Structural, Functional and Dynamical Responses of Protein in Restricted Environment Imposed by Macromolecular Crowding

Abstract

The inter-cellular environment is known to be very different from the environment where most of the elementary biological processes are studied in the laboratory. As a result, there was a considerable effort on cell mimicking either by confinement or by introducing macromolecular crowding. In the present study dextran of varying sizes has been used to crowd the environment of a protein, human serum albumin (HSA), and its structure, dynamics, and activity were studied as a function of crowder concentration. By employing bulk and single molecular level spectroscopic measurement, we elucidate the overall structure and local microsecond dynamics of HSA. Further, we have attempted to correlate these structural changes with its activity.

Full text

Structural, Functional, and Dynamical Responses of a Protein in a
Restricted Environment Imposed by Macromolecular Crowding
Nilimesh Das and Pratik Sen*
Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur, Uttar Pradesh 208 016, India
*
SSupporting Information
ABSTRACT: The intercellular environment is known to be very
different from the environment where most of the elementary
biological processes are studied in the laboratory. As a result, there
was a considerable effort on cell mimicking either by confinement
or by introducing macromolecular crowding. In the present study,
dextran of varying sizes has been used to crowd the environment
of a protein, human serum albumin (HSA), and its structure,
dynamics, and activity were studied as a function of crowder
concentration. By employing bulk and single molecular level
spectroscopic measurements, we elucidate the overall structure
and local microsecond dynamics of HSA. Further, we have
attempted to correlate these structural changes with its activity.
Most of the biological studies are being carried out in vitro
as performing experiments inside a living cell is always a
challenge.
1,2
Although researchers maintain a physiological pH
for in vitro studies, for every practical purpose, the cellular
environment is different from the dilute buffer solutions.
3
One
of the main differences is the presence of high concentrations
(up to 300−400 g L−1) of macromolecules in the biological
environment, creating a restricted environment.
4,5
It has been
estimated that up to 30% of the cellular volume is occupied by
such macromolecules that make the free space inside the cell
rather limited.
6−8
This is known as the excluded volume effect.
Also, inside a cell, the viscosity is about three times higher than
that of the dilute buffer solutions.
13,14
For all of these reasons,
it is necessary to modify the buffer solution to closely resemble
the cell environment. This could be done either by offering a
confined environment to the system or by adding some
synthetic substance, commonly known as a crowder, to the
solution. There have been several theoretical and experimental
investigations in the past on how crowders can affect the
protein structure and stability,
15−19
folding−unfolding path-
way,
20,21
binding kinetics,
22
aggregation behavior,
23
etc.
Usually, the eccentric behavior of proteins in the crowded
environment is explained on the basis of this excluded volume
effect. However, recent works suggest that the protein−
crowder interaction may also be responsible for the unique
structural, dynamical, and functional response of protein in the
crowded environment.
5,9,10
While the excluded volume effect is
purely stabilizing, the chemical interaction may be stabilizing
or destabilizing. Using E. coli cytoplasm as a model crowding
agent and chymotrypsin inhibitor 2 (CI2) as the protein, the
Pielak group suggests that the destabilizing chemical
interactions between the cytoplasm of E. coli and CI2
overcome the stabilizing hard-core repulsions.
11
Molecular
dynamics simulation by Feig and co-workers on the same
system showed that the effect of crowding primarily depends
on the nature of protein−crowder interactions.
12
Biological macromolecules are of various sizes, shapes, and
properties, which have a profound effect on the behavior of a
protein.
24,25
Therefore, it is essential to investigate the effect of
crowding on protein as exerted by biological macromolecules
in in vivo conditions. Note that the cell environment is
crowded with macromolecules of different sizes. Ribosomes,
molecular chaperons, GroEL, GroES, etc. are of comparatively
higher sizes, whereas substances like nucleic acids, carbohy-
drates, etc. are small in size. On the other hand, the size of
proteins present in the cellular environment may vary from a
few kDa to several hundreds of kDa. Thus, to understand their
effect, crowding by different sized crowders is necessary. Feig’s
group reported the influence of crowders of various sizes on
the hydration structure and dynamics of proteins.
26
In another
report, they have shown that the size distributions of
macromolecules are important factors for in vivo crowding
effects.
27
In this context, we intend to investigate the behavior
(structure, dynamics, and function) of proteins in the presence
of crowders of various sizes, taking human serum albumin
(HSA) as the model protein and differently sized dextrans as
the model crowder. Dextran is one of the most commonly used
synthetic crowder, which is a rod-shaped polysaccharide with
multiple numbers of glucose molecules.
28,29
The molecular
weight of dextran depends on the number of constituent sugar
moieties, and dextran is usually named based on the molecular
weight.
Received: May 28, 2018
Revised: September 10, 2018
Published: September 28, 2018
Article
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Cite This: Biochemistry 2018, 57, 6078−6089
© 2018 American Chemical Society 6078 DOI: 10.1021/acs.biochem.8b00599
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HSA is the most abundant transport protein in the human
body.
30,31
It is a large multidomain protein with three distinct
domains, namely, domains I, II, and III.
32
All of these domains
are responsible for binding and transporting different classes of
molecules like drugs, enzymes, hormones, carbohydrates,
etc.
33−37
There are several reports on the behavior of HSA
in the crowded environment in recent years.
38−44
Singh et al.
monitored the tryptophan fluorescence intensity of HSA as a
function of the extent of crowding.
38
They have concluded that
the quenching of tryptophan fluorescence by a crowder is
mainly static in nature and that dextran-6 exhibits maximum
quenching owing to its highest excluded volume by dextran-6.
Biswas et al. have studied the domain movement of HSA
through FRET analysis as a function of crowder concentration
and observed a gradual decrease of the interdomain distance
between domain-I and domain-II and between domain-II and
domain-III for dextran-40 and dextran-70.
