Triblock-Copolymer-Assisted Mixed-Micelle Formation Results in the

Aug 25, 2017 - On the basis of our experiments, we conclude that the formation of mixed micelles between SDS and P123 plays a pivotal role in refoldin...
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Triblock-Copolymer-Assisted Mixed-Micelle Formation Results in the Refolding of Unfolded Protein Ramakanta Mondal, Narayani Ghosh, Bijan K. Paul, and Saptarshi Mukherjee* Department of Chemistry, Indian Institute of Science Education and Research Bhopal, Bhopal Bypass Road, Bhauri, Bhopal 426 066, Madhya Pradesh, India

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S Supporting Information *

ABSTRACT: The present work reports a new strategy for triblock-copolymer-assisted refolding of sodium dodecyl sulfate (SDS)-induced unfolded serum protein human serum albumin (HSA) by mixed-micelle formation of SDS with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer EO20PO68EO20 (P123) under physiological conditions. The steady-state and time-resolve fluorescence results show that the unfolding of HSA induced by SDS occurs in a stepwise manner through three different phases of binding of SDS, which is followed by a saturation of interaction. Interestingly, the addition of polymeric surfactant P123 to the unfolded protein results in the recovery of ∼87% of its α-helical structure, which was lost during SDS-induced unfolding. This is further corroborated by the return of the steady-state and timeresolved fluorescence decay parameters of the intrinsic tryptophan (Trp214) residue of HSA to the initial nativelike condition. The isothermal titration calorimetry (ITC) data also substantiates that there is almost no interaction between P123 and the native state of the protein. However, the mixed-micelle formation, accompanied by substantial binding affinities, removes the bound SDS molecules from the scaffolds of the unfolded state of the protein. On the basis of our experiments, we conclude that the formation of mixed micelles between SDS and P123 plays a pivotal role in refolding the protein back to its nativelike state.

1. INTRODUCTION The refolding of proteins to their native states under in vitro conditions is one of the greatest challenges in biotechnology.1 The native state of protein that is biologically active in nature is generally characterized by a highly ordered, tightly folded, and thermodynamically stable conformation.2 The denaturants are chemical or physical agents that may unfold the protein by direct interaction or by modifying the properties of the surrounding aqueous milieu of the protein, which leads to perturbation of the native protein structure.3 It has been reported that ionic surfactants, at millimolar concentrations, can denature the structure of native proteins through strong interactions involving the charged and hydrophobic side chains.4,5 However, chemical denaturants such as guanidine hydrochloride (GdHCl) and urea, which are found to denature the protein at molar concentrations, probably act through weak binding interactions with proteins.4,5 Under physiological conditions, protein molecules may undergo spontaneous reversible conformational modification(s) and fold into a unique three-dimensional complex structure, which is crucial to its biological functioning.1 Inappropriate folding and improper aggregation of proteins have been documented as the reasons for several age-related diseases, including Alzheimer’s and Parkinson’s diseases along with other neurodegenerative disorders.6 During refolding, protein aggregation is a common phenomenon,6 and thus preventing proteins from aggregating © 2017 American Chemical Society

has always been a topic of growing interest. The in vitro refolding of a model protein has been established to understand the mechanisms by which a polypeptide chain folds its native conformation in the cellular environment.7 Also, if we can understand the certain rules that govern protein unfolding and folding, how the process takes place under both in vitro and in vivo conditions, and the driving forces involved, we can successfully design small peptides and enzymes. Over the past decade or so, many methods of protein refolding were developed to increase the refolding efficiency and yield.8−10 The refolding of a protein into its ordered biologically active state from the structureless denatured state is reported to be an intrinsically complex process owing to the lack of information about the intermediates involved in the folding pathway.11 The refolding process for multidomain proteins is found to be more complex, where each domain may undergo refolding independently and/or via a complex interplay.12,13 The mechanism of protein refolding to its biologically active state from its denatured state also depends on the type of denaturants used.14,15 The Special Issue: Early Career Authors in Fundamental Colloid and Interface Science Received: July 7, 2017 Revised: August 13, 2017 Published: August 25, 2017 896

