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 Enzyme catalysis in organic solvents has been an important and interesting area of research in biotechnology and biochemistry in the past two decades.1−4 The stability of the proteins and 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 the polar organic solvents.5 Hence, enzyme catalyzed reactions 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.6,7 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.8 The ionic liquids are also being explored as refolding agents and to improve the enzyme activity and selectivity in biotransformations.9 Yamamoto et al. studied the effect of N-alkylpyridinium chlorides and N-alkyl-N-methyl pyrrolidinium chlorides on hen © 2013 American Chemical Society
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.10 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 [bmim][NO3], whereas it exists in a partially globular state when the concentration of the ionic liquid exceeds 10 M.11 Mann et al. used near-UV CD measurements to understand the effect of alkyl ammonium formate ionic liquids on the thermal stability and activity of lysozyme.5 Horseradish peroxidase (HRP) activity was increased in [bmim][BF4], whereas a 50% loss in activity was observed in the presence of [bmpy][BF4], indicating that cation variation influences the stability of the enzyme.9 Noritomi et al. studied the thermal stability and activity of lysozyme in presence of [emim][BF4], [emim][Tf2N], and [emim][Cl]. A much lower activity of the protein was observed in the presence of [emim][Cl] when compared with that in [emim][BF4] and [emim][Tf2N].12 Recent NMR, CD, and fluorescence experiments have shown that protic ionic liquids can refold urea-denatured chymotripsin and thus can attenuate the action of urea on it. 13 Very recently, Received: October 3, 2013 Revised: December 7, 2013 Published: December 9, 2013 16587
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2.2. Synthesis of [Mor1,2][Br] and [Mor1,4][Br]. These compounds were synthesized following standard procedure.22 Briefly, the alkyl bromide solutions were added to the Nmethylmorpholine 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 vacuum. [Mor1,2][Br]. 1H NMR (400 MHz, D2O, δ/ppm): 4.05−4.00 (s, 4H), 3.60−3.50 (m, 2H), 3.45−3.40 (t, 4H), 3.1 (s, 3H), 1.38−1.30 (t, 3H); 13C NMR (100 MHz, D2O, δ/ppm): 77.0, 60.2, 59.2, 46.6, 7.9. [Mor1,4][Br]. 1H NMR (400 MHz,CDCl3,δ/ppm): 4.1−4.0 (m, 4H), 3.9−3.8 (m, 4H), 3.7−3.6 (t, 2H), 3.5 (s, 3H), 1.8− 1.7 (m, 2H), 1.5−1.4 (m, 2H), 1.0−0.9 (t,3H); 13C NMR (100 MHz, CDCl3, δ/ppm): 77.0, 64.5, 59.5, 46.4, 22.9, 18.9, 13.1. 2.3. Sample Preparation and Labeling of Lysozyme. 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 coefficients of the dye (71 000 M−1 cm−1 at 495 nm) and lysozyme (37 680 M−1 cm−1 at 280 nm).23 The dye/protein 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. 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 in 3D is given by14
Bhattacharyya and co-workers have studied the effect of [pmim][Br] on human serum albumin and cytochrome c by using fluorescence correlation spectroscopy.14,15 To the best of 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 combination of the ionic liquids.16 However, as the proteinionic 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 [Mor1,4][Br], and studied their influence on chicken egg white lysozyme (Chart 1) in its native and unfolded states using the Chart 1. Structure of (A) Lysozyme (RCSB Protein Data Bank ID: 1LYZ), (B) [Mor1, 2][Br], and (C) [Mor1,4][Br]
fluorescence correlation spectroscopy. This technique allows monitoring of the size of the protein in the presence of a high concentration of cosolutes17 and is shown to be ideally suited for the study of conformational dynamics of proteins, protein− protein, and protein−lipid interactions.18−21 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. EXPERIMENTAL SECTION 2.1. Materials. Acetone, acetonitrile, and alkyl bromides 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. Na2HPO4 (anhydrous) was obtained from Loba Chemie. NaH2PO4 was procured from local suppliers.