39,40
On the other
hand, for dextran-6, there was a surprising increase in the
distance between domain-I and domain-II.
39
In another
contribution, they reported that the solvation dynamics of
domain-I of HSA depends highly on the concentration of the
crowder, owing mainly to the stiffness of the protein matrix.
41
In a recent article, Samanta et al. concluded that in the
presence of polyethylene glycol (PEG), another type of
popular crowder, the α-helicity of HSA increases to a
considerable extent.
42
Examining the intrinsic tryptophan
fluorescence, they also concluded that HSA becomes more
prone to unfold in the presence of PEG.
43
In spite of the considerable amount of studies on the
structural features of protein in the crowded environment, the
conformational fluctuation dynamics and activity of HSA in the
presence of a crowder are not explored. To note here, it is well-
accepted that the structure of a protein is not rigid; rather, it is
an average of several closely related structures known as
different conformational states of the protein.
45
The
interconversion between these states is known to be conforma-
tional fluctuation dynamics of the protein.
45−47
This conforma-
tional fluctuation dynamics serves a key role in protein
functions like catalysis, drug binding, hormonal activities,
etc.
48,49
Naturally, it is also important to study such structural
dynamics of a protein in the crowded environment. As this
conformational motion of the proteins is uncorrelated, a bulk
measurement cannot provide information about the dynamics,
and one has to perform the single molecular level study. In our
previous contributions, we have reported the conformational
dynamics of domain-III of HSA has a time constant of 8 μs,
50
and that of domain-I has a time scale of 27 μs using single
molecular level fluorescence correlation spectroscopy
(FCS).
51,52
We have also studied the effect of thermal and
chemical denaturation on the dynamics using the same
technique.
50
In the present contribution, we have employed FCS to
investigate the effect of crowders on the structural transition
and conformational fluctuation dynamics and attempted to
relate it to the activity of HSA inside the crowding milieu. For
this purpose, we have selectively tagged tyrosine-411 residue
located in domain-III of HSA by a fluorescent marker p-
nitrophenyl coumarin ester (NPCE). We have used three
different macromolecular crowders, namely, dextran-6, dex-
tran-40, and dextran-70, for the present study.
■MATERIALS AND METHODS
Materials. We purchased human serum albumin (HSA,
fatty acid-free), coumarin-343, 4-dimethylamino pyridine
(DMAP), N,N-dicyclohexylcarbodiimide (DCC), 4-nitrophe-
nol, 4-nitrophenyl acetate, dextran-6, dextran-40, and dextran-
70 from Sigma-Aldrich and used as received. Analytical grade
disodium hydrogen phosphate and sodium dihydrogen
phosphate were purchased from Merck, India, and used to
prepare 50 mM buffer (pH 7.4). Dialysis membrane tubing (14
kDa cutoff) was obtained from Sigma-Aldrich and used after
removing the glycerol and sulfur compounds according to the
procedure given by Sigma-Aldrich. Centrifugal filter units
(Amicon Ultra, 10 kDa cutoff) have been purchased from
Merck Millipore, Germany. HPLC grade dimethyl sulfoxide
(DMSO) and dichloromethane (DCM) were purchased from
S. D. Fine Chemicals Limited, India, and used after distillation.
Synthesis of p-Nitrophenyl Coumarin Ester (NPCE).
The synthesis of NPCE was done according to the reported
procedure
51
following common esterification methodology.
53
Briefly, an equimolar amount (0.38 mmol) of coumarin-343, 4-
nitrophenol, and 4-dimethylamino pyridine (DMAP) was
taken in 5 mL of dichloromethane, and the mixture was
stirred in an ice bath for 10 min under a nitrogen atmosphere.
Then 0.38 mmol of N,N-dicyclohexylcarbodiimide (DCC) was
added slowly. The solution was stirred at 0 °C in an ice bath
for an additional 20 min and then at 20 °C for 24 h. The
organic layer was then washed first with 35 mL of 1.2 M HCl
and then with 35 mL of saturated NaHCO3solution. It was
then dried over MgSO4. The residue was suspended in
methanol, and the precipitate was washed with methanol. The
precipitate was collected in dichloromethane, dried under a
vacuum, and characterized by 1H NMR, IR, and mass
spectrometric methods.
51
Protein Labeling and Sample Preparation. NPCE was
tagged to HSA following the procedure by Wang et al. (see
Scheme 1).
54
Briefly, 36 mg of HSA was dissolved in 9 mL of
50 mM phosphate buffer (pH = 8.0), and 0.3 mg of NPCE was
dissolved in a minimum volume of DMSO. Both were mixed
slowly under stirring conditions. The reaction mixture was
then stirred at the room temperature for 30 h. Dialysis was
done against 500 mL of 1:15 (v/v) DMSO/buffer (pH 7.4, 50
mM phosphate buffer) solution at 5 °C for 4 days and then
against only phosphate buffer for another 4 days to remove any
untagged NPCE. The dialysis medium was replaced every 12 h.
Scheme 1. Tagging of Human Serum Albumin (HSA) by
NPCE
a
a
The molecular structure of NPCE is also shown. The structure file of
HSA is downloaded from the Protein Data Bank (PDB ID: 1ha2).
Biochemistry Article
DOI: 10.1021/acs.biochem.8b00599
Biochemistry 2018, 57, 6078−6089
6079

The labeled protein was concentrated by centrifuging the
sample using a 10 kDa cutofffiltration unit. For steady-state
and lifetime measurements, the protein concentration was
maintained around 5 μM, and for circular dichroism
measurements, the concentration was about 3 μM. For single
molecular level measurements, the protein concentration was
kept around 50 nM. To thermal denaturation, HSA has been
kept at 75 °C for 30 min.
