FCS study of the structural stability of lysozyme in the presence of morpholinium salts

Article abstract of DOI:10.1021/jp409842d

Ability of the ionic liquids to alter the structural stability of proteins in aqueous solution is a topic of considerable interest in modern bioscientific research because of possible applications of these substances in protein purification and as refolding agents. A few early studies involving the imidazolium ionic liquids have demonstrated their role as both denaturants and refolding agents. As the influence of an ionic liquid on a given protein depends on the identity of both species, it is necessary to extend the studies to a wider number of ionic liquids and proteins to obtain insight into the mechanism of interaction between the two and to arrive at a comprehensive picture. It is in this context that we have studied the effect of two morpholinium salts, [Mor1,2][Br] and [Mor1,4][Br], differing in the alkyl chain length of cation, on chicken egg white lysozyme in its native and chemically denatured states employing primarily the fluorescence correlation spectroscopy (FCS) technique. Fluorescence signal of Alexa488-labeled lysozyme (A488-Lysz) has been used to determine the changes in hydrodynamic radius of protein in the presence of additives. The results reveal a conformational dynamics of lysozyme with a time constant of 56 ?± 10 ??s in its native state. It is observed, when in its native state, both the morpholinium salts induce structural changes of lysozyme. However, when in its unfolded state, [Mor1,4][Br] at low concentration compacts the protein, but at higher concentration, it stabilizes the unfolded state, unlike [Mor1,2][Br], which compacts lysozyme at both low and high concentrations. A comparison of the effect of these salts and arginine, a protein stabilizer, on lysozyme indicates that [Mor1,2][Br] is a superior compacting agent for the unfolded state of the protein compared to arginine. ? 2013 American Chemical Society.

Full text of DOI:10.1021/jp409842d

Article
pubs.acs.org/JPCB
FCS Study of the Structural Stability of Lysozyme in the Presence of
Morpholinium Salts
Ashok Pabbathi, Shalini Ghosh, and Anunay Samanta*
School of Chemistry, University of Hyderabad, Hyderabad 500046, India
*
S Supporting Information
ABSTRACT: Ability of the ionic liquids to alter the structural stability of proteins in aqueous
solution is a topic of considerable interest in modern bioscientific research because of possible
applications of these substances in protein purification and as refolding agents. A few early
studies involving the imidazolium ionic liquids have demonstrated their role as both
denaturants and refolding agents. As the influence of an ionic liquid on a given protein depends
on the identity of both species, it is necessary to extend the studies to a wider number of ionic
liquids and proteins to obtain insight into the mechanism of interaction between the two and
to arrive at a comprehensive picture. It is in this context that we have studied the effect of two
morpholinium salts, [Mor1,2][Br] and [Mor1,4][Br], differing in the alkyl chain length of
cation, on chicken egg white lysozyme in its native and chemically denatured states employing
primarily the fluorescence correlation spectroscopy (FCS) technique. Fluorescence signal of
Alexa488-labeled lysozyme (A488-Lysz) has been used to determine the changes in
hydrodynamic radius of protein in the presence of additives. The results reveal a
conformational dynamics of lysozyme with a time constant of 56 ± 10 μs in its native state.
It is observed, when in its native state, both the morpholinium salts induce structural changes of lysozyme. However, when in its
unfolded state, [Mor1,4][Br] at low concentration compacts the protein, but at higher concentration, it stabilizes the unfolded
state, unlike [Mor1,2][Br], which compacts lysozyme at both low and high concentrations. A comparison of the effect of these
salts and arginine, a protein stabilizer, on lysozyme indicates that [Mor1,2][Br] is a superior compacting agent for the unfolded
state of the protein compared to arginine.