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different kinds of mixed aggregates depending on the temperature and the concentrations of the interacting partners.42,43 Initially, the binding of SDS to triblock copolymer occurs below the critical micelle concentration (CMC) of pure SDS, and upon increasing the concentration of SDS, the size and the number of aggregates increase.42 At high surfactant concentrations, only the small surfactant-rich complexes are present in the systems.42,43 The interaction of surfactant molecules with block copolymers can be considered to be the solubilization of the hydrocarbon chains of the surfactants into the PPO core of the copolymer micelle.44 Hence, in our case P123 has been used as a refolding agent to recover the unfolded state of HSA that has been otherwise unfolded by SDS. Thus, the current work demonstrates that the combination of two surfactants (SDS and P123) of varying structures can serve as refolding agents for key protein HSA, which happens to be the largest circulatory protein inside our body. Such strategies might be used to maintain the activity and extend the shelf life of many critical proteins.

chemically denatured proteins can be made to refold using the technique of many-fold dilution.16,17 Also additives such as poly(ethylene glycol), salts, short-chain alcohols, sugars, and surfactants have been reported to bring about effective refolding of the denatured proteins.18,19 Extensively used anionic surfactant SDS has been reported to denature proteins,20 and the addition of β-cyclodextrin (βCD) has been instrumental in bringing about subsequent refolding.21,22 The mechanism involves the stripping of SDS by βCD as a result of the formation of inclusion complexes.21,22 In a pioneering work, Gellman and co-worker have used artificial chaperone βCD as a stripping agent for the refolding of the unfolded protein.23,24 Herein, we report the subsequent refolding of the circulatory protein, human serum albumin (HSA, partially denatured by SDS), by the addition of block copolymeric surfactant P123 using several spectroscopic and calorimetric approaches. HSA, an α-helix-rich (66%) circulatory plasma protein accounting for ∼60% of the total protein content in blood plasma, is composed of 585 amino acid residues.20,25−27 A single tryptophan (Trp214) residue in domain IIA serves as the major intrinsic fluorophore for HSA, and this makes the protein rather unique in being investigated by fluorescence spectroscopy.20,28 The fluorescence monitoring of the interaction of SDS with proteins has been reported earlier by several researchers.20,21,29−31 In solution, surfactants are found to interact with proteins in monomers, shared-micelles, micelles, and vesicle forms, and the binding isotherm of the protein−surfactant interaction is mainly divided into three regions, i.e., monomeric, submicellar, and postmicellar domains.29 In the monomeric binding region, SDS is found to stabilize the structure of serum proteins thermally whereas it denatures the proteins in the submicellar region.29−31 Cationic surfactants such as cetyltriethylammonium chloride, dodecyltriethylammonium bromide, and gemini surfactants denature proteins even in the monomeric binding regions, primarily driven by strong electrostatic interactions.31−34 However, the effect of nonionic surfactants on the conformation of protein is very insignificant.35,36 It is reported that pluronic block copolymer F127 interacts with HSA at very high concentrations (∼17%), and at low concentration, its effect on the protein secondary structure is insignificant.36 In a seminal contribution, Pedersen and co-workers have studied the refolding of four SDS-induced denatured proteins: (bovine serum albumin, α-lactalbumin, β-lactoglobulin, and lysozyme) by the addition of nonionic surfactants, octaethylene glycol monododecyl ether, and dodecyl maltoside.37 They concluded that the nonionic surfactant extracted SDS from the protein-SDS complex and thereby allowing the proteins to refold to their nativelike structures.37 Our results suggest that although nonionic triblock copolymer P123 does not have any significant effect on the conformation of HSA in the low-concentrations regime, the addition of P123 in the postmiceller concentration region (∼1 mM) causes the SDS-induced refolded protein to regain a substantial extent of its secondary structure. Pluronic block copolymers have a broad range of applications in the pharmaceutical industry, targeted drug delivery, and the food industry as a result of their low toxicity, high biodegradability, and high solubility.36,38−41 Triblock copolymers are composed of one hydrophobic poly(propylene oxide) (PPO) block and two hydrophilic poly(ethylene oxide) (PEO) blocks having the general formula PEOx-PPOy-PEOx, and they interact with ionic surfactants.38,42−44 Earlier studies on the interaction of a block copolymer with ionic surfactants in aqueous media have been shown to result in the formation of either mixed micelles or