Instrumentation. A commercial UV−visible spectropho-
tometer (UV-2450, Shimadzu, Japan) and spectrofluorimeter
(FluoroMax-4, Jobin-Yvon, USA) were used for recording
steady-state absorption and emission spectra, respectively.
Circular dichroism spectra were recorded on a commercial CD
spectrometer (J-815, Jasco, Japan). Time-resolved fluorescence
was collected using a commercial TCSPC setup (LifeSpec II,
Edinburgh Instruments, U.K.). For lifetime measurements,
peak counts of 8000 were collected at the magic angle
condition by exciting the samples with a 442 nm diode laser
(EPL-445, Edinburgh Instruments, U.K.). Instrument response
function (IRF), which is measured to be 110 ps, is used for
deconvolution of the fluorescence transients. All of the
experiments were done at 298 K unless stated otherwise.
The transients are fitted with Fast software (Edinburgh
Instruments, U.K.) using the following equation:
55
I
tf t
() exp
i
n
ii
1τ
=Σ −
=
i
k
j
j
j
j
j
y
{
z
z
z
z
z(1)
In eq 1,I(t) is the fluorescence intensity at the time t, and fiis
the relative contribution to the lifetime component τi. The
average fluorescence lifetime is calculated using the following
equation:
f
f
i
n
ii
i
n
i
avg 1
1
τ
τ
=
Σ
Σ
=
=(2)
For the activity measurement, we have monitored the
formation of p-nitrophenol (λabs = 400 nm, ε400 = 17 700 M−1
cm−1)
56−58
as a function of time as produced by the enzymatic
action of the HSA on p-nitrophenyl acetate. One unit of
activity is defined as the liberation of 1.0 μMp-nitrophenol per
minute for a 3 min experiment.
Fluorescence Correlation Spectroscopic Measure-
ments. A home-built FCS set up has been used for the single
molecular level fluorescent measurement. The detailed
description of the set up can be found in our previous
reports.
50,51,59,60
The excitation source is a 405 nm continuous
wave laser (5 mW, Optoelectronics Tech. Co. Ltd., China). A
60×water immersion objective with a numerical aperture
(NA) of 1.2 was used to focus the light into the sample placed
on a clean coverslip onto the sample platform of an inverted
microscope (IX-71, Olympus, Japan). For all of the measure-
ments, the light was focused at a distance of 40 μm from the
surface of the coverslip. The same objective collects the
emitted photons, and after passing through a dichroic mirror
(ZT405rdc, Omega Optical Inc., USA) and an emission filter
(FSQ-GG455, Newport, USA), these photons are focused on a
multimode fiber patch chord of 25 μm diameter (M67L01 25
mm 0.10 NA, ThorLabs, USA). The optical fiber carries the
photons to the single photon counting avalanche photodiode
(SPCM-AQRH-13-FC, Excelitas, USA). These photon counts
are then sent to the autocorrelator card (FLEX990EM-12D,
Correlator.com, USA) to generate the autocorrelation
function, G(τ), and displayed on a LabView platform.
The autocorrelation is the self-similarity of fluorescence
intensity at different times and can be mathematically
described as
GFt Ft
Ft
() () ( )
()2
τδδ τ
=⟨+⟩
⟨⟩ (3)
In the above equation, ⟨F(t)⟩is the average fluorescence
intensity, and δF(τ) and δF(t+τ) are the fluctuations in
fluorescence intensity around the mean value at time tand (t+
τ).
55,61
For a single component system, where all of the
particles have the same diffusion coefficient, the diffusion time
(τD) can be obtained by fitting the autocorrelation function
G(τ) using the following equation:
55
GN
() 111
D
1
2D
1/2
ττ
τ
τ
ωτ
=+ +
−−
i
k
j
j
j
j
j
y
{
z
z
z
z
z
i
k
j
j
j
j
j
y
{
z
z
z
z
z(4)
In the above equation, Nis the number of particles in the
observation volume, and ω= l/r is the depth to diameter ratio
of the three-dimensional Gaussian volume. If the diffusing
species undergoes any other process leading to an additional
fluorescence fluctuation having an amplitude, A, and a time
scale, τR, the modified correlation function can be written as
55
GNA() 111 1exp
DDR
1
2
1/2
ττ
τ
τ
ωτ
τ
τ
=+ + +·−
−−
i
k
j
j
j
j
j
y
{
z
z
z
z
z
i
k
j
j
j
j
j
y
{
z
z
z
z
z
i
k
j
j
j
j
j
i
k
j
j
j
j
j
y
{
z
z
z
z
z
y
{
z
z
z
z
z
(5)
From the diffusion time (τD) and radius of the observation
volume (ωxy), the diffusion coefficient (Dt) and hydrodynamic
radius (rH) of the molecule can be calculated using the
following couple of equations:
D4
xy
t
2
D
ω
τ
=(6)
r
kT
D6
HB
t
πη
=(7)
Here, kBis the Boltzmann constant, Tis the temperature (298
K in the present study), and ηis the viscosity of the solution.
The structural parameter (ω) associated with the detection
volume was calibrated using a sample of a known diffusion
coefficient (rhodamine 6G, R6G, in water, Dt= 4.14 ×10−6
cm2s−1).
62
The detection volume of the present setup is
estimated to be 0.5 fL with a transverse radius of 280 nm.
Under the application of external agents, crowders in the
present case, the solution conditions such as refractive index
and viscosity may change significantly. In turn, the diffusion
properties of the molecule therein will also change. The effect
of the viscosity change has been nullified by doing control
experiments at every crowder concentration taking R6G as the
probe. R6G is a rigid molecule and will not undergo any
structural change when exposed to the crowder. So, any change
in its diffusion time with a varying crowder concentration will
be solely due to the change of viscosity of the medium. Using
this information and the reported value of the hydrodynamic
radius of R6G, we have calculated the hydrodynamic radius of
HSA at every crowder concentration according to eq 8.