1
. INTRODUCTION
egg white lysozyme and found that ionic liquid with short chain
length enhances the refolding of lysozyme. They also found
that ionic liquids with increasing chain length destabilize
lysozyme. Recently, with the help of small-angle X-ray
scattering and FTIR studies Takekiyo and co-workers have
shown that lysozyme unfolds in the presence of 6 M
Enzyme catalysis in organic solvents has been an important and
interesting area of research in biotechnology and biochemistry
in the past two decades.
solubility of the substrates are important considerations for
catalysis in such media. While the enzymes are active in water
and nonpolar solvents, they show hardly any activity in most of
10
1
−4
The stability of the proteins and
[
bmim][NO ], whereas it exists in a partially globular state
3
11
5
when the concentration of the ionic liquid exceeds 10 M.
the polar organic solvents. Hence, enzyme catalyzed reactions
Mann et al. used near-UV CD measurements to understand the
cannot be carried out for substrates that are soluble only in
polar organic solvents. Search is on for alternative media to
overcome this problem and the ionic liquids can be seriously
considered as a possible alternative in this regard. However,
considering the fact that structure of a protein determines its
activity, it is absolutely essential that prior to utilization of the
ionic liquids as media for catalytic reaction, the effect of the
ionic liquid on the structural stability of the protein is
investigated. This perhaps explains the recent interest in
studies aimed at understanding the protein−ionic liquid
interactions. We take note in this context that ionic liquids
are also being employed for the extraction, purification, and
preservation of proteins and as substitute media in biocatalysis.
The ionic liquids are also being explored as refolding agents
and to improve the enzyme activity and selectivity in
effect of alkyl ammonium formate ionic liquids on the thermal
5
stability and activity of lysozyme. Horseradish peroxidase
(
HRP) activity was increased in [bmim][BF ], whereas a 50%
4
loss in activity was observed in the presence of [bmpy][BF4],
indicating that cation variation influences the stability of the
9
enzyme. Noritomi et al. studied the thermal stability and
activity of lysozyme in presence of [emim][BF ], [emim]-
4
[
Tf N], and [emim][Cl]. A much lower activity of the protein
2
was observed in the presence of [emim][Cl] when compared
12
6
,7
with that in [emim][BF ] and [emim][Tf N]. Recent NMR,
4 2
CD, and fluorescence experiments have shown that protic ionic
8
liquids can refold urea-denatured chymotripsin and thus can
1
3
attenuate the action of urea on it.
Very recently,
9
biotransformations.
Yamamoto et al. studied the effect of N-alkylpyridinium
chlorides and N-alkyl-N-methyl pyrrolidinium chlorides on hen
Received: October 3, 2013
Revised: December 7, 2013
Published: December 9, 2013
©
2013 American Chemical Society
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Bhattacharyya and co-workers have studied the effect of
2.2. Synthesis of [Mor1,2][Br] and [Mor1,4][Br]. These
compounds were synthesized following standard procedure.
22
[
pmim][Br] on human serum albumin and cytochrome c by
1
4,15
using fluorescence correlation spectroscopy.
To the best of
Briefly, the alkyl bromide solutions were added to the N-
methylmorpholine solution in acetonitrile and refluxed for 6−8
h at 80 °C. Subsequently, acetonitrile was removed from the
reaction mixture and the resulting bromide salts were washed
with acetone. The white bromide salts were dried under high
our knowledge, the last two studies are the only ones where
fluorescence correlation spectroscopy technique has been
employed to understand the effect of ionic liquids on proteins.
The recent studies exploring the ionic liquids as denaturants
and refolding agents clearly show that the influence of ionic
liquids on enzymes is dependent on the cation−anion
vacuum.
1
16
[Mor1,2][Br]. H NMR (400 MHz, D O, δ/ppm): 4.05−4.00
2
combination of the ionic liquids. However, as the protein-
ionic liquid interaction is yet to be understood, it is necessary to
extend the studies to a wider variety of ionic liquids and
proteins. To obtain an improved understanding of the protein−
ionic liquid interactions, we have chosen two morpholinium
salts, differing in alkyl chain length of cation, [Mor1,2][Br] and
(
1
s, 4H), 3.60−3.50 (m, 2H), 3.45−3.40 (t, 4H), 3.1 (s, 3H),
13
.38−1.30 (t, 3H); C NMR (100 MHz, D O, δ/ppm): 77.0,
2
60.2, 59.2, 46.6, 7.9.
