopy

of

NiR


23

Experimental set-up

Detection pinhole

Single photon detector

ObjectiveSample plane

Point laser light source

PNAS (2008) 105, 3250.Lorentz/EdRox, 1 Nov 2010

24

Measuring single molecules at work

Background

Inactive and bleaches

Turning over and bleachesTurnover!


25

Intensity histogram

300 320 340 360 380 4000

5

10

15

20

25

Co

un

ts /

10 m

s

360 361 3620

5

10

15

20

25

Co

un

ts /

10 m

s

Time, s

Time, s 0 5 10 15 20 25

0

100

200

300

400

500

600

700

Nu

mb

er

of

bin

s

counts/bin

Binsize: 10 ms

Poissonian distributions


26

NiR - ATTO 655 turnovers with asc/PES

20mM HEPES pH710mM NO2

-

3mM ascorbate0.3 nM PES

25

360 361 362

0

5

10

15

20

highCou

nts

/ 10

ms

Time, s

bglow

high

300 320 340 360 380 4000

5

10

15

20

25

Cou

nts

/ 10

ms

Time, s

bglow

0 5 10 15 20 25

0

1000

2000

3000

4000

5000

6000

7000

Nu

mb

er

of

eve

nts

Fluorescence intensity, counts/10 ms


27

Autocorrelation:Correlation of a signal with its time-shifted image.

Fluorescence time trace: AUTOCORRELATION

Fluorescence

t1

t2

t

2)(I

)t(I)(I)t(G

0

0


28

)texp(A)texp(A)t(G 2211 )texp(A 22

λ1, λ2: f (ki)

S1S2

S3 k3

k-3

k1

k2

k-2

k-1

OO

RO

OR

k3

k1

k2

e

NO2-NO

k-3

Qian & Elson Biophys Chem 101-102 (2002) 565


29

[NO2-]-dependent autocorrelation decay timing

The autocorrelation curves

can be fitted to a

stretched exponential:

0/ tetG

=0.8

0.7

0.6

0.6ms

70342217

i/ti eA

0/ tetG X


30

de t /)(

Single exponential meansSingle rate:

Stretched exponential meansDistribution of rates:

/)( tetG

)/( 0)( tetG

τ/τ0

ρ

0.8

0.7 d)(

)/(Γ/0 1

k/1

Mumbai 4 Nov 2009

31

Why a "stretched" instead of a simple exponential?

In the stretched exponential is not a single value but a distribution

The distribution of depends on :if =1, there is no distribution in if <1, the distribution becomes broader

0.070 s0.034 s0.022 s0.017 s

[NO2-]

5M50M

500M5000M

0.810.720.600.61

= 0.6

1 order of magnitude distribution

WHY?

50.50

Mumbai 4 Nov 2009

32

A partial disorder at the catalytic heart of NiR First coordination sphere - Type-1 Cu site: Met150 is partially disordered

- Type-2 Cu site: The water ligand is disordered in the reduced state

Proton delivery - His255: is partially disordered

- Asp98 : has a large B-factor

- Network of water molecules

PNAS 105 (2008) 3250


33

How can we get the kinetics parameters?

Global fit:

Mumbai 4 Nov 2009

04/21/23 34

How can we get the kinetics parameters?

k1 = 3.5 x105 M-1s-1

k2 = 9.5 s-1

k3 = 21 s-1

k-3 = 14 s-1

Electron Transfer Ratebetween Cu1 and Cu2!

KM = k2 ( k3 + k-3 )

k1 ( k2 + k3 )= 31 M

Vmax = k2k3

( k2 + k3 )= 6.5 s-1

In good agreement with in-bulk measurements : 50 M and 8.0 s-1

Mumbai 4 Nov 2009

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