63
Biochemistry Article
DOI: 10.1021/acs.biochem.8b00599
Biochemistry 2018, 57, 6078−6089
6080

r
r
HHSA
HR6G
DHSA
DR6G
τ
τ
=
(8)
The change in the refractive index is taken care of by
changing the objective collar position and setting it to have the
minimum diffusion time for each sample. This way we
maintain the lowest detection volume attainable for each
sample.
■RESULTS
Circular Dichroism Spectroscopy. To proceed with the
NPCE-tagged HSA for any further experiment, it is necessary
to confirm that the tagging has not perturbed the secondary
structure of the protein, which we have verified by CD
spectroscopy (see figure S1 of the Supporting Information).
We have analyzed our CD data using CDNN software (http://
gerald-boehm.de) to get the information about the secondary
structural parameters of HSA and listed them in table S1 of the
Supporting Information. The CD spectra of untagged HSA was
recorded in the presence of different crowder concentrations as
shown in the figure S2 of the Supporting Information. At high
crowder concentrations (≥125 g L−1), the CD data cannot be
recorded beyond 210 nm because the output voltage becomes
greater than the instrument threshold due to the scattering.
From the measured CD spectra, the structural parameters, i.e.,
the α-helicity, β-sheet, β-turn, and random coil content, of
HSA have been estimated, which are depicted in Figure 1.
With an increasing concentration of dextran-6, we have
observed an almost 88% decrease in α-helicity of HSA from
67.5% to 8%. This decrease in α-helicity is primarily
compensated by the increase of random coil content, which
increases almost 66% from 15% in the absence of dextran-6 to
43.5% in the presence of 200 g L−1of dextran-6 (the highest
crowder concentration used in the study). On the contrary, for
both dextran-40 and dextran-70, the change of secondary
structural content of HSA with an increasing crowder
concentration is not that prominent. For both of the cases,
there is a slight but gradual increase of α-helicity with an
increasing crowder concentration. For dextran-40, there is
about a 7% increase in the α-helicity; whereas, for dextran-70,
there is about a 15% increase in α-helicity, mainly at the
expense of the random coil.
Steady-State Absorption and Fluorescence Spectro-
scopic Study. Free NPCE shows absorption and emission
maxima at 447.0 and 489.0 nm, respectively. Upon tagging to
HSA, the absorption and emission maxima of NPCE blue-
shifted to 440.0 and 480.0 nm (figure S3 of the Supporting
Information). These values are in accordance with our
previous report.
44
The emission spectra of NPCE-tagged
HSA at different crowder concentrations are shown in figure
S4 of the Supporting Information. As can be seen, the emission
maximum does not change in the presence of crowders.
Fluorescence Lifetime Measurement. All of the
fluorescence transients (in the presence and absence of a
crowder) are best fitted by a three exponential function. For
native HSA, the average lifetime of NPCE is found to be 3.59
ns with components of 0.95 ns (6.9%), 2.9 ns (47.9%), and
4.74 ns (45.2%). With the increase of crowder concentration,
the lifetime gradually decreases for all of the crowders used in
this study (see figure S5 of the Supporting Information for the
raw data and Figure 2 for the variation). In the presence of 200
gL
−1crowder, for all three cases, the average lifetime decreases
to ∼3.43 ns. Thus, for all cases, the extent of decrease is
negligible (less than 5%).
Fluorescence Correlation Spectroscopic Study. The
fluorescence autocorrelation curve of free NPCE in the buffer
is satisfactorily fitted with a single diffusion model (eq 4), and
the associated diffusion time is observed to be 24.3 μs (see
figure S6 of the Supporting Information). However, the
fluorescence autocorrelation of NPCE-tagged HSA cannot be
fitted satisfactorily with eq 4. This implies that some additional
process is contributing to the fluorescence fluctuation apart
from the simple diffusion. Upon incorporation of an additional
relaxation term (eq 5), the fitting quality improves significantly
(see Figure 3). The diffusion time of NPCE-tagged HSA is
found to be much higher (140 μs) compared to the free NPCE
in the buffer, as expected. The fluorescence autocorrelation
curves for NPCE-tagged HSA were recorded in the presence of
different crowder concentrations, and all of them were fitted
Figure 1. Variation of secondary structural parameters of HSA as a
function of (a) dextran-6, (b) dextran-40, and (c) dextran-70
concentrations. The filled circles represent the α-helicity. The open
circles represent the β-turn. The filled squares represent the random
coil. The open squares represent the β-sheet.
Figure 2. Variation of the average lifetime of NPCE-tagged HSA with
increasing concentrations of (a) dextran-6, (b) dextran-40, and (c)
dextran-70.
Biochemistry Article
DOI: 10.1021/acs.biochem.8b00599
Biochemistry 2018, 57, 6078−6089
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with eq 5. Some representative fluorescence autocorrelation
curves and the comparison of fitting by two different models
are shown in Figure 3. Such analysis gives two important
parameters about the system: the first one is the diffusion time,
and the other one is the time scale of the additional fluctuation.
From the diffusion time of NPCE-tagged HSA, the hydro-
dynamic radius of the native HSA is calculated to be 38.4 Å,
which is in good agreement with the previous reports.
