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Abstract and Figures

In the current study, ANS fluorescence was established as a powerful tool to study proteins in solid-state. Silk fibroin from Bombyx mori cocoons was used as a paradigm protein. ANS incorporated into the films of silk fibroin exhibits fluorescence with two-lifetime components that can be assigned to the patches and/or cavities with distinct hydrophobicities. Decay associated spectra (DAS) of ANS fluorescence from both sites could be fit to the single log-normal component indicating their homogeneity. ANS binding sites in the protein film are specific and could be saturated by ANS titration. ANS located in the binding site that exhibits the long-lifetime fluorescence is not accessible to the water molecules and its DAS stays homogeneously broadened upon hydration of the protein film. In contrast, ANS from the sites demonstrating the short-lifetime fluorescence is accessible to water molecules. In the hydrated films, solvent-induced fluctuations produce an ensemble of binding sites with similar characters. Therefore, upon hydration, the short-lifetime DAS becomes significantly red-shifted and inhomogeneously broadened. The similar spectral features have previously been observed for ANS complexed with globular proteins in solution. The data reveal the origin of the short-lifetime fluorescence component of ANS bound to the globular proteins in aqueous solution. Findings from this study indicate that ANS is applicable to characterize dehydrated as well as hydrated protein aggregates, amyloids relevant to amyloid diseases, such as Alzheimer's, Parkinson, and prion diseases.
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Biochemistry and Biophysics Reports 24 (2020) 100843
2405-5808/© 2020 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
ANS uorescence: Potential to discriminate hydrophobic sites of proteins in
solid states
Aytaj J. Guliyeva, Oktay K. Gasymov
*
Laboratory of Structure, Dynamics and Functions of Biomolecules, Institute of Biophysics of ANAS, 117 Z. Khalilov, Baku, AZ1171, Azerbaijan
ARTICLE INFO
Keywords:
ANS uorescence
Solid-state protein characterization
Biolms
Hydration
Decay associated spectra
ABSTRACT
In the current study, ANS uorescence was established as a powerful tool to study proteins in solid-state. Silk
broin from Bombyx mori cocoons was used as a paradigm protein. ANS incorporated into the lms of silk broin
exhibits uorescence with two-lifetime components that can be assigned to the patches and/or cavities with
distinct hydrophobicities. Decay associated spectra (DAS) of ANS uorescence from both sites could be t to the
single log-normal component indicating their homogeneity. ANS binding sites in the protein lm are specic and
could be saturated by ANS titration. ANS located in the binding site that exhibits the long-lifetime uorescence is
not accessible to the water molecules and its DAS stays homogeneously broadened upon hydration of the protein
lm. In contrast, ANS from the sites demonstrating the short-lifetime uorescence is accessible to water mole-
cules. In the hydrated lms, solvent-induced uctuations produce an ensemble of binding sites with similar
characters. Therefore, upon hydration, the short-lifetime DAS becomes signicantly red-shifted and inhomoge-
neously broadened. The similar spectral features have previously been observed for ANS complexed with
globular proteins in solution. The data reveal the origin of the short-lifetime uorescence component of ANS
bound to the globular proteins in aqueous solution. Findings from this study indicate that ANS is applicable to
characterize dehydrated as well as hydrated protein aggregates, amyloids relevant to amyloid diseases, such as
Alzheimers, Parkinson, and prion diseases.
1. Introduction
8-anilino-1-naphthalenesulfonic acid (ANS), as an extrinsic uores-
cent probe is widely utilized to characterize proteins in various states
[1]. In a molten globule state, enhancement of ANS uorescence in-
dicates that the hydrophobic clusters of proteins are exposed [25]. The
time-resolved uorescence of ANS bound to proteins provides a more
detailed description of the hydrophobic clusters. Long- (1015 ns) and
short- (25 ns) lifetimes of ANS correspond to the solvent shielded and
exposed hydrophobic clusters, respectively [510]. Fluorescent probes
thioavin T (ThT) and ANS are used together to study kinetics and
mechanisms of amyloid bril formations relevant to the serious diseases,
such as Alzheimers, Parkinsons, prion diseases, etc [1115]. ANS
uorescence has also been used to study the pH- and urea-dependent
structural transitions as well as to characterize the multiple binding
sites of the proteins [16,17].