1
[Mor1,4][Br]. H NMR (400 MHz,CDCl ,δ/ppm): 4.1−4.0
3
(m, 4H), 3.9−3.8 (m, 4H), 3.7−3.6 (t, 2H), 3.5 (s, 3H), 1.8−
13
[Mor1,4][Br], and studied their influence on chicken egg white
1
.7 (m, 2H), 1.5−1.4 (m, 2H), 1.0−0.9 (t,3H); C NMR (100
lysozyme (Chart 1) in its native and unfolded states using the
MHz, CDCl , δ/ppm): 77.0, 64.5, 59.5, 46.4, 22.9, 18.9, 13.1.
3
2
.3. Sample Preparation and Labeling of Lysozyme.
Chart 1. Structure of (A) Lysozyme (RCSB Protein Data
Bank ID: 1LYZ), (B) [Mor1, 2][Br], and (C) [Mor1,4][Br]
The solutions were prepared using phosphate buffer (pH 7.4,10
mM). Labeling of Alexa Fluor 488 carboxylic acid TFP ester to
lysozyme was carried out by following the procedure provided
by the manufacturer (Molecular Probes, Invitrogen). Labeling
reaction was carried out in sodium bicarbonate buffer (pH 9.0,
0
.1 M) for two hours at a molar dye/protein ratio of 2. The
labeled protein was separated from free dye using a sephadex
G-25 column (30 cm × 1.3 cm) equilibrated in sodium
phosphate buffer (pH 7.4, 10 mM). The concentrations of the
protein and dye were determined using known molar extinction
−
1
−1
coefficients of the dye (71 000 M cm at 495 nm) and
lysozyme (37 680 M− cm at 280 nm). The dye/protein
1
−1
23
ratio was found to be 1.2 in the labeled protein.
2
.4. Instrumentation and Methods. 2.4.1. Fluorescence
Correlation Spectroscopy. FCS experiments were carried out
using a time-resolved confocal microscope (MicroTime 200,
PicoQuant). Pulsed diode laser (λexc = 485 nm, fwhm 144 ps)
was used as the excitation source. The excitation light was
focused onto the sample using a water immersion objective
(60×/1.2 NA). The same objective was utilized to collect
fluorescence from the sample and directed through a dichroic
mirror and 510LP filter. A pinhole having diameter of 50 μm
was used for the spatial filtration of the signal and focused
through a 50/50 beam splitter before entering the two single
photon avalanche diodes (SPADs). The correlation functions
were generated using PicoHarp 300 TCSPC module, which
collects the data in time-tagged time-resolved (TTTR) mode.
Correlation curves were generated by cross correlating the
signal from two SPADs to remove the after-pulsing. The
excitation laser power was ∼4 μW and 20 nM solution of the
labeled protein was used in FCS experiments.
fluorescence correlation spectroscopy. This technique allows
monitoring of the size of the protein in the presence of a high
17
concentration of cosolutes and is shown to be ideally suited
for the study of conformational dynamics of proteins, protein−
18−21
protein, and protein−lipid interactions.
Herein, the FCS measurements have been performed on
Alexa488-labeled chicken egg white lysozyme. The influence of
the morpholinium salts on both the native and unfolded states
of the protein has been monitored by a combination of FCS
and near-UV CD measurements by following the change of the
hydrodynamic radii of the protein and the spectral changes,
respectively. The conformational dynamics of lysozyme is also
studied by utilizing the Alexa488 dye interaction with the
protein amino acids (tryptophan). The effect of a protein
stabilizer L-arginine on lysozyme is also studied and compared
with the stabilizing effect of the morpholinium salts on the
same.