34,64
It is
found that with an increasing dextran-6 concentration there
was almost no change of hydrodynamic radii of HSA. For
dextran-40, as we increase its concentration, the hydrodynamic
radius of HSA remains almost constant up to 75 g L−1,
followed by a sharp decrease to 29.3 Å at 200 g L−1. On the
otherhand,fordextran-70,agradualdecreaseinthe
hydrodynamic radius of HSA from 38.9 to 29.7 Å was
observed. The variation of hydrodynamics radii of HSA as a
function of crowder concentration is shown in Figure 4.
The additional fluctuation time component observed for
native HSA is found to be ∼8.3 μs, which is assigned as the
conformational fluctuation time of domain-III of HSA.
50
There
is almost no change of this time component with the addition
of dextran-6. For both dextran-40 and dextran-70, a gradual
increase of conformational fluctuation time was observed, as
shown in Figure 5. For dextran-40, the value reaches 16.9 μsat
200 g L−1, and for dextran-70, the value reaches 15.4 μs at 200
gL
−1.
Activity Measurement. The activity of native HSA in the
absence of a crowder is found to be 3.60 ±0.15 (see figure S7
of the Supporting Information and Figure 6). With the increase
in crowder concentration, a gradual decrease of activity was
observed for all three cases. At the highest concentration of
crowders used in this study, the activities of HSA are found to
be 1.2, 1.27, and 1.05 units for dextran-6, dextran-40, and
dextran-70, respectively (see Figure 6).
Figure 3. Normalized autocorrelation curve for NPCE-tagged HSA
(represented by circles) in (a) dextran-6, (b) dextran-40, and (c)
dextran-70 at different crowder concentrations and the comparison of
fitting of these fluorescence autocorrelation curves with a single
diffusion equation (in green) and after incorporation of a relaxation
time component (in black).
Figure 4. Variation of hydrodynamic radii of HSA with increasing
concentrations of (a) dextran-6, (b) dextran-40, and (c) dextran-70.
Solid red lines represent the best fit using eq 14.
Figure 5. Variation of conformational fluctuation time of HSA with
increasing concentrations of (a) dextran-6, (b) dextran-40, and (c)
dextran-70. Solid red lines represent the best fit using eq 14.
Figure 6. Estimated activity of HSA from a 3 min experiment with
increasing concentrations of (a) dextran-6, (b) dextran-40, and (c)
dextran-70.
Biochemistry Article
DOI: 10.1021/acs.biochem.8b00599
Biochemistry 2018, 57, 6078−6089
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■DISCUSSION
The fluorescent labeling of NPCE to HSA leads to a 9 nm blue
shift in its emission maximum due to the more hydrophobic
environment inside the protein matrix, thus confirming the
tagging. With increasing crowder concentrations, the emission
maxima of NPCE in HSA does not show any appreciable
change. Also, for all three different crowders used in this study,
we observed a gradual decrease in the fluorescence lifetime in
the experimental concentration range. However, the decrease is
very small for all cases (less than 5%). Taken together, these
two ensemble-averaged measurements hint that the local
environment around NPCE does not change significantly in
the presence of a crowder.
The increased diffusion time of NPCE-tagged HSA as
compared to the untagged NPCE also proves the tagging of
NPCE to HSA. The almost similar value of secondary
structural parameters for the tagged and untagged HSA
(table S1 of the Supporting Information) confirms a minimal
change of secondary structure of HSA upon tagging with
NPCE. The CD spectra of HSA in the presence of various
dextran molecules were recorded to have an idea on the change
in the overall secondary structure of HSA with increasing
crowder concentrations. We observed a huge decrease in the α-
helical content of HSA with increasing dextran-6 concen-
trations with a concomitant increase in the random coil
contribution. However, for dextran-40 and dextran-70, the
secondary structural parameters remain almost the same,
except for a slight increase in the α-helicity of HSA is observed
as the concentration of the crowder was increased. The general
expectation is that, with increasing crowder concentrations, the
exerted force on the protein surface would increase, making the
protein take a more compact structure. In fact, for larger
dextrans, this is the case observed. However, for the smallest
dextran, i.e., dextran-6, the observation is completely opposite,
suggesting the presence of some different type of interaction
between HSA and dextran-6. This interaction possibly
interferes with the stabilizing interactions that hold the
secondary structural content of HSA together.
FCS measurements reveal almost no change of hydro-
dynamic radii of HSA as the dextran-6 concentration is
increased from 0−200 g L−1, while, for dextran-40 and dextran-
70, there is an appreciable decrease in the hydrodynamic radius
with increasing crowder concentrations. The pressure on the
protein surface may modulate the protein structure, and we
have tried to make a rough estimation of the relative pressure
on the protein surface by various crowder molecules. To do
that, we have made some assumptions. First of all, we assumed
the ideal behavior of the solution and used the following
equation for calculating the pressure (P):
65
P
mnc
V
1
3
2
=(9)
where “m”and “n”are respectively the mass of a crowder
molecule and the total number of crowder molecules, cis the
root-mean-square speed of a crowder, and Vis the volume.
Second, we have assumed that there are no interactions
between the crowder molecules. The number of various
dextrans at a fixed concentration will be different owing to
their different molecular weights. In this study, the highest
concentration that we have used is 200 g L−1for all of the
crowders. For ensemble-averaged measurements, at this
concentration, the number of dextran-6, dextran-40, and
dextran-70 molecules is 6600, 1000, and 560, respectively,
for each HSA. For single molecular level experiments, this ratio
is 1:660 000, 1:100 000, and 1:56 000, respectively, for dextran-
6, dextran-40, and dextran-70. From these numbers, it is
evident that the number density of dextran-6 molecule is the
highest followed by dextran-40 and dextran-70, and the
number of crowder molecules is much higher as compared
to the protein molecule. Thus, we may visualize that a protein
is surrounded by a large number of dextran molecules. At this
point, we assume that only the first layer of the crowder around
the protein will be effective to give a pressure on the protein
surface. Thus, in eq 9,“n”will be the number of dextran
molecules that form the immediate first layer around an HSA
molecule. Hydrodynamic radii of dextran-6, dextran-40, and
Scheme 2. Rough Estimation of Pressure on an HSA Molecule by the Crowders
Biochemistry Article
DOI: 10.1021/acs.biochem.8b00599
Biochemistry 2018, 57, 6078−6089
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dextran-70 (rc) are reported by Masuelli et al., and the values
are 18.6 Å, 47.8 Å, and 64.9 Å, respectively.