2. EXPERIMENTAL SECTION 2.1. Materials. Human serum albumin (HSA), Tris buffer, guanidine hydrochloride (GdHCl), p-nitrophenyl acetate (PNPA), and P123 were used as purchased from Sigma-Aldrich Chemical Co. Tris-HCl buffer (0.01 M, pH 7.4) was prepared in deionized triply distilled Millipore water. The protein concentration was kept at 2.5 μM throughout all of the experiments. All experiments were carried out at 25 ± 0.5 °C unless otherwise specified. 2.2. Instrumentation and Methods. 2.2.1. Circular Dichroism (CD) Measurements. Far-UV CD measurements were performed on a Jasco J- 815 spectropolarimeter using a cylindrical quartz cuvette of 0.1 cm path lengh.45 Each CD spectrum was recorded with the appropriate baseline correction and was the average of four successive scans collected at a scan rate of 20 nm min−1. 2.2.2. Steady-State Spectral Measurements. The emission spectra were recorded on a Fluorolog 3-111 fluorometer. All spectra were recorded in a 10 mm path length quartz cuvette with appropriate background corrections. The samples were excited at 295 nm to minimize the contribution from tyrosine amino acid residues.45 2.2.3. Time-Resolved Fluorescence Measurements. Fluorescence lifetimes were recorded by the time-correlated single photon counting (TCSPC) technique.45,46 The samples were excited using an IBHNanoLED-295 light source (fwhm ∼810 ps), and the signals were collected using a Hamamatsu MCP photomultiplier (model R-3809U50) at the magic angle polarization of 54.7°.45 The decays were deconvoluted on DAS-6 decay analysis software. 2.2.4. Zeta Potential Measurements. The zeta potential for the various micellar systems was determined using a Delsa Nano-C (Beckman Coulter Instruments) with a flow cell at 25 °C in 0.01 M Tris-HCl buffer at pH 7.4. 2.2.5. Isothermal Titration Calorimetry (ITC) Measurements. The ITC experiments were carried out on a Nano ITC from TA Instruments at 25 °C. Degassed solutions of HSA, SDS, and P123 were used to eliminate the possibility of any bubbles within the solutions. A total of 25 aliquots of P123 solutions ([P123] = 10 mM, 2 μL for each injection) were injected from a syringe rotating at 300 rpm into the ITC sample cell containing the protein−surfactant (mixture of HSA 2.5 μM and 4 mM SDS) solution at an interval of 120 s between successive injections. Corresponding control experiments were done to determine the heat of dilution of P123 solution by injecting an identical volume of the same concentration of P123 into the buffer kept in the sample cell. Similarly, the thermodynamics of the P123-HSA interaction was evaluated by the titration of 10 mM P123 with 2.5 μM HSA under the same experimental conditions mentioned above. The ITC data were analyzed using the NanoAnalyze v2.4.1 software supplied with the instrument according to a model for multiple binding sites. It is relevant to mention in this context that according to the convention of the accompanying software with our Nano ITC from TA Instruments, the exothermic and endothermic heat burst curves are 897