2.4.2. FCS Data Analysis. Individual correlation curves were
analyzed using the SymphoTime software provided by Pico
Quant. The correlation function for a molecule freely diffusing
2. EXPERIMENTAL SECTION
14
in 3D is given by
2.1. Materials. Acetone, acetonitrile, and alkyl bromides
−
1
−1/2
were purchased from Merck. N-Methylmorpholine, sephadex
G-25 gel filtration medium, and lysozyme from chicken egg
white were obtained from Sigma Aldrich. Alexa Fluor488
carboxylic acid TFP ester was purchased from Invitrogen.
⎛
⎞ ⎛
⎞
⎟
G(τ) = 1 ⎜1 +
τ
D
⎟ ⎜1 +
τ
2
N ⎝
τ ⎠ ⎝
κ τ ⎠
D
(1)
Na HPO4 (anhydrous) was obtained from Loba Chemie.
The correlation function for a molecule undergoing conforma-
2
24
NaH PO was procured from local suppliers.
tional fluctuation along with diffusion is given by
2
4
1
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Article
−
1
⎛
⎞
⎟
G(τ) = 1
− F + F exp(−τ/τR)
N(1 − F)
⎜1 +
τ
D
⎝
τ ⎠
−
1/2
⎛
⎞
τ
⎜
⎝
1 +
⎟
2
κ τ ⎠
D
(2)
In the above correlation functions, N represents the number of
molecules in the observation volume, τ is the diffusion time
D
and τ is the lag time. κ is the structure parameter of the
observation volume and is given by κ = ω /ω , where ω and
z
xy
z
ω are the longitudinal and transverse radii of the observation
xy
volume, respectively. τ is the relaxation time and F is the
R
associated amplitude. Rhodamine 6G (diffusion coefficient 426
2
25
μm /s) was used to estimate the excitation volume, which
was found to be 0.8 fL.
The hydrodynamic radius of lysozyme was estimated using
1
5,17,21
the following equation
refractive index mismatches.
correcting for both viscosity and
protein
protein
Rh
τ
D
=
Rh6G
Rh6G
Rh
τ
(3)
D
The τ values were also corrected for the viscosity according
R
1
5
to the literature.
.4.3. CD Spectroscopy. The near-UV CD spectra were
2
recorded using a Jasco J-810 spectropolarimeter. A quartz
cuvette with path length of 10 mm was used for the
measurements. Each spectrum was obtained from the average
of three scans. All the spectra were corrected for respective
blank spectrum. The far-UV measurements were not performed
in view of strong absorption due to morpholinium salts in this
region.
3
. RESULTS AND DISCUSSION
Figure 1. Correlation data of A488-Lysz in phosphate buffered
solution (pH 7.4) along with the fit to (a) simple diffusion and (b)
simple diffusion along with conformational dynamics models. The
quality of the fits is illustrated by the plots of the residuals as well.
3.1. Size and Conformational Dynamics of Native
Lysozyme. The fluorescence correlation data of Alexa488-
labeled lysozyme (A488-Lysz) is shown in Figure.1. The
correlation data was fit to both eq 1 (simple diffusion) and eq 2
(
simple diffusion with an exponential term). As is evident from
affect the interaction between Alexa488 and the amino acids
leading to fluctuations of the fluorescence intensity, an
additional term in the correlation function apart from the
simple diffusion term explain the data better.
the quality of the fits (depicted in Figure 1), eq 2 clearly is a
better model for this correlation data, and hence, we have used
this model for subsequent analysis. We have also analyzed the
correlation data using a two component diffusion model, but
the diffusion time obtained for the second component from this
model is not matching with the diffusion time of free dye (53 ±
The hydrodynamic radius (R ) of lysozyme estimated from
h
the FCS data is 2.1 ± 0.1 nm, which is in agreement with the
2
8
29
literature. Wilkins et al. proposed an empirical formula, R =
h
4.75N0 Å, where N is the number of amino acid residues in
.29
2
μs). Hence, the presence of free dye in solution and its
contribution to the faster component is ruled out. The fact that
the correlation data of free Alexa488 dye is very well described
by eq 1 (Figure S1, Supporting Information) clearly indicates
that under experimental conditions (excitation power of 4 μW)
the fluctuation of the fluorescence intensity of the dye due to
intersystem crossing is negligible. Majima et al. have also
shown that the contribution from intersystem crossing of free
A488 dye is negligible at an excitation power of 45 μW, which is
much higher than the power used in the present study.