Mechanisms by which ANS contributes to uorescence enhancement
processes are broadly studied. ANS free in aqueous solution demon-
strates a negligible uorescence intensity with a maximum of around
540 nm. It has been suggested that both restricted mobility and hydro-
phobicity of the nearest environment of ANS contribute signicantly to
the blue shift of the uorescence spectra and increased uorescence
intensity and lifetime. The application of steady-state uorescence
provides limited information about hydrophobic sites of the proteins. In
the presence of multiple binding sites, the contribution of external hy-
drophobic sites exposed to solvent is masked by more intense uores-
cence from buried sites. The time-resolved uorescence of ANS offers a
more detailed description of the hydrophobic sites associated with
different degrees of solvent exposure.
ANS uorescence depends on ion pairing with positively charged
side chains, a variation of solvent polarity and viscosity [1821]. Two
distinct excited states contribute to the emission of ANS [22,23].
Non-polar (NP) state localized on the naphthalene moiety of ANS is the
rst step of the excitation event. NP state is the uorescent state for ANS
in hydrophobic solvents. The wavelength of uorescence maximum of
ANS from this state moderately depends on polarity. In polar solvents,
NP state relaxes to form the intramolecular charge-transfer state (ICT),
which is dynamically stabilized through molecular distortion and
* Corresponding author.
E-mail addresses: ogassymo@g.ucla.edu, oktaygasimov@gmail.com (O.K. Gasymov).
Contents lists available at ScienceDirect
Biochemistry and Biophysics Reports
journal homepage: www.elsevier.com/locate/bbrep
https://doi.org/10.1016/j.bbrep.2020.100843
Received 1 August 2020; Received in revised form 12 October 2020; Accepted 25 October 2020
Biochemistry and Biophysics Reports 24 (2020) 100843
2
solvent relaxation. Along with intermolecular electron transfer (ET),
ionization and subsequent electron salvation were also detected in the
aqueous solution of ANS. ET is considered to be a major quenching
mechanism for ANS uorescence yielding a very low quantum yield [22,
23]. Fluorescence of ANS in polar solvents is attributed to the emission
from charge transfer state (CT).
Until now, however, ANS has been applied only to characterize the
various protein states in solution. No efforts have been made to show the
feasibility of ANS to characterize protein in solid states. In this study,
ANS was incorporated into silk broin (SF) lms to characterize the
hydrophobic cluster of the protein in solid-state. The time-resolved
uorescence of ANS incorporated into the SF lm indicates that it has
the potential to characterize protein sites with different hydrophobic-
ities linked to the distinct uorescence lifetimes. Partial hydration of the
SF lms further discriminates hydrophobic sites by accessibility to the
water molecules. The results unambiguously reveal the origin of the
protein sites where ANS uorescence shows inhomogeneously broad-
ened spectra with the lifetimes of about 25 ns. These hydration induced
spectral features have also been demonstrated for globular proteins in
solution. SF was used as a paradigm protein. Steady-state and time-
resolved uorescence can be utilized with ANS to characterize other
proteins in ber, lm, and dehydrated amyloid bril forms. The appli-
cation by using the ANS uorescence can also be used to monitor the
hydration dynamics of amyloid brils that are relevant to neurodegen-
erative disease propagation.
2. Materials and methods
2.1. Preparation of aqueous solution of silk broin
Silk cocoons from the silkworm of Bombyx mori were used to obtain
silk broin using a widely employed degumming procedure [24,25].
Silk cocoons cut into small pieces boiled for 30 min in 0.02 M Na
2
CO
3
(Fisher Sci.) and then rinsed extensively with deionized water to remove
sericin. The air-dried SF was dissolved in 9.5 M LiBr (Sigma-Aldrich)
solution and then dialyzed (molecular weight cut-off 10 kDa) against
deionized water 48 h at 4 C. Then the silk solution was centrifuged for
10 min at 9000 rpm, and the supernatant was collected and stored at
4 C. The concentration of the stock solution of SF was determined by
spectrophotometer using
ε
275nm
=1.064 cm
1
(mg/ml)
1
[26]. The
molar concentration of the solution was calculated using 390 kDa for the
molecular weight of SF. The nal concentration of the stock solution of
SF was determined to be 95
μ
M.
2.2. Preparation of ANS incorporated lms of silk broin
The stock solution of ANS was prepared with deionized water and its
concentration was determined using the molar extinction coefcient of
ε
350nm
=4950 M
1
cm
1
(provided by Sigma-Aldrich). The solution of
SF (95
μ
M) was used to prepare the mixtures with various molar ratios of
SF:ANS (1:0.13, 1:0.35, 1:0.72, 1:0.87, 1:1). The lms were obtained by
slow evaporation of the mixture solutions on polystyrene substrates. The
formation of ANS aggregates is not expected in the lms with SF:ANS
above shown ratios. Aggregate formation of the ligand (rhodamine 6G)
in the SF matrix was examined previously. It has been shown that the
ligand binding to the protein prevents aggregate formation even in mM
concentrations of the ligand [25]. Besides, the lifetimes of DAS com-
ponents of ANS uorescence are not changed much in the lms with
various SF:ANS ratios. Usually, aggregate formations of the chromo-
phores result in decreased uorescence lifetimes.
