Download (a) (b)

Supporting Information
A Comparative Study of the Influence of Sugars Sucrose, Trehalose and Maltose on the
Hydration and Diffusion of DMPC Lipid Bilayer at Complete Hydration: Investigation
of Structural and Spectroscopic Aspect of Lipid-Sugar Interaction
Arpita Roy, Rupam Dutta, Niloy Kundu, Debasis Banik and Nilmoni Sarkar*
Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, WB, India
E-mail: [email protected]
Fax: 91-3222-255303
1. Instrumentations:
1.2. Dynamic Light Scattering (DLS) Measurements.
Dynamic light scattering (DLS) measurements were performed using a Malvern Nano ZS
instrument employing a 4 mW He-Ne laser (λ = 632.8 nm) and equipped with a thermostatic
sample chamber. In the Malvern Zetasizer Nano ZS DLS instrument, the detector angle is
fixed at 173° and we have used this instrument for DLS measurement. In DLS measurements
the used water was first filtered through a syringe filter (0.2 µm) to make solutions dust-free.
All measurements were performed at 298 K.
1.3. Transmission Electron Microscopy Measurements.
Transmission electron microscopy (TEM) analysis was carried out for the structural
characterization of DMPC vesicles and DMPC vesicles in presence of sucrose was carried
out by using the JEOL model JEM 2010 transmission electron microscope at an operating
voltage of 200 kV. TEM images of the vesicles were taken by using 0.5 wt % of uranyl
acetate as the staining agent.
1.4. Fluorescence Correlation Spectroscopy (FCS) Measurement:
1
3D diffusion model have been used in order to fit the correlation curves. For K fraction of
dyes which are diffused within the system with distinct diffusion coefficients, the correlation
function G(τ) is defined as,1
1
1
1
1 1+ 1+ = 1
In the above equation, N denote the number of fluorescent species with the focal volume, is the fractional weighting factor for the i-th contribution to the autocorrelation curve and τi is
the diffusion time of the fluorescent species within the observation volume and τ is the delay
or lag time. S denotes the structure parameter of the excitation volume and it is defined as
(l/r), where l is the longitudinal radii and r is the transverse radii. Transverse radii (r) can be
determined through the fitting of an autocorrelation curve of a fluorescent species with
known diffusion constant. We have used R6G in water for this purpose and the diffusion
coefficient is (426µm2 S-1).2The curves are fitted with the following equation in order to
determine the global parameter r and S.
= 1 + !" # &
1
$%
+ ' % $" % &(.*
! #
(2)
Where the diffusion coefficient of the fluorescent species and τ is the diffusion time and in
the fitting analysis r and S are kept as linked global parameter. S= 5 is obtained after fitting
the correlation curves of R6G in water and observation volume (Veff) is obtained from the
following equation,
+,-- = . // 1 / 2
(3)
The final value of r is obtained as 365 nm and Veff is 1.35 fl. All the FCS experiments were
performed at 250C and the diffusion coefficient can be obtained from the following equation,
2
= (4)
$%
!#
Usually, diffusion is characterized by the ensemble averaged mean square displacements
(MSD) (i.e., < 1 4 >= 64, where D is normal time-dependent Stoke-Einstein diffusion
constant, and this type of diffusion is termed as Fickian diffusion).3 However, in our case,
some of the FCS traces cannot be fit by the commonly used equation describing the transport
of molecules through the focal volume via normal diffusion (i.e., α = 1 in eq 2). Thus, an
equation which is related to the anomalous diffusion is used to fit the FCS traces.
< 1 4 >= 674 (5)
The value of α in eq 5 and eq1 denotes the extent of deviation from normal diffusion (α = 1).
For α > 1, the process is termed as superdiffusive and for α < 1, the process is known as
subdiffusion.
1.5. Fluorescence Lifetime Imaging Microscopy (FLIM) Measurement
Fluorescence lifetime images of the DMPC vesicular solutions in presence of different sugars
were taken using the same DCS 120 confocal laser scanning FLIM system (Becker & Hickl
DCS-120) equipped with an inverted optical microscope of Zeiss. Detection of fluorescence
lifetime was achieved with a polarized dual channel confocal scanning instrument (Becker &
Hickl DCS-120) attached to an output port of the microscope and controlled by a galvo-drive
unit (Becker & Hickl GDA- 120). The DCS-120 is equipped with a polarizing beam splitter
and two single photon avalanche photodiode (SPAD) detectors for the acquisition of
fluorescence lifetime images. Polarized fluorescence transients used in the generation of
images presented in this work are acquired using time correlated single photon counting
detection electronics (Becker & Hickl). This system is characterized by an instrument
response function of less than 100 ps fwhm (full width half maximum).
3
1.6. Viscosity (η) Measurements. Viscosities of DMPC solution in absence and presence of
different sugar were measured using a Brookfield DV-II+Pro viscometer at 298 K. The
corresponding viscosities are given below:
Table S1: Diffusion Coefficient of C480 and C153 inside DMPC Vesicles in Presence
and absence of Various Sugars.