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Figure 1. (A) Far-UV CD spectra of HSA in the presence of various concentrations of SDS and P123. Native HSA 2.5 μM, −■−; 2.5 μM HSA + 4 mM SDS (unfolded HSA), −●−; unfolded HSA + 0.11 mM P123, −▲−; unfolded HSA + 0.15 mM P123, −○−; unfolded HSA + 0.4 mM P123, −□−; and unfolded HSA + 1 mM P123, −◊−. (B) Variation of the α-helix percentage (−●−) and random coil (−○−) in the unfolded state of HSA (in the presence of 4 mM SDS) as a function of [P123]. presented by upward and downward directions, respectively. However, the upward/downward directions of heat changes as shown by any ITC enthalpogram are not universal and depend on the instrument used. The sign of ΔH as obtained from the fitting of the experimental data provides the genuine thermodynamic signature of the concerned process, and we have interpreted our data accordingly.38,45,47 2.2.6. Esterase-Like Activity. The esterase-like activity of HSA has been investigated by the action of HSA on the p-nitrophenyl acetate (PNPA) substrate. Here, the absorbance of the released product, pnitrophenol, has been monitored on a Cary-100 UV−vis spectrophotometer (λabs = 400 nm, molar absorption coefficient, ε = 17 700 M−1 cm−1).45 The reaction conditions for these experiments were kept as [HSA] = 2.5 μM, [PNPA] = 30 μM, and pH 7.4, and the temperature was kept constant at 37 ± 0.5 °C.

Table 1. Variation of the Secondary Structure of HSA with Increasing Concentrations of SDS and P123 system HSA 2.5 μM +4 mM SDS +[P123] (mM) 0.007 0.04 0.11 0.15 0.18 0.25 0.40 0.81 1.00 1.35

3. RESULTS AND DISCUSSION 3.1. Circular Dichroism Spectroscopy. The far-UV CD profile of native HSA (Figure 1A) displays two negative peaks at ∼208 nm and ∼222 nm, which are a typical characteristic of the α-helix-rich secondary content of the protein.21,48 The mean residue ellipticity (MRE) was estimated from the recorded ellipticity (θobs in mdeg at 222 nm) value using the following equation21 MRE(deg· cm 2· dmol−1) =

θ c pnl × 10

a

α-helix (%)a

antiparallel β-sheet (%)a

parallel β-sheet (%)a

β-turn (%)a

random coil (%)a

65.5 48.9

3.3 5.2

3.4 5.6

12.4 14.2

16.0 23.6

49.0 49.6 50.8 53.6 55.1 55.4 56.1 56.8 56.7 56.9

5.2 5.2 5.0 4.6 4.5 4.4 4.4 4.3 4.3 4.3

5.6 5.5 5.3 4.9 4.7 4.6 4.6 4.4 4.4 4.5

14.5 14.4 14.3 13.9 13.7 13.6 13.6 13.5 13.5 13.5

23.6 23.3 22.7 21.4 20.7 20.5 19.9 19.9 19.9 20.2

±0.5%.

Interestingly, a completely reversed trend in the CD signal of HSA was obtained when we added block copolymer P123 to the partially denatured HSA (Figure 1). To monitor the refolding process by P123, we did not use a concentration of SDS beyond 4 mM (please refer to Figure 2B for an estimation of the saturation point) in order to avoid the presence of excess micellar SDS that would have consumed P123 beyond what would have been required to initiate and execute the refolding mechanism. From Figure 1, it is evident that the addition of P123 to the unfolded state of HSA brings about structural compactness and the refolding process results in an 87% recovery of α-helicity and correspondingly decreases the random coil content in the presence of 1 mM P123 (Figure 1B). Beyond this concentration of P123, there was no considerable change in its α-helical content, which indicates that almost all of the labile SDS molecules that induced the unfolding of HSA by forming the protein−surfactant assembly are removed from the protein scaffolds and form a mixed micelle with P123. In a control experiment, we have also checked the interaction of block copolymer P123 with native HSA (i.e., in the absence of SDS) and observed that there was no appreciable change in the CD signals, which indicates that P123 has an almost insignificant effect on the protein conformation in this concentration range (Figure S1 in Supporting Information, SI). Hence, the modulation in the CD signal of HSA upon addition of P123 to the SDS-induced unfolded HSA is solely due to the refolding of

