padua 15 april 20101 interface physics group biophysics the fluredox principle: biosensors and...
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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
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Fluorescence detection of redox state
No FRET FRET
+e-
-e-
Energy
Reduced Oxidized
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4
+ ++
Proof of principle
Anal. Biochem. 350 (2006) 52
Now for:
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Single Molecules
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6Proc. Natl. Acad. Sci. (1961), 47. 1981Lorentz/EdRox, 1 Nov 2010
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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)
+
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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
Lorentz/EdRox, 1 Nov 2010
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FRET & Electrochemistry:
Fluorescent CVThe quest for single molecules
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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
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Fluorescence image (32x32 μm) of WT azurin
200 mV/s
Cyclic Voltammetry
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-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
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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
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
Potential vs. SCE (V)
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
)
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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
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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)
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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
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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
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Nitrite Reductase
NiR
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Cu-containing Nitrite Reductase - NiR
NO2-
e-
e-
Xox
NO
Xred
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What will happen during turnover?
e-
NO2-NO
Ex Em Ex ExEm Em
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21NO2- + e- + 2H+ NO + H2O
Nitrite Reductase
J. Biol. Chem. 281 (2006) 16340Lorentz/EdRox, 1 Nov 2010
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Confocal Fluorescence Spectroscopy
of
NiR
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Experimental set-up
Detection pinhole
Single photon detector
ObjectiveSample plane
Point laser light source
PNAS (2008) 105, 3250.Lorentz/EdRox, 1 Nov 2010
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Measuring single molecules at work
Background
Inactive and bleaches
Turning over and bleachesTurnover!
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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
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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
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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
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)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
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[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
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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
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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