Hydration of the lms was performed using two quartz plates sealed
with a spacer. The lm was placed in the vertical position between the
quartz plates. The bottom of the quartz plates was lled with water that
did not touch the lm. The system was left to equilibrate for one day.
The hydration value was determined immediately after the experiment
by weight.
2.3. Absorption spectroscopy
UV absorption spectra of broin solutions and lms with various SF:
ANS ratios were measured using Shimadzu UV-2700 spectrophotometer.
Spectral bandwidth was set to1.0 nm.
2.4. Steady-state and time-resolved uorescence spectroscopy
Steady-state and time-resolved uorescence were recorded using a
spectrouorometer FluoTime 300 (PicoQuant, Germany). The steady-
state measurements were performed in front-face mode using an exci-
tation wavelength of 375 nm. Bandwidths for excitation and emission
were 3 nm. In the time-resolved experiments, a picosecond laser with a
wavelength of 375 nm was used for excitation. The emission bandwidth
was 2.5 nm.
2.5. Construction of decay-associated spectra (DAS)
To construct DAS for various SF:ANS lms, the ANS uorescence
decay measurements were performed for the wavelengths across the
emission spectra with a 5 nm interval. The emission intensity was
measured with a front-face setup. The instrument response function
(IRF) was determined by measuring scattered light from pristine SF lm.
The intensity decay data were analyzed using the software supplied with
the PicoQuant instrument. For a multiexponential decay law:
I(t) = aiexp( − t/
τ
i)
where I, a
i
and
τ
i
are uorescence intensity, the normalized pre-
exponential factors, and decay time, respectively. The fractional uo-
rescence intensity of each component is dened as fi=ai
τ
i/aj
τ
j.
Decay-associated spectra were assembled using global multi-
exponential decay analysis across the emission spectra of the lms
with various SF:ANS ratios [27].
I(λ,t) = ai(λ)exp( − t/
τ
i)
Then a
i
(λ) values were used to construct the DAS for each global
τ
i
parameter
Ii(λ) = ai(λ)
τ
iI(λ)
aj(λ)
τ
j
=fi(λ)I(λ)
DAS components were t to the bi-parametric log-normal function.
The parameters specic for ANS uorescence were established previ-
ously [6].
3. Results and discussion
3.1. The absorption spectra of ANS bound to proteins in lm and solution
states show similar features
Properties of the ANS absorption band positioned in the longer
wavelength side of the spectrum have been studied in solvents with
various polarities. This band is composite and can be characterized by
the superposition of two spectra with the absorption maximum of about
375 nm and 355 nm. The substitution of solvent from water to dioxane
leads to an increase of the ratio of intensities R =λ
max
(1)/λ
max
(2) (~375
nm and ~355 nm, respectively) from 0.90 to 1.17 [22]. In aqueous
solution, the absorption band of ANS can be characterized by two
Gaussian components with λ
max
(1) =377.3 nm and λ
max
(2) =338.3 nm
(Supplementary Fig. 1). The inclusion of ANS molecules into SF lms
exhibits a signicant red-shift for both absorption bands (377.3 nm385
nm and 338.3 nm348.9 nm). The ratio of intensities λ
max
(1)/λ
max
(2)
increases from 0.95 to 1.20, which closely matches the changes observed
from the substitution of water with dioxane (Supplementary Fig. 1).
Almost identical spectral changes have been observed between ANS free
A.J. Guliyeva and O.K. Gasymov
Biochemistry and Biophysics Reports 24 (2020) 100843
3
and bound to the protein in solution [28]. Observed changes in the
absorption spectra are most likely to have resulted from the modication
of the electron density distribution of ANS molecules. This indicates a
complex formation between ANS molecules and some groups of protein
molecules. ANS binding characteristics in the lm are strikingly similar
to the binding in aqueous solution. ANS in the SF lm can be described
as ANS molecules bound to binding sites of the protein. Below we show
the evidence for two distinct specic binding sites for ANS binding in the
SF lm.