Considering these aspects, a better description of the
correlation data of A488-Lysz in terms of eq2 can be
rationalized as follows. When bound to a protein, the
fluorescence intensity of Alexa488 is known to be influenced
the protein, for the calculation of the R value of a native
h
protein. The R value of native lysozyme using this formula is
h
estimated to be 1.9 nm, which agrees reasonably with the value
estimated from our FCS experiment. It clearly suggests that,
after conjugation with dye, the structure of protein is not
26
30
affected much, which was shown earlier by Melo et al. The
time constant (τ ) of the conformational dynamics is measured
R
to be 56 ± 10 μs and can be attributed to the chain dynamics of
1
5,21
the protein.
3.2. Effect of Additives on Native Lysozyme.
3.2.1. GdnHCl. The variation of the hydrodynamic radius
(R ) of native lysozyme with increasing amounts of guanidine
h
hydrochloride (GdnHCl), a common denaturant, is shown in
(
Figure S2, Supporting Information) by its interactions with the
Figure 2. As can be seen, the R value increases steadily and it
h
intrinsic amino acids (tryptophan and tyrosine) of the
protein. As the conformational dynamics of protein can
reaches a value of 3.3 ± 0.2 nm for 6 M GdnHCl. The R value
h
2
7
for the unfolded state of lysozyme calculated using the
1
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constancy of the relaxation time with variation of the amount of
GdnHCl. A similar trend is observed for the other additives
(
arginine and both the morpholinium salts). While this
observation is similar to those made earlier by Chattopadhyay
and co-workers in their study of pH-induced unfolding of
3
1
32
IFABP protein and by Sherman et al. for protein-L,
Bhattacharyya et al. found an increase in the relaxation time
15
with unfolding of cytochrome c. We now attempt to find out
the reason for this near-constancy of the conformational
relaxation time despite denaturation of the protein. Considering
that lysozyme consists of six lysine residues, all located on the
33
97
surface of lysozyme, the highest chemical reactivity for Lys
34
47%) and Lys33 (40%) and the labeling ratio of close to
unity (1.2) in our A488-Lysz conjugate, we can assume that
(
Figure 2. Variation of the measured Rh value of lysozyme with
increasing concentration of GdnHCl.
97
33
Alexa488 dye is attached covalently to Lys or Lys . We also
take note of the fact that lysozyme contains six tryptophan
residues, four of which are exposed to the solvent and two are
buried into the hydrophobic interior of the protein. Under
these circumstances, one can conclude that the four tryptophan
residues, which are exposed to water, interact with the Alexa488
dye and contribute to the fluctuations. As the tryptophan
residues and the dye moiety are already on the surface of the
A488-Lysz conjugate, the average distance between the probe
and tryptophans may not change much during the unfolding
process. Another possibility is that the effect due to change in
distance is compensated by a faster motion of the protein in the
unfolded state.
0
.57
29
empirical formula (R = 2.21N Å) of Wilkins et al. is 3.51
h
nm. A comparison of this value with the one measured by us
from the FCS studies clearly suggests complete unfolding of
lysozyme in the presence of 6 M GdnHCl. The disappearance
of the CD signal in the presence of 6 M GdnHCl, as can be
seen from the near-UV CD spectra of lysozyme with and
without GdnHCl, shown in Figure 3, also confirms the
unfolding of lysozyme.
3
.2.2. [Mor1,2][Br] and [Mor1,4][Br]. The effect of
morpholinium salts on the near-UV CD spectra of lysozyme
is shown in Figure 5. A shift (1−2 nm) of the 295 nm peak is
observed which can be attributed to the change in the
microenvironment of the aromatic amino acid residues of
Figure 3. Near-UV CD spectra of lysozyme (90 μM) in phosphate
buffered (pH 7.4) aqueous solution without and with 6 M GdnHCl.