−1/2 −1 1⎛ τ ⎞ ⎛ τ ⎞ G (τ ) = ⎜1 + ⎟ ⎜1 + 2 ⎟ N⎝ τD ⎠ ⎝ κ τD ⎠
(1)
The correlation function for a molecule undergoing conformational fluctuation along with diffusion24 is given by 16588
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1 − F + F exp( −τ /τR ) ⎛ τ ⎞ ⎜1 + ⎟ N (1 − F ) τD ⎠ ⎝
−1
G (τ ) =
−1/2 ⎛ τ ⎞ ⎜1 + 2 ⎟ κ τD ⎠ ⎝
(2)
In the above correlation functions, N represents the number of molecules in the observation volume, τD is the diffusion time and τ is the lag time. κ is the structure parameter of the observation volume and is given by κ = ωz/ωxy, where ωz and ωxy are the longitudinal and transverse radii of the observation volume, respectively. τR is the relaxation time and F is the associated amplitude. Rhodamine 6G (diffusion coefficient 426 μm2/s)25 was used to estimate the excitation volume, which was found to be 0.8 fL. The hydrodynamic radius of lysozyme was estimated using the following equation15,17,21 correcting for both viscosity and refractive index mismatches. R hprotein R hRh6G
=
τDprotein τDRh6G
(3)
The τR values were also corrected for the viscosity according to the literature.15 2.4.3. CD Spectroscopy. The near-UV CD spectra were 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 3.1. Size and Conformational Dynamics of Native Lysozyme. The fluorescence correlation data of Alexa488labeled 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 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 ± 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.26 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 (Figure S2, Supporting Information) by its interactions with the intrinsic amino acids (tryptophan and tyrosine) of the protein.27 As the conformational dynamics of protein can
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.
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 hydrodynamic radius (Rh) of lysozyme estimated from the FCS data is 2.1 ± 0.1 nm, which is in agreement with the literature.28 Wilkins et al.29 proposed an empirical formula, Rh = 4.75N0.29 Å, where N is the number of amino acid residues in the protein, for the calculation of the Rh value of a native protein. The Rh value of native lysozyme using this formula is 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 affected much, which was shown earlier by Melo et al.30 The time constant (τR) of the conformational dynamics is measured to be 56 ± 10 μs and can be attributed to the chain dynamics of the protein.15,21 3.2. Effect of Additives on Native Lysozyme. 3.2.1. GdnHCl. The variation of the hydrodynamic radius (Rh) of native lysozyme with increasing amounts of guanidine hydrochloride (GdnHCl), a common denaturant, is shown in Figure 2. As can be seen, the Rh value increases steadily and it reaches a value of 3.3 ± 0.2 nm for 6 M GdnHCl. The Rh value for the unfolded state of lysozyme calculated using the 16589
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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 IFABP protein31 and by Sherman et al.32 for protein-L, Bhattacharyya et al. found an increase in the relaxation time with unfolding of cytochrome c.15 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 surface of lysozyme,33 the highest chemical reactivity for Lys97 (47%) and Lys33 (40%)34 and the labeling ratio of close to unity (1.2) in our A488-Lysz conjugate, we can assume that Alexa488 dye is attached covalently to Lys97 or Lys33. 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. 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 2. Variation of the measured Rh value of lysozyme with increasing concentration of GdnHCl.
empirical formula (Rh = 2.21N0.57 Å) of Wilkins et al.29 is 3.51 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.
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 τR value with increasing concentration of GdnHCl.
Figure 5. Near-UV CD spectra of lysozyme (120 μM) with increasing concentration of (a) [Mor1,2][Br] and (b) [Mor1,4][Br]. 16590
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lysozyme upon addition of the these salts.35 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 completely. The variation of the Rh value of lysozyme, as estimated from the FCS studies, with increasing concentration of the two salts, are shown in Figure 6. Small change of the
Chattopadhyay et al.37,39 The near-UV CD spectra shown in Figure 8 also confirms a more compact structure of native lysozyme in the presence of arginine.
Figure 8. Near-UV CD spectra of lysozyme (90 μM) with increasing concentration of arginine.