(1)

where cp is the molar concentration of HSA, n is the number of amino acid residues present in protein (for HSA, n is 585), and l is the path length of the cuvette (here 0.1 cm).21,45 For a deeper understanding of the nature of the interaction of surfactant with HSA, the quantitative changes in secondary structural content (α-helical, β-sheet, random coil, etc.) were calculated using CDNN secondary structure analysis software.49 The secondary structure of HSA in its native state consists of 65.5% α-helix, 3.3% antiparallel β-sheet, 3.4% parallel β-sheet, 12.4% β-turn, and 16% random coil (Table 1), which is in good agreement with previous literature reports.20,49 Following the addition of SDS to HSA, the CD signal was found to decrease at all wavelengths, indicating induced perturbation of the HSA secondary structure as depicted in Figure 1A. Table 1 indicates the reduction of the α-helicity of HSA upon addition of SDS; consequently, the random coil content increases. The α-helicity of HSA decreases by ∼25%; correspondingly, the random coil content increases by ∼48% in the presence of 4 mM SDS (Table 1). With the further addition of SDS to HSA, the decrement of the CD signal becomes insignificant, which indicates a saturation of interaction of SDS with HSA (details in Section 3.2). 898

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Figure 2. (A) Fluorescence spectra of HSA in the presence of various [SDS]: 1 → 9 correspond to [SDS] = 0.0, 0.004, 0.008, 0.6, 0.9, 2.0, 2.8, 3.6, and 4.2 mM. (B) Plot of fluorescence intensity of HSA (monitored at 347 nm) against [SDS] for the unfolding process.

Figure 3. (A) Emission profiles of unfolded HSA (in the presence of 4 mM SDS) with increasing concentrations of P123 for the refolding process: 1 → 14 correspond to [P123] = 0.0, 0.0025, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.15, 0.2, 0.4, 0.6, and 1.0 mM. (B) Plot of fluorescence intensity (monitored at 338 nm) for the unfolded HSA (in the presence of 4 mM SDS) against [P123] for the refolding process.

crowding, which in turn swells the protein, thereby opening and exposing more binding sites to be accessed by the incoming surfactant molecules. The second region (0.6 mM SDS, up to C2 in Figure 2B) is termed noncooperative binding. In the third region (up to 3.6 mM, up to C3 in Figure 2B), an extreme diminution in fluorescence intensity was observed, and beyond that, there was almost no change; these are identified as the cooperative binding region and saturation phase, respectively.20,21,45 Thus, the unfolding of HSA induced by SDS proceeds through a stepwise mechanism characterized by three distinct regions: specific, noncooperative, and cooperative binding regions followed by a saturation phase. Figure 3 illustrates the effect of increasing P123 on the fluorescence properties of the unfolded HSA in the presence of 4 mM SDS. As seen in the figure, an increasing concentration of P123 results in a significant increase in the fluorescence intensity of the protein; a completely opposite pattern of modulation of the fluorescence intensity of HSA was observed as compared to that of the unfolding process (Figure 2A). However, the emission maximum of Trp214 does not show any considerable shift with increasing concentration of P123. The complete recovery of fluorescence intensity of HSA is not possible as the SDS molecules get attached to the active sites of the protein with a very high binding affinity.20 Therefore, it may not be possible for the added P123 molecules to remove those strongly bound SDS molecules from the protein scaffolds. Upon addition of 0.8 mM P123, 91% of the intrinsic fluorescence intensity of unfolded HSA was regained. Similar observations were reported previously by using bovine serum albumin (BSA, an analogue of HSA), where about ∼75% of the fluorescence intensity had been recovered by using βCD as a refolding agent. The mechanism of