66
The
hydrodynamic radius of HSA (rHSA) is 38.9 Å. The radius
(rt) and the volume (vt) of the overall system comprising one
HSA surrounded by a single layer of crowder molecule are
defined by the following equation (see Scheme 2):
r
rr
tHSA
c
=+ (10)
v
r
4
3
tt
3
π=
(11)
The volume occupied by the crowder molecules in the first
layer around HSA will be given by
Vv v
tHS
A
=− (12)
Also, the approximate number of crowder molecules forming
the first layer around HSA (Nc) is therefore
N
V
v
c
c
=(13)
Using eq 13 in calculating the approximate numbers of
crowders in the first layer around HSA suggests that we are
neglecting the presence of any void space. Using eq 9, the ratio
of the effective pressure on HSA by different dextrans are
estimated to be Pdextran‑40/Pdextran‑6/Pdextran‑70 = 1.00:0.38:0.27.
The exerted pressure is found to be largest for dextran-6
followed by dextran-40 and dextran-70, which suggests that
HSA should take a more compact structure in the presence of
dextran-6 compared to dextran-40 and dextran-70. However,
our FCS result shows a completely reversed picture. The
reason could be the smallest size of dextran-6 as discussed
above. The volume of one dextran-6 molecule is only 11% of
that of one HSA, whereas the volume of the other two
crowders is higher as compared to that of HSA. Owing to the
small volume, some of the dextran-6 molecule may go inside
the protein matrix. We know that dextran is a branched
polysaccharide chain, and this branching increases with an
increasing molecular weight. Lower molecular weight dextrans,
like dextran-6, are more like rod-shaped, and with an increasing
molecular weight, the dextran molecule tends to be more and
more spherical. This difference of shape may also cause a
dextran-6 molecule to penetrate the protein matrix, while the
other two cannot. From our CD data, it is clear that, in the
presence of dextran-6, the secondary structure of HSA is
disrupted. Due to the breakdown of the secondary structure of
HSA, the polypeptide chain may unfold and the size may tend
to increase. This tendency is neutralized by the pressure
exerted on the surface of HSA by dextran-6, which tries to
decrease the overall size of the HSA molecule. These two
forces balance themselves in such a way that there is almost no
change in the size of the HSA when increasing the dextran-6
concentration from 0 to 200 g L−1. For dextran-40 and
dextran-70, we observed a decrease of the hydrodynamic radius
of HSA, proposed solely due to the exerted pressure effect by
these crowders. Dextran-40 causes an approximate 25%
decrease in the hydrodynamic radius, and dextran-70 causes
a 23% decrease in the hydrodynamic radius at the highest
concentration of the crowder used. The relative change of the
hydrodynamic radii of HSA with three different dextrans is
plotted in Figure 7. A slightly higher effect of dextran-40 is
attributed to the higher pressure exerted by it on the HSA
surface as compared to dextran-70 as we have estimated.
To further illustrate the mechanism of crowder-induced
changes, we have done a companion experiment involving
thermally and chemically denatured HSA. Thermally dena-
tured HSA (at 75 °C) and chemically denatured HSA
(denatured by 7.5 M of urea) have α-helicities of 38% and
16.6% and hydrodynamic radii of 59.3 and 57.7 Å, respectively.
The relative change of the α-helicity and hydrodynamic radius
of such a way denatured HSA in the presence of three different
crowders are shown in Figure 8. In the presence of 100 g L−1
dextran-6, the α-helicity of thermally and chemically denatured
HSA decreases 58% and 42.5% from its original value, whereas
for both dextran-40 and dextran-70, at the same concentration
of crowder, the α-helicity increases. For thermally denatured
HSA, this increase is minimal, whereas for chemically
denatured HSA, the increase is significant. On the other
hand, all of the three crowders used in this study compact the
structure of the denatured HSA. For dextran-6, the decrease in
the hydrodynamic radius is only 10.5% for thermally denatured
HSA and 1.5% for chemically denatured HSA, whereas, for
dextran-40 and dextran-70, the changes are, respectively, 24.1%
Figure 7. Relative change of the hydrodynamic radius of HSA in the
presence of three dextrans with an increasing concentration.
Figure 8. Relative change of the α-helicity and hydrodynamic radius
of (a) thermally denatured HSA at 75 °C and (b) chemically
denatured HSA at 7.5 M urea, in the presence of 100 g L−1dextran-6,
dextran-40, and dextran-70.
Biochemistry Article
DOI: 10.1021/acs.biochem.8b00599
Biochemistry 2018, 57, 6078−6089
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and 19% for thermally denatured HSA and 22.8% and 18% for
urea denatured HSA. The extent of change in α-helicity and
hydrodynamic radius in the presence of crowders for thermally
and chemically denatured HSA can be exploited to understand
how dextrans of different molecular weights interact with HSA.