3.2. Steady-state uorescence and DAS components of ANS
ANS uorescence spectra in various solvents can be satisfactorily
characterized by the single log-normal component indicating the ho-
mogeneous nature of the ANS environment [6]. A single log-normal
component, also named as an elementary component, is characterized
by two parameters, wavelength of uorescence maximum and scaling
factor. Other parameters of the log-normal function are xed and
satisfactory for all ANS uorescence spectra in different solvents [6].
The log-normal function was used to characterize the DAS components
of ANS uorescence in the lms with different SF:ANS ratios (Fig. 1).
Three lifetimes were necessary for satisfactory tting in the global
analysis. Fig. 1AE shows the steady-state uorescence spectra and
resolved DAS components for ANS uorescence in the representative SF:
ANS lms with ratios of 1:0.13, 1:0.35, 1:0.72, 1:0.87 and 1:1, respec-
tively. As an example, the global triple-exponential decay ts are shown
for the SF:ANS (1:0.35) lm at various wavelengths (Fig. 1F). The dis-
similar contributions of the fast decay components are evident at the
beginning of the decay curves. The decay curves for selected wave-
lengths are plotted separately for better judgment (Supplementary
Fig. 2). All lms with different SF:ANS ratios show a similar set of life-
times (Fig. 2). To test the validity of the triple-exponential decay model,
a representative ANS uorescence decay was also analyzed by a
model-free maximum entropy method (MEM). In the MEM, a series of
200 exponentials, logarithmically spaced lifetimes and variable
pre-exponential terms were used. The goal of the tting procedure was
to have the minimized
χ
2
and maximized ShannonJaynes entropy
function [29]. The results of the ANS uorescence decay analysis by the
MEM are shown in Supplementary Fig. 3. The lifetime components from
the MEM analysis can be grouped into three classes with the maximum
values of 1.0 ns, 8.6 ns and 15.6 ns. These data are in good agreement
with the uorescence lifetimes obtained from the triple-exponential
decay model (1.1 ns, 7.8 ns, 15.2 ns). Thus, MEM analysis validates
the use of the triple-exponential decay model for ANS uorescence
decay and DAS in the different SF:ANS lms.
The steady-state uorescence spectra of ANS in various SF:ANS are
inhomogeneously broadened and, therefore, could not be t to the single
Fig. 1. Steady-state emission spectra and DAS
components of ANS incorporated into SF lms at
various SF:ANS ratios. (A)(E) represents SF:ANS
lms with ratios of 1:0.13, 1:0.35, 1:0.72, 1:0.87
and 1:1, respectively. Solid circles represent
steady-state uorescence spectra. Open square,
triangle and circles denote DAS components for
very short- (0.19 ns- 1.08 ns), short- (5.62 ns-
7.79 ns) and long (14.41 ns- 15.18 ns) lifetime
components. The solid lines of DAS components
show the best t to the single log-normal function
with parameters specic for ANS. (F) Fluores-
cence intensity decay curves of ANS in SF:ANS
(1:0.35) lm at various wavelengths. Circles are
experimental data points. The dashed line is an
instrument response function. Solid curves
denote the best t for global triple-exponential
decay.
Fig. 2. The steady-state and time-resolved uorescence parameters of ANS in
the lms with various SF:ANS ratios. The concentration of ANS indicates their
corresponding values in the solution from which lms were made. Actual
concentrations are about ×40 as estimated previously. Open squares, triangles
and circles represent the parameters of the DAS corresponding to the very short-
, short- and long lifetimes shown in (C). Solid symbols show the parameters of
the DAS of the hydrated samples of the corresponding counterparts.
A.J. Guliyeva and O.K. Gasymov
Biochemistry and Biophysics Reports 24 (2020) 100843
4
log-normal component. As an example, a single and double log-normal
component t of the ANS uorescence spectrum in the lm SF:ANS
(1:0.35) is shown in Supplementary Fig. 4. At least two log-normal
components are required for satisfactory tting of the spectrum. The
values of the wavelength of uorescence maximum obtained from the
log-normal t do not match that of DAS components (Fig. 1B and Sup-
plementary Fig. 4.). Therefore, the multicomponent log-normal t is not
adequate to resolve a DAS component. However, it is valuable to show
the presence of the inhomogeneous broadening in the ANS uorescence
spectra.