3.3. Effect of Additives on Unfolded Lysozyme. 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 GdnHCl, when its Rh value increases to 3.3 nm. Under this condition, addition of [Mor1,2][Br] and [Mor1,4][Br] leads to a decrease of the Rh value of the unfolded protein (Figure 9)
Figure 6. Dependence of the measured Rh value of native lysozyme on the concentration of [Mor1,2][Br ] and [Mor1,4][Br].
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][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 the case of imidazolium ionic liquids.36 3.2.3. Arginine. Arginine, an α-amino acid, is a versatile additive in biological studies. It serves as a refolding agent for proteins,37 it can compact the native protein,38 and is also known to destabilize the protein because of its structural similarity with GdnHCl.37 The effect of arginine on the native state of lysozyme is best captured in FCS measurements by the change in the Rh value of the protein with increasing arginine 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 Rh value of unfolded lysozyme (in the presence of 6 M GdnHCl) on the concentrations of [Mor1,2][Br], [Mor1,4][Br], and arginine.
indicating a significantly compact state of the protein in the presence of the morpholinium salts. The Rh value of lysozyme 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 concentration, 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 the presence of propyl alkyl ammonium formate5 and [bmim][Cl].36 We attribute the effect to more favorable interactions between the unfolded protein and [Mor1, 4][Br] at high concentration.
Figure 7. Plot of the Rh value of lysozyme, estimated from the FCS measurements, against the concentration of arginine. 16591
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3.3.2. Arginine. A steady decrease of the Rh value of unfolded lysozyme is observed with increase in concentration of added arginine (Figure 9). The Rh value reaches 2.1 nm, which is close to that of the native state, in the presence of 0.7 M arginine, suggesting that it also compacts the unfolded lysozyme. However, it is evident that a compact state of the protein that is reached using 0.7 M arginine can be achieved at a much lower concentration (0.3 M) of [Mor1,2][Br] indicating that this salt is more effective in compacting the denatured lysozyme compared to arginine. Hence, like the imidazolium ionic liquids,36 these morpholinium salts are also efficient refolding enhancers (with respect to size), and on occasion, the latter can be more efficient than arginine. We also note that like in the case of GdnHCl, the conformational dynamics of lysozyme is hardly affected in the presence of the morpholinium salts and arginine. Hence, the explanation offered earlier for the invariance of the time constant of the process is applicable here as well.
ASSOCIATED CONTENT
S Supporting Information *
Correlation curve of free Alexa488 and its fit to simple diffusion model (Figure S1), and steady state fluorescence spectra of Alexa488 and Lysz-A488 in the presence and absence of GdnHCl (Figures S2−S4). This material is available free of charge via Internet at http://pubs.acs.org.
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REFERENCES
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4. CONCLUSION Effect of morpholinium salts on the native and unfolded states of lysozyme is studied using fluorescence correlation spectroscopy and near-UV CD spectral measurements. A conformational dynamics of lysozyme with a time constant of 56 μs, which was not reported earlier, has been detected. It is found that both [Mor1,2][Br] and [Mor1,4][Br] destabilize the native state of lysozyme. However, the prominence of the effect in case of [Mor1,4][Br] suggests the possible role of the hydrophobic effect in the process. As far as the effect of morpholinium salts on unfolded lysozyme is concerned, we found that [Mor1,2][Br] compacts the protein, but the [Mor1,4][Br], with a cation having longer alkyl chain length, compacts the protein at low concentration and stabilizes the unfolded state at high concentration. This study also reveals that arginine compacts both the native and unfolded states of lysozyme, but it is significantly less effective compared to [Mor1,2][Br] in compacting the unfolded state.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is supported by the J. C. Bose Fellowship (to A.S.) and PURSE grant (to the University of Hyderabad) of the Department of Science and Technology, Government of India. A.P. thanks Council of Scientific and Industrial Research for a Fellowship and S.G. thanks University Grants Commission for Dr. D. S. Kothari Postdoctoral Fellowship (No.F-4-2/2006/ (BSR)/13-993/2013). 16592
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The Journal of Physical Chemistry B
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dx.doi.org/10.1021/jp409842d | J. Phys. Chem. B 2013, 117, 16587−16593
