Transcript Solvent relaxation - UFCH JH

Effects of fluorophore’s
environment on its spectra
Lenka Beranová, Martin Hof, Radek Macháň
The fluorescence spectrum
The fluorophore’s spectrum is
determined by the spacing of its
energy levels and the
probabilities of transitions
between them (Jablonski
diagram).
S1
Absorption
Fluorescence
kf ~ 107 – 109 s-1
The fluorophore’s environment
influences its lifetime (transitions
kinetic constants) and also its
spectrum (spacing of levels)
S2
S0
To explain that we need to regard the fluorophore and the molecules
surrounding as one quantum system and look at its energy states.
Dipole-dipole interactions are the most important source of the
interactions  polar solvents have most pronounced effects
Fluorophore in a polar solvent
The molecules of the polar solvent are oriented in such a way that their
dipole moments compensate for the dipole moment of the fluorophore in
order to minimize the total energy of the system fluorophore + solvation
envelope
S1FC
S0
Emission
Excitation
S1Rel
S0FC
Franck-Condon principle: redistribution of electron density caused by an
electronic transition happens on a much faster scale than reorientation of
nuclei  Reorientation of the fluorophore’s dipole moment upon excitation
leads to en energetically unfavourable Franck-Condone state from which
the system relaxes through reorientation of fluorophore’s solvation
envelope to a state of lower energy.
Similar situation upon emission of photon from relaxed state
Fluorophore in a polar solvent
The solvent relaxation introduces an additional red shift to the Stokes shift
of the fluorophore  spectra of fluorophores in more polar solvents tend
to be shifted more to the red
S1FC
S0
Emission
Excitation
S1Rel
S0FC
The red shift is the bigger:
• the more polar the solvent is,
• the bigger the dipole moment of the fluorophore is and
• the bigger its change upon excitation is.
Lifetime vs. solvent relaxation
The time-scale of the solvent relaxation depends on the mobility of
fluorophore’s solvation envelope (local viscosity). If it is slower or
comparable to the fluorescence lifetime, emission from non-relaxed state
contributes largely to the spectrum.
S1FC
S0
Emission
Excitation
S1Rel
S0FC
The lower the temperature:
• the higher the local viscosity is,
• the smaller the red shift of the emission spectrum is.
Lifetime vs. solvent relaxation
The centre of mass of the emission spectrum is shifting to red side with
advancing relaxation (molecules which have stayed longer in the excited
state emit photons of higher wavelength). For a homogeneous sample a
mono-exponential decay of emission spectrum centre of mass can be
assumed.
 (t)     ( 0    ) exp(t /  SR )
Assuming a mono-exponential decay of fluorescence intensity (lifetime ), we
can write for the centre of mass of the steady-state spectrum:

S 
  (t ) exp(t /  ) dt
0

 exp(t /  ) dt
0
 SR     S   0
    ( 0    )
 SR
 SR  
 SR     S   
Note that the steady-state spectrum of a fluorophore, whose lifetime is
sensitive to the polarity of environment, is an interplay between the effect of
solvent on total red shift D and fluorescence lifetime 
Fluorescence spectra of Prodan
H3C
E1
CH3
N
Emission spectra of prodan in different solvents:
heptane
water
C O
CH3
Increase of solvent polarity leads to larger red-shift
Fluorescence spectra of Prodan
H3C
E1
CH3
N
Emission spectra of prodan at different temperatures:
100 K
300 K
C O
CH3
1.0
0.8
0.6
0.4
0.2
400
440
480
520
560
600
wavelength (nm)
Decrease of temperature → increase of viscosity → increasing
fluorescence contributions of non-relaxed states → blue-shift
Experimental characterization of solvent relaxation
The most comprehensive information is obtained form
Time Resolved Emission Spectra (TRES)
1,0
S1FC
0.1 ns
10 ns
S0
Intensity
S1Rel
Emission
Excitation
0,8
0,6
0,4
0,2
S0FC
0,0
480
520
560
600
wavelength (nm)
640
Fluorescence is excited by short pulses (like in lifetime measurements),
photons emitted shortly after excitation pulse come from molecules in
nonrelaxed state (had not enough time to relaxed). The longer after
excitation pulse, the more relaxed the molecules are.
The measurement requires spectral and time resolved photon detection –
can be achieved by a streak camera combined with imaging spectrograph
(2-dimensional detector, one dimension arrival time, other wavelength).
Most often measured indirectly
Time Resolved Emission Spectra (TRES)
0.1 ns
10 ns
Intensity
decays
(TRES)
D(t,λ)
5 ns
Steady-state emission
spectrum S0(λ)
500 nm
470 nm
440 nm
400 nm
2 ns
S(, t ) 
D(t ,  )  S0 ( )