Individual DAS of ANS uorescence in the lms with various SF:ANS
ratios can be satisfactorily t the single log-normal component. This is in
contrast to the data observed for ANS bound to the protein in solution
[6]. ANS bound to apo-tear lipocalin (apo-TL) and its mutant apo-G59W
shows two DAS components relatively short (4.09 and 4.64 ns) and the
longer lifetime (17.33 and 15.82 ns) components, respectively [6]. In
both cases, relatively short lifetime DAS could not be t to a single
log-normal component. However, long lifetime DAS of ANS-apoG59W,
but not ANS-apoTL, can be satisfactorily t a single log-normal
component. It has been concluded that the heterogeneous nature of
ANS binding sites necessitates the tting of the DAS to double
log-normal components. Interestingly, both short- and long lifetime DAS
components of ANS uorescence in the lms with various SF:ANS ratios
can be characterized by single log-normal components. This indicates
that in the solid protein lm, ANS is bound to the sites with two different
hydrophobicities, but environments of the binding sites in both cases are
homogeneous.
The steady-state spectra and the resolved DAS components of ANS
uorescence in the lms with various SF:ANS ratios reveal the nature of
ANS-SF interaction. Despite signicant differences in wavelengths of
uorescence maxima of between DAS, the same DAS component ex-
hibits a minor variation at different ANS concentration (Fig. 2). The
same statement is also true for the lifetime components of DAS. Data
indicate that even in solid-state ANS binding sites of SF are distinct and
specic. The increased concentrations of ANS saturate the specic
binding sites (Fig. 2 and Supplementary Fig. 5). Thus, as in solution
studies, ligand (ANS)- protein (SF) interaction in solid states can be
described in similar terms, saturations of specic binding sites.
Compared to solution studies, SF in solid-state assumes much less
conformational states that are supported by homogeneous DAS com-
ponents of ANS uorescence.
ANS bound to proteins in solution shows blue-shifted uorescence
maxima, the increase uorescence intensity and lifetimes that are
attributed to the hydrophobicity of a protein site and restriction of
mobility of ANS. Incorporation of ANS into the solid lm of SF elimi-
nates one of these factors, i.e., mobility. In the solid protein lm, the
mobility of all sites is signicantly restricted. Therefore, it is expected
that the lifetimes of ANS incorporated into the solid protein lms will be
mainly inuenced by the hydrophobicity of the sites. ANS in the SF:ANS
lms shows uorescence with three lifetimes, very short- (0.19 ns- 1.08
ns), short- (5.62 ns- 7.79 ns) and long- (14.41 ns- 15.18 ns). Very short-
lifetimes are associated with very low uorescence intensities. There-
fore, DAS and the lifetimes for these components were not consistent in
the lms with various SF:ANS ratios. The short- and long-lifetime
components could be compared to that of ANS bound to TL and beta-
lactoglobulin in solution [6,9,10]. The long-lifetime of ANS in the SF:
ANS lms (14.41 ns- 15.18 ns) are very similar to that of ANS-TL and
ANS-G59W (17.33 ns and 15.82 ns, respectively, and ANS-BLG (~14 ns)
complexes. Thus, long-lifetimes of ANS report high hydrophobicity and
restricted mobility in both incorporated into the protein lms and
complexed with globular proteins in solutions. The situation is different
with the short-lifetimes. In the solid protein matrix, the short-lifetimes of
ANS uorescence are in a range of 5.62 ns 7.79 ns. It can be compared
with the short-lifetimes of ANS-TL and ANS-G59W (4.09 ns and 4.46 ns,
respectively) and ANS-BLG (~3 ns) complexes in solution [6,9,10].
Short-lifetimes of ANS in the SF:ANS lms are appreciably higher than
that of the proteins in solution (Figs. 1 and 2). This fact can be ratio-
nalized by the solvation of the ANS binding sites responsible for short
uorescence lifetimes in solution. ANS is expected to have relatively
higher mobility in the solvated states.
To test this hypothesis we performed the experiments with the hy-
drated SF:ANS lms. Fig. 3 shows the steady-state and DAS component
of ANS uorescence in the SF:ANS (1:0.83) lm without and with hy-
dration. The hydrated samples demonstrate red-shifted steady-state and
DAS components. However, the shifts are not uniform among the DAS
components. The DAS component with long-lifetime shows the smallest
shift, the spectrum of which could be t to the single log-normal
component (Figs. 2 and 3). Hydration of the lm induces a slight
decrease (~9%) of the uorescence lifetime, from 15.18 ns to 13.84 ns.