the protein induced by the removal of SDS as a result of mixedmicelle formation between the P123 and SDS molecules. 3.2. Steady-State Fluorescence Study. To understand the mechanism of SDS-induced protein denaturation and subsequent refolding by triblock copolymer P123, we utilized the intrinsic fluorescence of HSA rendered by the Trp214 amino acid residue by exciting HSA at 295 nm.20,45,50 Because tryptophan fluorescence is highly sensitive to small changes in its microenvironment, it generally reports the alterations of protein conformation(s) and, consequently, the changes in polarity due to the interaction with surfactant molecules.35 It is shown in Figure 2 that the fluorescence intensity of HSA decreases in a stepwise manner upon addition of SDS. Beyond 0.006 mM SDS, the emission maximum of HSA changes, and at 0.008 mM, it is blue-shifted by 4 nm from 347 nm (in the native state). Upon increasing the concentration of SDS, the emission maximum is further blue-shifted, centered around 332 nm at 0.2 mM SDS, and remains almost constant up to 3.2 mM (Figure 2A). The blue shift in the emission maxima signifies that the Trp214 residue experiences more hydrophobic environments as a result of the presence of hydrophobic alkyl chains of the surfactant molecules as well as the internalization of the Trp residue into the more hydrophobic protein scaffolds as a result of the structural loss of the protein.20,21 Furthermore, when the SDS concentration is increased up to 3.6 mM, the emission maximum gets red-shifted to 338 nm (6 nm red shift), indicating the exposure of the Trp214 residue in the bulk solvent compared to what was observed in the presence of 3.2 mM SDS. At low concentrations of the surfactant (0.05 mM SDS, up to C1 in Figure 2B), SDS binds to the high-energy sites of HSA, and the fluorescence intensity decreases sharply. This is referred to as the specific binding of SDS with HSA.20,21 Further addition of SDS results in 899

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Figure 4. Fluorescence lifetime decay transients of (A) native HSA in the presence of various concentrations of SDS: 1 → 6 correspond to [SDS] = 0.0, 0.006, 0.08, 2.8, 3.2, and 4 mM. (B) Unfolded HSA (HSA + 4 mM SDS) in the presence of various concentrations of P123: 1 → 5 correspond to [P123] = 0, 0.015, 0.075, 0.12, and 0.8 mM. Insets show the variation of the average lifetime of (A) native HSA with increasing concentrations of SDS and (B) unfolded HSA with increasing concentrations of P123.

unfolding has been attributed to the formation of inclusion complexes between βCD and monomers of SDS.21 We propose that the mixed micelles formed between SDS and P123 are responsible for the refolding of the unfolded protein. Thus, we have examined the effect of P123 on HSA unfolded by commonly used denaturant guanidine hydrochloride (GdHCl) by monitoring the fluorescence spectra and conformational changes that the protein undergoes using CD spectroscopy. To our surprise, we found that P123 has no effect on the GdHCldenatured HSA, which confirms that P123 does not recover the secondary structure of the unfolded protein (Figure S2, SI). Also, the intrinsic fluorescence of the unfolded HSA changes insignificantly upon addition of P123 (Figure S2, SI). Thus, our experiments using GdHCl substantiate the mechanism of refolding initiated and implemented through the formation of mixed micelles between P123 and SDS. In the present investigation, we have also tried to establish mixed-micelle formation in this experimental concentration regime between P123 and SDS by steady-state fluorescence spectroscopy using phenosafranine (PSF) as an extrinsic fluorescence probe. As seen in Figure S3 in the SI, the emission profile of PSF undergoes a significant enhancement of emission intensity with a 15 nm blue shift following the addition of 4 mM SDS (λmax em ∼ 582 nm in aqueous buffer to ∼567 nm in the presence of 4 mM SDS). This blue shift in the emission maximum of PSF in the presence of SDS is attributed to the hydrophobic environment experienced by the fluorophore upon incorporation into the micelles. Figure S3 in the SI exemplifies a notable enhancement in emission intensity followed by an additional 3 nm blue shift in the emission maximum in the presence of 0.9 mM P123 to the SDS-bound PSF compared to that in SDS micelles alone. This result evidently shows that the probe is buried in the more hydrophobic interior of the mixed micelles compared to the SDS micelles alone. Hence, when P123 is added to the unfolded state of the protein, the mixed micelles remove the surfactant molecules, resulting in a structural recovery for the protein. To confirm the formation of the mixed micelle between P123 and SDS, we have also performed zeta potential measurements. As expected, SDS, being an anionic surfactant having a negative surface charge, exhibits a negative zeta potential value (Table S1 in the Supporting Information). As seen from Table S1, the values of the zeta potential becomes more positive (less negative) as we add nonionic pluronic P123 to 4 mM SDS in a titrimetric manner. This significant change in the zeta potential due to the addition of P123 to a micellar solution of SDS indicates that the