System
Sugars
89
: ;9
C 480
(<=> ?&9 )
8>
:;>
89
(<=> ?&9 )
: ;9
C 153
(<=> ?&9 )
8>
:;>
(<=> ?&9 )
0.33
374.23
0.67
41.63
0.27
370.07
0.73
39.18
Maltose
0.40
290.66
0.60
13.53
0.67
330.39
0.33
33.29
DMPC
Sucrose
0.16
253.14
0.84
3.82
0.39
165.84
0.61
13.32
Vesicle
Trehalose
0.37
208.16
0.63
8.83
0.50
220.82
0.50
28.64
Table S2: Viscosity of DMPC vesicular solutions in presence and absence of different
sugars.
Sugars
Viscosity
(cP)
-
1.09
DMPC
Maltose
1.77
Vesicle
Trehalose
1.78
Sucrose
1.75
4
(a)
(b)
(c)
Figure S1. Confocal fluorescent images of single sugar intercalated DMPC vesicle with
different sugars (a) maltose, (b) trehalose and (c) sucrose. In each case samples are taken in
gray mode and in FLIM mode.
5
(a)
(b)
Figure S2. Confocal fluorescent images of single maltose intercalated DMPC vesicle at a
height (z) of (a) 3 µm and (b) 5 µm above the glass surface. In both cases samples are taken
1.0
Normalized Fluorescence Intensity (a.u)
Normalized Fluorescence Intensity (a.u)
in gray mode and in FLIM mode.
DMPC
(a)
DMPC+Maltose
0.8
DMPC+Trehalose
DMPC+Sucrose
0.6
0.4
0.2
0.0
450
500
550
600
650
Wavelength(nm.)
1.0
DMPC
(b)
DMPC+Maltose
DMPC+Trehalose
0.8
DMPC+Sucrose
0.6
0.4
0.2
0.0
450
500
550
600
650
Wavelength(nm.)
Figure S3. Steady-state fluorescence spectra of (a) C480 and (b) C153 in DMPC vesicle and
in presence of different sugars.
6
1.2
(a)
1.00
C480 in DMPC
C480 in DMPC+Maltose
C480 in DMPC+Trehalose
C480 in DMPC+Sucrose
C153 in DMPC
C153 in DMPC+Maltose
C153 in DMPC+Trehalose
C153 in DMPC+Sucrose
(b)
1.0
G (τ)
G(τ)
0.8
0.75
0.50
0.6
0.4
0.25
0.2
0.30
0.15
0.00
-0.15
-0.30
0.30
0.15
0.00
-0.15
-0.30
0.30
0.15
0.00
-0.15
-0.30
0.30
0.15
0.00
-0.15
-0.30
0.0
100
1000
Time (µs)
10
10000
neat DMPC
4
4
1x10
2x10
4
3x10
4
4x10
DMPC+Maltose
4
4
1x10
2x10
4
3x10
4
4x10
DMPC+Sucrose
4
4
1x10
2x10
4
3x10
4
4x10
DMPC+Trehalose
4
100
1000
10000
100000
Time (µs)
Residual
Residual
0.00
10
4
2.0x10
4.0x10
Time (µs)
4
6.0x10
0.30
0.15
0.00
-0.15
-0.30
0.30
0.15
0.00
-0.15
-0.30
0.30
0.15
0.00
-0.15
-0.30
0.30
0.15
0.00
-0.15
-0.30
neat DMPC
3
4
8.0x10
7
2.4x10
neat DMPC+Maltose
3
3
2.0x10
3
4.0x10
6.0x10
3
8.0x10
neat DMPC+Sucrose
5
5
1x10
5
2x10
3x10
neat DMPC+Trehalose
3
5.0x10
4
1.0x10
Time (µs)
Figure S4. FCS traces of (a) C480 and (b) C153 in DMPC vesicle and in presence of
different sugars.
4
1.6x10
4
1.5x10
4
2.0x10
6
7
(b)
(a)
DMPC+Trehalose
5
Number of Event
DMPC+Sucrose
5
4
3
2
4
3
2
1
1
0
0
0
2
4
6
8
10
12
2
-1
Diffusion Coefficient, (D , µm s )
0
14
t
5
10
15
20
25
2
-1
Diffusion Coefficient, (Dt, µm s )
6
(c)
DMPC+Maltose
5
Number of Event
Number of Event
6
4
3
2
1
0
0
5
10
15
20
25
30
Diffusion Coefficient, (Dt, µm2s-1)
Figure S5. Distribution of Dt values of C480 obtained in DMPC in presence of (a) Sucrose,
(b) Trehalose and (c) Maltose.
8
30
References:
(1) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New
York, 2006.
(2) Petrasek, Z.; Schwille, P. Precise Measurement of Diffusion Coefficients using
Scanning Fluorescence Correlation Spectroscopy. Biophys. J. 2008, 94, 1437.
(3) Gorenflo, R.; Mainardi, F.; Moretti, D.; Pagnini, G.; Paradisi, P. Discrete Random
Walk Models for Space−Time Fractional Diffusion. Chem. Phys. 2002, 284, 521.
9