Consequently, it can be concluded that the hydrophobic sites responsible
for long-lifetimes of ANS uorescence are not solvated and homoge-
neous. In contrast, upon the hydration, the DAS component with short-
lifetime shows a large red-shift and inhomogeneous broadening (Figs. 2
and 3). As a result, the short-lifetime DAS requires at least two log-
normal components for satisfactory tting (Fig. 3). Compared to the
long-lifetime, upon hydration of the lm the short-lifetime of ANS
uorescence exhibits a considerable (~26%) decrease, from 7.27 ns to
5.4 ns. All parameters, spectral FWHM, lifetime and wavelength of
uorescence maximum, of ANS uorescence in the SF lm corre-
sponding short-lifetime DAS upon hydration become similar to that of
ANS complexed with globular proteins in solution. The inhomogeneous
broadening of the short-lifetime DAS indicates that hydration generates
multiple conformational states. In the protein lm, hydration disrupts
homogeneously distributed ANS binding sites responsible for the short-
lifetime DAS and uctuation induced by hydration enables it to sample
an ensemble of patches and/or cavities with similar hydrophobicity.
Slow rate desiccation of the lms removes hydration related dynamics
and an ensemble of substates becomes trapped into the energetically
minimum state. ANS incorporated into these states yields homoge-
neously broadened DAS (short-lifetime component).
Thus, the results observed for the partially hydrated (45%) lms
reveal the origin of the inhomogeneously broadened DAS with lifetimes
of about 35 ns observed in solution studies. It is important to notice that
binding sites with different hydrophobicities revealed in the SF lms by
ANS uorescence are specic. Indeed, titration of SF lms with various
concentrations of ANS exhibits saturation behavior for the steady-state
intensity and DAS of short- and long-lifetimes (Supplementary Fig. 5).
The DAS component for very short-lifetime does not saturate. Most
likely, this uorescence originates from ANS molecules localized in void
volumes of the protein lms, analogous to free ANS in solution.
The relationship between the wavelengths of uorescence maximum
and lifetimes of ANS complexed with various proteins is revealing
(Fig. 4). It is evident that there is a linear relationship between the
wavelengths of uorescence maximum and lifetimes for both short- and
long lifetimes in the desiccated SF lms. However, in the solution and
hydrated protein lms, only long lifetime components follow that trend.
The short lifetimes of ANS uorescence do not depend on the wave-
lengths of uorescence maximum values in the hydrated SF lm and
globular proteins in solution.
4. Conclusion
Thus, ANS has the potential to characterize proteins in various solid
states to reveal the hydrophobic sites with distinct features. ANS in the
solvent accessible sites exhibits relatively short-lifetime DAS that in
hydrated states becomes very similar to that of ANS-globular protein
complexes in solution. ANS in binding sites characterized by long life-
time DAS is not solvent accessible. ANS uorescence properties revealed
in the protein lms indicate that it can be used to characterize dehy-
drated as well as hydrated protein aggregates, amyloids relevant to the
Alzheimers, Parkinson, and prion diseases.
A.J. Guliyeva and O.K. Gasymov
Biochemistry and Biophysics Reports 24 (2020) 100843
5
Funding
This work was supported by the program of Azerbaijan National
Academy of Sciences (ANAS).
Declaration of competing interest
The authors declare that there is no conict of interest.
Acknowledgment
None.
Appendix A. Supplementary data
Supplementary data related to this article can be found at https://
doi.org/10.1016/j.bbrep.2020.100843.
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Fig. 3. The inuence of hydration (45%) on the
DAS component of ANS in the lm with SF:ANS
(1:0.83). (A), the steady-state spectra of ANS in the
lm with desiccated (open circles) and hydrated
(solid circles) states. (B), (C) and (D) represent very
short-, short- and long lifetime DAS components of
ANS in the lm with desiccated (open circles) and
hydrated states (solid circles). Solid lines in (C) and
(D) represent a single log-normal t for the lm with
desiccated forms. (C) and (D) for the hydrated state,
dashed- and solid lines show single- and double log-
normal component tting.
Fig. 4. Fluorescence lifetimes versus uorescence maximum wavelength for
ANS in different conditions. The lifetimes represent the resolved DAS compo-
nents. Data for nano-SF are from unpublished data. The red ellipse highlights
the region for short-lifetime DAS components in solution. (For interpretation of
the references to colour in this gure legend, the reader is referred to the Web
version of this article.)
A.J. Guliyeva and O.K. Gasymov
Biochemistry and Biophysics Reports 24 (2020) 100843
6
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A.J. Guliyeva and O.K. Gasymov
Supplementary Data
ANS fluorescence: Potential to discriminate hydrophobic sites of protein in solid states
Aytaj Guliyevaa, Oktay K. Gasymova*
a Laboratory of Structure, dynamics and functions of biomolecules, Institute of Biophysics of
ANAS, 117 Z. Khalilov, Baku, AZ1171, Azerbaijan
*Corresponding author. E-mail address: ogassymo@g.ucla.edu; oktaygasimov@gmail.com
Supplementary Fig. 1. The absorption spectrum of ANS incorporated into silk fibroin film.