 D(t, )dt
0
Time-zero estimation
Measurements:
1. Emission and absorption spectra of the dye in non-polar solvent (hexan,...)
2. Absorption spectrum of the dye in the polar system of interest (liposomes,...)
Data treatment:
Absorption or Intensity
1.0
3. Calculation of the so called
lineshape functions f(), g()
from the non-polar reference
spectra
0.8
0.6
4. Finding shift distribution p(δ)
by fitting convolution of p(δ)
and g() with polar absorption
spectrum Ap()
0.4
0.2
5. Calculation of time-zero
spectrum using f(), g(), p(δ)
0.0
18
20
22
24
26
28
3
30
32
-1
Wavenumber ( 10 cm )
Spectra of DTMAC 4-[(n-dodecylthio)methyl]-7-(N,N-dimethylamino)coumarin
J. Sykora et al. Chem. Phys. Lipids (2005) 135 213
TRES and description of the relaxation
static (spectral shift)
D   (0)   ()
fully relaxed state
Frank-Condon
state
-1
TRES centre of mass  (cm )
the change in position of the centre of
mass of the spectrum is proportional
to the polarity of the fluorophore’s
environment
24000
23000
D
22000
21000
20000
0
5
10
Time (ns)
15
20
Dependence of spectral shift on
fluorophoe’s environment polarity
CF3
Coumarin 153
N
O
O
D [cm-1]
D  is directly proportional to the
polarity function F
example:
C1OH: F = 0.71; D  = 2370 cm-1
C5OH: F = 0.57; D  = 1830 cm-1
F
= [(s-1)/ (s+2)] - [(n2-1)/ (n2+2)]
Horng et al., J Phys Chem 1995 99:17311
E2
TRES and description of the relaxation
Kinetic (correlation function and relaxation time)
 (t )   ()
C (t ) 
D
Reflects local viscosity of the
fluorophore’s surroundings
1,0

 SR   C(t ) dt
0,8
C(t)
0
0,6
C (t ) 
0,4
 (t )   ( )
 (0)   ( )
0,2
0,0
0
5
10
time (ns)
15
20
Kinetics of the relaxation reflect local viscosity
surrounding the fluorophore
E3
dyes in tetrahydrofuran 90-170 K
N
P = 0.25 s
N
Ru(bpy)2(CN)2
τCT = 4 s
O
τF = 20 ns
H
N H
N
H
O
92 K
}
}
}
Probed by
phosphorescence
Probed by chargetransfer emission
Probed by S1S0
fluorescence
170 K
R. Richert et al. Chem. Phys. Lett. (1994) 229:302
TRES and width (FWHM) of the spectra
Width (FWHM) of emission spectra changes during relaxation process. In
ideal case (all fluorophores in identical environment) it would decrease
monotonically to the width of the fully relaxed spectrum. In real samples
a maximum is observed (differences in local environment  relaxation
not “in phase”).
6000
relaxation too slow
compared to lifetime
-1
FWHM (cm )
5500
5000
4500
4000
3500
3000
0
2
4
time (ns)
6
relaxation too fast
compared to
experimental time
resolution
8
Together with the time-zero estimation it can be used to estimate how
much of the relaxation process is observable in the experiment.
Furthermore, more complex dependence suggests fluorophore
populations located in distinct environments
Red-edge excitation spectra
The emission spectra are known to be independent on the excitation
wavelength. However, that is not exactly so in polar environments of
sufficient viscosity (SR ≈> )
S1FC
S1Rel
F
S0
R
S0R*
In the equilibrium state, a small fraction of molecules in the ground state
have solvation envelopes like excited molecules in the relaxed state.
They can be excited by photons of lower energy R (located at the red
edge of the excitation spectrum)
Red-edge excitation spectra
The effect of excitation wavelength depends on ration SR / (whether the
emission spectrum is closer to 0 or ∞ )
S1FC
emission spectra excited by
F or R
S1Rel
F
S0
SR << 
R

S0R*
SR >> 
red-edge excitation spectra can
be used to estimate the
characteristic timescale of solvent
relaxation SR