mixed micelle formation between P123 and SDS causes the transfer of the negatively charged SDS into the micelle core. This reduces the overall surface charge of the mixed micelles so formed. Also, the zeta potential of mixed micelles is more negative compared to that of pure P123 micelles, which indicates that the properties of the mixed micellar system are different from those of both pure P123 and SDS. We have also investigated the interaction of native HSA with P123 (in the absence of SDS) by monitoring the modulation of the intrinsic fluorescence of HSA as depicted in Figure S4 in the SI. As seen in Figure S4, the intensity and the emission maxima of Trp214 do not change prominently with the addition of even 1 mM P123 copolymer, which corroborates the fact that nonionic block copolymer P123 does not interact prominently with HSA in this concentration range. 3.3. Time-Resolved Measurements. The fluorescence lifetime decay transients of HSA in the presence of increasing concentrations of SDS and with the subsequent addition of P123 to SDS-denatured HSA are shown in Figure 4A,B, respectively. The biexponential fluorescence lifetime decay of the native HSA in the absence of surfactant is consistent with previous literature reports.20,45 The nature of the decay transients of HSA can be explained by the existence of different rotational conformational isomers of tryptophan, called rotamers.20,46,51 In the present work, we have studied the modulations of the Trp214 lifetime during the unfolding of HSA upon addition of SDS and the refolding of the unfolded protein by P123 (Figure 4). It is noted that the relative amplitude (α2) of the slower decay time constant (τ2) gradually decreases (from about 83 to about 51%), and simultaneously the relative amplitude (α1) of the faster decay time constant (τ1) progressively increases (from about 17 to about 49%) with increasing SDS concentration (Table 2). In doing so, the average lifetime of HSA decreases from 6.37 ns (for native HSA) to 2.80 ns in the presence of 4 mM SDS. According to the reported literature, the decrease in the average lifetime of HSA is due to the quenching of Trp214 fluorescence by SDS molecules, internal quencher moieties, and/or solvent accessibility due to partial unfolding of the native protein conformation by SDS.20,21 Also, the variation of the average lifetime follows the same stepwise mechanism of unfolding as encountered in steadystate measurements (Figure 4A inset). The variations of the relative contributions of the lifetime may originate from the facile interconversion from conformers due to the unfolding of HSA, resulting in a decrement in structural compactness that provides the freedom of interconvertion of the rotameric forms of Trp214 discussed elsewhere.20,21 900

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3.5. Isothermal Titration Calorimetry (ITC). Having established that the SDS unfolded HSA can be refolded by the SDS/P123 mixed-micellar system, we were interested in estimating the energetics of the concerned processes in terms of the associated thermodynamic parameters. Figure 5 displays

Table 2. Time-Resolved Fluorescence Decay Parameters of HSA with Increasing Concentrations of SDS SDS (mM)

τ1 (ns)

α1 (%)

τ2 (ns)

α2 (%)

⟨τ⟩ (ns)a

χ2

0 0.006 0.01 0.08 0.2 0.6 1.2 2.4 2.8 3.2 3.6 4

2.68 2.31 2.25 2.10 1.91 1.83 1.69 1.31 1.28 1.25 1.18 1.18

17.12 24.39 26.91 30.83 30.78 30.23 31.67 35.95 37.93 43.89 46.41 48.72

7.13 6.62 6.37 5.77 5.73 5.73 5.33 4.45 4.19 4.15 4.15 4.32

82.88 75.61 73.09 69.17 69.22 69.77 68.33 64.05 62.07 56.11 53.59 51.28

6.37 5.57 5.26 4.64 4.55 4.55 4.18 3.32 3.09 2.88 2.77 2.80

1.04 1.00 1.06 1.04 1.03 1.17 1.10 1.14 1.13 1.16 1.15 1.18

a

±5%.

Figure 5. ITC profile for the titration of unfolded HSA (HSA 2.5 μM + 4 mM SDS) with P123 at 25 °C (pH 7.4). (A) Raw data for the integrated heat after the correction of the heat of dilution. (B) ITC enthalpograms obtained at 25 °C. The solid line is obtained on fitting the data to a multiple-sites binding model. See the Instrumentation and Methods section for the power sign convention.