(A) red and black lines represent the spectra of pristine SF and SF: ANS (1:1) films,
respectively. The blue line is the difference spectrum (black-red) that is shown on the right-
hand ordinate scale for easy to follow. (B) The absorption spectrum of the ANS in the SF:
ANS (1:1) film and its Gaussian components. (C) The absorption spectrum of ANS in water
and its Gaussian components. Sick gray and green lines in (B) and (C) show experimental and
best fit to three Gaussian components. The third component, the absorption band of which is
out of the spectral band of interest, gives a better fit to the blue side of the spectra.
ANS molecules incorporated into the SF films exhibit the spectral components that are
significantly red-shifted compared to that of free in aqueous solution. Almost identical
spectral changes are observed for ANS bound to the protein in solution [1]. Unlike changes
observed in fluorescence, spectral changes in the absorption band of ANS, resulted from the
250 300 350 400 450
0.5
1.0
1.5
2.0
2.5
3.0
B
Absorbance (O.D.) Absorbance (O.D.) Absorbance (O.D.)
A
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Absorbance (O.D.)
320 340 360 380 400 420 440
0.00
0.05
0.10
0.15
0.20
0.25
348.9
385.5
320 340 360 380 400 420 440
0.00
0.05
0.10
0.15
0.20
0.25
C
Wavelength (nm)
338.3 377.3
250 300 350 400 450
0.5
1.0
1.5
2.0
2.5
3.0
B
Absorbance (O.D.) Absorbance (O.D.) Absorbance (O.D.)
A
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Absorbance (O.D.)
320 340 360 380 400 420 440
0.00
0.05
0.10
0.15
0.20
0.25
348.9
385.5
320 340 360 380 400 420 440
0.00
0.05
0.10
0.15
0.20
0.25
C
Wavelength (nm)
338.3 377.3
modification of the electron density distribution, indicate complex formation between ANS
and protein molecules.
Supplementary Figure 2. Time-resolved fluorescence decay of ANS in the SF:ANS (1:0.35) at
selected wavelength values along the steady-state emission spectrum. The wavelength values
at which the measurements were performed are shown in the Figure. The solid black circles
represent experimental data. Dashed lines are the instrument response function. Solid cyan
lines represent the best fit from the triple-exponential global analysis, where lifetimes are the
global parameter. The goodness of the fit (χ2) for individual fits is shown in the Figure.
Model-free maximum entropy model (MEM) analysis to show the validity of triple-
exponential decay analysis for ANS fluorescence incorporated in SF films.
Supplementary Figure 3. The fluorescence lifetime distribution for the SF:ANS (1:0.35) film
by MEM decay analysis. (A) The black line represents the ANS fluorescence intensity decay
curve; the blue line is the decay fit using MEM; the red line is the instrument response
function. The wavelength for monitoring of the fluorescence decay was 470 nm. The χ2 value
in the fitting procedure was 1.12. (B) The lifetime distribution for ANS fluorescence in the
SF:ANS (1:0.35) film obtained by MEM decay analysis.
Supplementary Figure 4. Log-normal fitting the fluorescence spectrum of ANS in SF:ANS
(1:0.35). (A): Open cirls represent the experimental data. The blue and red lines show the best
fit to the single- and double log-normal components, respectively. The dashed red lines are
components of the spectra resolved from double log-normal function. (B) and (C) are
residuals for the fitting for single- and double log-normal components, respectively.
Single log-normal component fitting yields fluorescence wavelength maximum at
463.5± 0.3 nm. Double log-normal component fitting yields fluorescence wavelength maxima
at 455.4± 0.5 and 482.9± 1.1 nm. Although the log-normal fit to the double component is
perfect, however, data do not reflect DAS. Therefore, the log-normal fit is good to determine
the heterogeneity of the spectra but not appropriate to resolve the DAS components.
Supplementary Figure 5. Titration of SF film with ANS. Open squares are the values of
steady-state fluorescence intensity. Red circles, blue triangles and black squares are the
intensities of DAS components for long-, short- and very short-lifetimes. The DAS
component for very short-lifetime does not show saturation behavior. Fluorescence intensities
were measured at 464 nm.