We carefully monitored variation of the relaxation time as a
function of the concentration of GdnHCl (and other additives).
The results for GdnHCl shown in Figure 4 indicate near-
Figure 4. Variation of the τ value with increasing concentration of
Figure 5. Near-UV CD spectra of lysozyme (120 μM) with increasing
R
GdnHCl.
concentration of (a) [Mor1,2][Br] and (b) [Mor1,4][Br].
1
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35
37,39
lysozyme upon addition of the these salts. It is however
clearly evident from the decrease in the CD signal that in the
concentration range of 0−1 M, these salts induce changes in
the tertiary structure of lysozyme, but do not unfold it
Chattopadhyay et al.
The near-UV CD spectra shown in
Figure 8 also confirms a more compact structure of native
lysozyme in the presence of arginine.
completely. The variation of the R value of lysozyme, as
h
estimated from the FCS studies, with increasing concentration
of the two salts, are shown in Figure 6. Small change of the
Figure 8. Near-UV CD spectra of lysozyme (90 μM) with increasing
concentration of arginine.
3
.3. Effect of Additives on Unfolded Lysozyme.
Figure 6. Dependence of the measured R value of native lysozyme on
the concentration of [Mor1,2][Br ] and [Mor1,4][Br].
3.3.1. [Mor1,2][Br] and [Mor1,4][Br]. We have shown earlier
that lysozyme is denatured completely in the presence of 6 M
h
GdnHCl, when its R value increases to 3.3 nm. Under this
h
condition, addition of [Mor1,2][Br] and [Mor1,4][Br] leads to
values observed in the presence of both salts is in accordance
with the CD results. It also appears from both CD signal and Rh
values that the changes induced by [Mor1,4][Br] are somewhat
greater compared to [Mor1,2][Br], indicating that [Mor1,4]-
a decrease of the R value of the unfolded protein (Figure 9)
h
[
Br] comprising a higher alkyl chain length destabilizes the
protein structure more than the one with a shorter chain. This
finding is in accordance with the observation made earlier in
36
the case of imidazolium ionic liquids.
.2.3. Arginine. Arginine, an α-amino acid, is a versatile
additive in biological studies. It serves as a refolding agent for
3
3
7
38
proteins, it can compact the native protein, and is also
known to destabilize the protein because of its structural
37
similarity with GdnHCl. The effect of arginine on the native
state of lysozyme is best captured in FCS measurements by the
change in the R value of the protein with increasing arginine
h
concentrations, shown in Figure 7. It can be seen that the Rh
value of native lysozyme decreases by nearly 30% in the
presence of 0.5 M arginine, indicating that arginine compacts
native lysozyme. In the case of cytochrome c and bovine serum
albumin, a similar observation was made earlier by
Figure 9. Dependence of the R value of unfolded lysozyme (in the
presence of 6 M GdnHCl) on the concentrations of [Mor1,2][Br],
h
[
Mor1,4][Br], and arginine.
indicating a significantly compact state of the protein in the
presence of the morpholinium salts. The R value of lysozyme
h
comes down to 2.1 nm for 0.3 M [Mor1,2][Br] and it remains
there for concentration up to 0.7 M. In the presence of
[
Mor1,4][Br], the size of the denatured protein also decreases
initially by ∼25% for a concentration of 0.3 M. Interestingly,
with further increase in concentration of [Mor1,4][Br], the size
was found to be closer to that of the unfolded state. Thus,
[Mor1,4][Br] compacts the unfolded protein at low concen-
tration, but at higher concentration it favors the unfolded state.
Observation of this kind was made earlier during the
investigation of thermal stability and activity of lysozyme in
5
the presence of propyl alkyl ammonium formate and
36
[
bmim][Cl]. We attribute the effect to more favorable
Figure 7. Plot of the R value of lysozyme, estimated from the FCS
measurements, against the concentration of arginine.
interactions between the unfolded protein and [Mor1, 4][Br]
at high concentration.
h
1
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.3.2. Arginine. A steady decrease of the Rh value of
Article
3
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