Considering only the crowder exerted pressure, dextran-6
should impose a maximum decrease of the hydrodynamic
radius, but the result is just the opposite. This is the same trend
that we have observed for the native protein. Thus, it is
obvious that there is some other way in which dextran-6
interacts with HSA. Moreover, the % α-helicity of HSA, either
in the native or denatured state, decreases sharply with the
addition of dextran-6. These observations unambiguously
prove that dextran-6 induces a structural change in HSA not
only through the pressure effect but also through some soft
chemical interaction, which is destabilizing in nature. At this
point, we hypothesize that, owing to the smaller size of
dextran-6, it may access the protein interior that facilitates the
destabilizing interactions within HSA. This causes the
breakdown of weak chemical forces that stabilize HSA. For
dextran-40 and dextran-70, the changes are in line with what is
expected from the pressure effect only.
The FCS experiment reveals an additional time component
of 8.3 μs apart from the diffusion time scale for the native HSA.
The origin of this extra time component arises because of the
local environmental change around the fluorescent probe due
to the conformational fluctuation of the protein.
51
Such
conformational fluctuation consists of the breathing motion
and the motion of the side chains of the protein.
55
The time
required for this type of conformational fluctuation is termed
as conformational fluctuation time, which is typically in the
range 1−100 μs.
67−69
This fluctuation is a very local
phenomenon and represents the ease of the concerted chain
motion dynamics. The conformational fluctuation time in the
present case is 8.3 μs, which is in good agreement with
previous reports.
51
The effect of the crowder on this
fluctuation time is more prominent for dextran-40 and
dextran-70 than for dextran-6. For dextran-6, there is almost
no change of the conformational fluctuation time even at the
highest concentration. The protein interior can be accessed
through dextran-6, and the higher pressure exerted by this
probably nullified its effect on the conformational dynamics.
On the other hand, for dextran-40 and dextran-70, there is
almost a 2-fold increase in the conformational fluctuation time,
which is probably because of the pressure effect by these
crowders on HSA. The relative change of the conformational
fluctuation time of domain-III of HSA with an increasing
crowder concentration is shown in Figure 9. One interesting
point to note is that ensemble-averaged bulk measurement
cannot detect this local environmental change and thus
signifies the importance of the single molecular level
measurement over the bulk measurement.
FCS data have been fitted to estimate the thermodynamic
parameters related to crowder-induced structural changes of
HSA. Fitting of hydrodynamic radius data gives us an insight
into the thermodynamics of the overall structural change of
HSA, whereas the fitting of the conformational fluctuation time
sheds light on the thermodynamics of the crowder-induced
change of the domain-III of HSA. All transitions related to
structural and dynamical changes are fitted with a two-state
model as
NCF
where “N”stands for the native and represents the protein in
the absence of any crowder and “C”stands for the crowder and
represents the protein in the presence of 200 g L−1crowder.
The variation of RHand τRwith an increasing crowder
concentration was fitted using the following two-state model:
70
Y
YYe
1e
x
x
NC
=+×
+
−
−(14)
In the above equation, “Y”is the spectroscopic signal at
some crowder concentration, YNand YCare the spectroscopic
signals for native and crowded states, respectively. Here, xis
defined as
70
x
Gm
RT
(crowder)
=Δ−[ ]
°
(15)
Here, ΔG0is the free energy change associated with the
concerned transition, mdenotes the slope of the free energy
change plotted against crowder concentration, and Rand Tare
the universal gas constant and temperature in K, respectively.
The midpoint of the transition was calculated using
71
G
m
C
1/2
[
]=
Δ
°
(16)
Both the hydrodynamic radius and conformational fluctua-
tion time of HSA remain almost the same with the changing
dextran-6 concentration. Thus, in this case, “N”and “C”states
are virtually the same, and therefore, the fitting of dextran-6-
induced structural and dynamical change is meaningless. The
most important observation from this analysis is that in the
case of the overall structural change, dextran-40 shows a higher
value of ΔG0than that of dextran-70, whereas, for the
dynamical transition, which is a local phenomenon within the
domain-III of HSA, the order is reversed (see Table 1),
suggesting that, for a big multidomain protein, all domains and
the overall protein may not respond in a similar fashion.
From the activity measurements, we have observed that the
activity of HSA decreases with increasing crowder concen-
trations for all three crowders under investigation. There are
conflicting reports on the origin of the catalytic activity of
HSA. Some reports suggest that Tyr-411 in domain-III is
mainly responsible for this esterase activity of HSA.
72,73
A
more recent article reports that the pseudo esterase activity of
HSA is a result of multiple irreversible chemical modifications
rather than of the catalytic activity occurring at a single reactive
Figure 9. Relative change of conformational fluctuation time of
domain-III of HSA for three dextrans with increasing concentrations.
Biochemistry Article
DOI: 10.1021/acs.biochem.8b00599
Biochemistry 2018, 57, 6078−6089
6085

site.
74
Whatever the case may be, the structural change of HSA
should have a profound effect on this activity. Additionally, the
conformational fluctuation dynamics of domain-III could have
a major role to play.
75,76
It has also been reported that, during
the initial hydrolysis, only Tyr-411 is important.
74,77
In our
study, we have estimated the activity during the initial
hydrolysis period (up to 3 min) and have considered the
only involvement of Tyr-411. Naturally, we expect a direct
correlation between the esterase activity of HSA and
conformational fluctuation time of domain-III of HSA. For
all three crowders used in this study, we observe a gradual
decrease of esterase activity of HSA with the increase in
crowder concentration. Here it is to be noted that the activity
study has been performed with untagged HSA where the Tyr-
411 residue is free. This decrease may be attributed to several
factors. First, with the increase in crowder concentration, the
viscosity of the solution increases. This makes the acetylation
of the Tyr-411 residue by PNPA more difficult. The second
factor that could be attributed to the retardation of the activity
of HSA is that with the increasing crowder concentration the
number of crowder molecules around a protein molecule
increases, making the accessibility of the Tyr-411 residue more
difficult. Third, for the two higher dextrans used in this study,
i.e., dextran-40 and dextran-70, with an increasing crowder
concentration, the size of the protein molecule decreases, and
because of this gradual compactness of HSA, it becomes
progressively harder for the PNPA molecule to reach the Tyr-
411 site. However, the most astonishing observation is the
decrease of the activity of HSA with an increasing dextran-6
concentration. The general expectation is exactly the opposite;
i.e., with a high dextran-6 concentration, HSA is denatured,
and as a consequence, Tyr-411 should be accessible. However,
our data suggests that it is indeed inaccessible even in this
denatured state. Note that a denatured state of a protein might
take such a confirmation that a particular amino acid sequence
does not need to be more exposed compared to that in its
native state.