References:
[1]. I.M. Kuznetsova, A.I. Sulatskaya, O.I. Povarova, K.K. Turoverov, Reevaluation of ANS
Binding to Human and Bovine Serum Albumins: Key Role of Equilibrium Microdialysis in
Ligand – Receptor Binding Characterization, PLoS ONE 7 (2012) e40845,
http://doi:10.1371/journal.pone.0040845
020 40 60 80 100 120 140 160 180 200
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Kd ~ 55 µM
Kd ~ 58 µM
Normalized fluorescence intensity
[ANS] (µM)
K
d ~ 80 µM
no binding
020 40 60 80 100 120 140 160 180 200
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
K
d
~ 55 µM
K
d
~ 58 µM
Normalized fluorescence intensity
[ANS] (µM)
K
d
~ 80 µM
no binding
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The exposed hydrophobic patches of protein are widely detected through the binding by the fluorescent probes such as 1-anilino-8-naphthalene sulfonate (ANS), Nile Red (NR) and 1-(N-phenylamino) naphthalene, N-(1-Naphthyl) aniline (1NPN). Interestingly, at pH 4, where the Toxoplasma gondii Ferredoxin-NADP(+) reductase (TgFNR) is stable, an exclusive binding and fluorescence emission was observed for ANS. To understand the underlying difference in the binding of ANS, NR and 1NPN; their effect on the protein structure was studied in detail. ANS was found to interact with TgFNR via electrostatic as well as hydrophobic interactions at pH 4. NR and 1NPN did not show any such binding to TgFNR in the similar conditions, however showed strong hydrophobic interaction in the presence of NaCl or DSS (2, 2-dimethyl-2-silapentane-5-sulfonate). The subsequent structural studies suggest that ANS, NaCl and DSS induced partial unfolding of TgFNR by modulating ionic interactions of the enzyme, leading to the exposure of buried hydrophobic patches amicable for the binding by NR and 1NPN. The induced unfolding of TgFNR by ANS is unique and thus cautions to use the fluorescent dye as simple indicator to probe the exposed hydrophobic patches of the protein or its folding intermediates.
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Silks are natural fibrous protein polymers that are spun by silkworms and spiders. Among silk variants, there has been increasing interest devoted to the silkworm silk of B. mori, due to its availability in large quantities along with its unique material properties. Silk fibroin can be extracted from the cocoons of the B. mori silkworm and combined synergistically with other biomaterials to form biopolymer composites. With the development of recombinant DNA technology, silks can also be rationally designed and synthesized via genetic control. Silk proteins can be processed in aqueous environments into various material formats including films, sponges, electrospun mats and hydrogels. The versatility and sustainability of silk-based materials provides an impressive toolbox for tailoring materials to meet specific applications via eco-friendly approaches. Historically, silkworm silk has been used by the textile industry for thousands of years due to its excellent physical properties, such as lightweight, high mechanical strength, flexibility, and luster. Recently, due to these properties, along with its biocompatibility, biodegradability and non-immunogenicity, silkworm silk has become a candidate for biomedical utility. Further, the FDA has approved silk medical devices for sutures and as a support structure during reconstructive surgery. With increasing needs for implantable and degradable devices, silkworm silk has attracted interest for electronics, photonics for implantable yet degradable medical devices, along with a broader range of utility in different device applications. This Tutorial review summarizes and highlights recent advances in the use of silk-based materials in bio-nanotechnology, with a focus on the fabrication and functionalization methods for in vitro and in vivo applications in the field of tissue engineering, degradable devices and controlled release systems.
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The ANS− (1-anilino-8-naphthalene sulfonate) anion is strongly, dominantly bound to cationic groups of water-soluble proteins and polyamino acids through ion pair formation. This mode of ANS− binding, broad and pH dependent, is expressed by the quite rigorous stoichiometry of ANS− bound with respect to the available summed number of H+ titrated lysine, histidine, and arginine groups. By titration calorimetry, the integral or overall enthalpies of ANS− binding to four proteins, bovine serum albumin, lysozyme, papain, and protease omega, were arithmetic sums of individual ANS−–polyamino acid sidechain binding enthalpies (polyhistidine, polyarginine, polylysine), weighted by numbers of such cationic groups of each protein (additivity of binding enthalpies). ANS− binding energetics to both classes of macromolecules, cationic proteins and synthetic cationic polyamino acids, is reinforced by the organic moiety (anilinonaphthalene) of ANS−. In a much narrower range of binding, where ANS− is sometimes assumed to act as a hydrophobic probe, ANS− may become fluorescent. However, the broad overall range is sharply dependent on electrostatic, ion pair formation, where the organic sulfonate group is the major determinant of binding.