Applications of solvent relaxation
Investigation of local polarity and viscosity at specific sites of
macromolecules and supramolecular complexes (biomembranes, proteins)
A. Solvent relaxation in biomembranes
polarity  amount of clustered water (forming solvation envelopes)
viscosity  restrictions to its motion – packing of molecules
a) External interface: from sub ps to ns.
b) Headgroup region:
pure ns process;
mobility of hydrated
functional groups
c) Backbone
region: several
ns; water
diffusion
bulk water: sub-ps
A
Defined localization of the fluorophores
+
H
-
O
+
N
N
-
Cl
O
O
P
O
O
DOPC
O
N
O
N
O
O
OH
O
O
S
O N
O
+
O
O
O
N
O
H
F
N
F
OH
S
N
O
NH
OH
O
O
O
O
O
O
HO
Prodan
Dauda
O
O
O
DTMAC
Laurdan
O
O
9-AS
16-AP
backbone
C17DiFU
ABA-C15
2-AS
Patman
headgroup region
local polarities and viscosities in all regions
external interface
A1
Headgroup labels (DOPC - fluid bilayer)
N
-
O
Cl
P
N
D: 3750 cm-1 (Prodan); 3000 cm-1 (Patman)
 Prodan probes larger polarity
O
O
O
+
DOPC
N
+
O
H
N
O
τSR: 1.0 ns (Prodan); 1.7 ns (Patman)
 Prodan probes lower “micro-viscosity“
O
O
O
Prodan
O
1,0
Prodan
Patman
0,8
Patman
C (t)
0,6
0,4
0,2
0,0
0
2
4
time (ns)
6
8
A1
Summary of SR in DOPC vesicles
+
H
O
N
O
-
O
O
P
O
+
N
Cl
DOPC
N
O
H
O
-
O
+
O
O
N
O
OH
O
F
O
O
DOPC
O
N
O
O
OH
F
O
H
O
O
P
O
+
-
O
O
NH
O
O
Prodan
O
O
O
Sykora, Kapusta,
Fidler, Hof (2002)
Langmuir 18 571
O
C 16 DiFU
ABA-C
9-AS
15
2-AS
Patman
D (cm-1)
τSR(average) (ns)
2100
3.4
2750
2.1
3000
1.7
3750
1.0
3100
0.5
% SR (<50 ps)
75
95
95
95
85
•
•
•
•
1700
n.d.
50
Deeper localisation means probing lower polarity and higher “vicosity”
Significant part within the external interface < 50 ps; partially “bulk” water
Head group labels: “pure” ns SR: bound water to charged and polar groups
Backbone: SR slows down with depth of location: water diffusion
A2
Membrane curvature and headgroup hydration
Motivation: membrane fusion, vesiculation, formation of new organelles,
... are intermediated via highly deformed bilayer structures.
Large unilamellar vesicles
(LUV) = low curvature
d≈200nm
Small unilamellar vesicles
(SUV) = high curvature
d≈30nm
A2
Membrane curvature and headgroup hydration
d≈30nm
d≈200nm
• degree of hydration remains
constant
• relaxation becomes faster with
increasing curvature
mobility of the dye
microenvironment increased when
the bilayer is more bent – different
packing of the bilayer
J. Sýkora et al. Chem. Phys. Lipids (2005) 135 213
τSR = 1.2 ns
τSR = 0.9 ns
B
Solvent relaxation in proteins (haloalkan dehalogenases)
Proteins substituting halogens in haloalkans with hydroxyl. Reaction in a
tunnel shaped active site
The active site of two mutations is investigated by SR – fluorescently
labelled substrate + inhibition of enzymatic activity  the fluorophore
stays for a long time in the active site
DbjA
Jesenská et al. JACS (2009) 131 494
DhaA
B
Solvent relaxation in proteins (haloalkan dehalogenases)
DbjA
D (cm-1)
SR (ns)
% observed
1300
2.8
70
DhaA
950
4.1
90
The difference correlates with molecular modelling – more polar and
mobile in wider tunnel mouth. DbjA has higher enzymatic activity
Pure ns dynamics  no “bulk” water, water in enzyme active site is
structured (like solvation envelope)
Dinitrostilbene in different solvents
Dissolved in:
a)Cyclohexane
(nonpolar)
b)Diethyl ether
(medium polar)
c)Ethyl acetate (polar)