78−80
Thermal denaturation of HSA is one of such
example. In thermally denatured HSA, Tyr-411 is more
buried.
78−80
In the present case, it is also possible that the
disruption of α-helicity does not lead to the increase in the
accessibility of the Tyr-411 residue.
For all of the cases, the extent of decrease is almost similar,
although the extent of size compactness and conformational
fluctuation are not same. This may be due to the first two
factors that we have stated as the possible reason for the
decrease of activity of HSA. In such a scenario, it is very hard
to find any correlation between the conformational fluctuation
time and the activity. In our next study, we will try to establish
a clear relation between the activity of a protein and its
structure and dynamics by eliminating the first two factors
while measuring the activity.
■CONCLUSION
The effect of macromolecular crowding on the structure,
function, and dynamics of HSA has been investigated by bulk
and single molecular level studies, which led us to conclude the
following. (i) For higher molecular weight dextrans, i.e., for
dextran-40 and dextran-70, with an increasing concentration of
the crowder, the overall size of the protein decreases with an
increase in the helical content of the protein (though slightly).
Thus, the secondary structural change and overall structural
change are in line with each other. However, for dextran-6, the
helical content of the protein decreases significantly with no
change of the hydrodynamic radius. For dextran-40 and
dextran-70, the effect can solely be explained in terms of the
pressure exerted by the crowders. The disparity of the behavior
of dextran-6 compared to that of dextran-40 and dextran-70
confirms the presence of a destabilizing soft chemical
interaction between dextran-6 and HSA. We hypothesized
that the smaller size of dextran-6 allows it to go inside the
protein matrix and facilitates the destabilizing interaction. Due
to a fine balance between the denaturating effect and pressure
exerted by the dextran-6 onto the protein surface, there is
almost no change of the hydrodynamic radius with increasing
dextran-6 concentrations. (ii) To the best of our knowledge,
this is the very first report on the modulation of conforma-
tional fluctuation dynamics in a macromolecular crowded
environment. Smaller sized dextran-6 can not impose a
significant constraint in the movement of the side chains of
HSA; on the other hand, relatively large crowders can
significantly hinder the side-chain movement of HSA, thus
increasing the conformational fluctuation time. The difference
in the spatial distribution of these three different crowders
around HSA and their effect to modulate the structure and
dynamics of protein is summarized in Scheme 3. (iii) The
presence of crowders affect the activity of HSA, which
decreases with an increasing crowder concentration and is
probably due to the lower accessibility of the Tyr-411 residue
in the restricted environment imposed by macromolecular
crowding. As a whole, the global and local changes of HSA
Table 1. Thermodynamic Parameters for the Structural and
Dynamical Change of NPCE-Tagged HSA in Phosphate
Buffer (pH 7.4, 50 mM) by Dextran-40 and Dextran-70
a
analysis of rHchange analysis of τRchange
ΔG0m[C]1/2 ΔG0m[C]1/2
dextran-40 3700 26 140 1400 16 90
dextran-70 970 12 80 2100 16 130
a
ΔG0is in cal mol−1;mis in cal mol−1g−1L, and [C]1/2 is in g L−1.
Scheme 3. Profile for the Hydrodynamic Radius and
Conformational Fluctuation Time of HSA in the Presence
of Various Crowders
a
a
Our hypothesis of the asymmetry in the spatial distribution of
dextran-6 around HSA has been emphasized.
Biochemistry Article
DOI: 10.1021/acs.biochem.8b00599
Biochemistry 2018, 57, 6078−6089
6086

induced by the macromolecular crowding have been
summarized, and the plausible explanations have been related
to the difference in size of the crowders.
■ASSOCIATED CONTENT
*
SSupporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
chem.8b00599.
Circular dichroism (CD) spectra of untagged and
NPCE-tagged HSA, CD spectra of HSA with increasing
concentrations of dextran-6, dextran-40, and dextran-70,
absorption and emission spectra of free NPCE and
NPCE-tagged HSA, emission spectra and fluorescence
transient of NPCE-tagged HSA with varying concen-
trations of dextran-6, dextran-40, and dextran-70,
fluorescence autocorrelation curve of free NPCE and
NPCE-tagged HSA in buffer, activity of HSA with
increasing concentrations of dextran-6, dextran-40, and
dextran-70, CD signal of the thermally denatured HSA
at 75 °C in the absence and presence of dextran-6,
dextran-40, and dextran-70, and fluorescence autocorre-
lation curve for the thermally denatured HSA at 75 °Cin
the absence of a crowder and in the presence of dextran-
6, dextran-40, and dextran-70 (PDF)
■AUTHOR INFORMATION
Corresponding Author
*E-mail: psen@iitk.ac.in. Fax: +91-512-259-6806.
ORCID
Pratik Sen: 0000-0002-8202-1854
Funding
N.D. acknowledges the Council of Scientific and Industrial
Research (CSIR, Government of India) for providing a
fellowship. This work is financially supported by the Science
and Engineering Research Board, Government of India (EMR/
2016/006555), and IIT Kanpur.
Notes
The authors declare no competing financial interest.
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