In the refolding process (Figure 4B, Table 3), the amplitude of the faster lifetime component (α1) decreases gradually from Table 3. Time-Resolved Fluorescence Decay Parameters of Unfolded HSA (in the Presence of 4 mM SDS) with Increasing Concentrations of P123 P123 (mM)

τ1 (ns)

α1 (%)

τ2 (ns)

α2 (%)

⟨τ⟩ (ns)a

χ2

0 0.01 0.015 0.075 0.12 0.3 0.4 0.5 0.6 0.8

1.18 1.41 1.48 2.17 2.35 2.29 2.31 2.48 2.42 2.31

48.72 41.48 37.32 18.28 19.68 18.96 19.16 21.61 22.22 21.01

4.32 4.57 4.66 4.95 5.02 5.10 5.11 5.19 5.24 5.21

51.28 58.52 62.68 81.72 80.32 81.04 80.84 78.39 77.78 78.99

2.80 3.26 3.47 4.44 4.49 4.57 4.58 4.61 4.61 4.62

1.18 1.14 1.14 1.12 1.02 1.03 1.08 1.06 1.02 1.05

a

the calorimetric titration data (after subtraction for the heat of dilution), and the thermodynamic parameters as obtained from the fitting of the integrated heat data to a multiple-sites binding model are summarized in Table S2 in the SI. It was reported earlier20 that the binding of SDS to the native state of HSA occurs in a sequential manner characterized by three-stage binding (with a very high binding affinity especially for the cooperative stage). Herein, we try to establish the fact that the binding of P123 to the SDS molecules within the protein scaffolds results in mixedmicelle formation that in turn subsequently refolds the protein to its nativelike conformation. To establish this, we have carried out ITC experiments using varying concentrations of P123 and HSA in the native state (Figure S6, SI). As seen, the heat of interaction is almost negligible, and hence we can rationally conclude that the block copolymer does not interact with the native state of the protein. Hence, our ITC results are in good agreement with the steady-state emission and CD data. However, when P123 was added to the unfolded state of the protein (resulting in the addition of 4 mM SDS), the heat changes are quite dramatic, which substantiates the fact that the interaction of P123 with the unfolded state of the protein leads to the formation of mixed micelles, resulting in the protein refolding. The existence of exothermic as well as endothermic heat changes in the ITC curves (Figure 5) indicates that the interaction between anionic surfactant SDS and triblock copolymer P123 is mainly controlled by the balance of two binding forces. The initial exothermic heat change (ΔH < 0, Table S2 in the SI) can be ascribed to the breakup of the SDS micelles induced by P123 and the accompanying hydration of SDS monomeric units.53 In the second region, the appearance of the endothermic heat change (ΔH > 0, Table S2 in SI) with high positive entropy change signifies the change in hydration from hydrated SDS monomers that form compact aggregates with the PPO units of P123, resulting in the release of water molecules.53 This large increase in entropy can be attributed to the tendency of the hydrophobic groups of the surfactant molecule to transfer themselves from the solvent environment to the hydrophobic aggregate of the nonpolar core of the micelle38 with an accompanying release of water molecules (water of hydration). It is to be noted that such an entropy-driven process is usually

±5%.

about 49 to about 21%, whereas the amplitude of the slower lifetime component (α2) progressively increases from about 51 to about 79% and shows no significant variation thereafter. The average lifetime of HSA increases up to 4.62 ns as a result of the addition of 0.8 mM P123. These results indicate the partial refolding of HSA because the lifetime parameters are not fully recovered compared to the native state. Thus, the conclusions drawn from our time-resolved measurements are in excellent agreement with those obtained from steady-state emission and CD measurements. (Refer to the similar nature of the plots as seen from the insets in Figures 2B, 3B, and 4A,B.) 3.4. Esterase-like Activity HSA. We have investigated the esterase-like activity of HSA by monitoring the catalytic reactivity HSA using p-nitrophenyl acetate (PNPA) as the substrate.45,52 The esterase-like activity of HSA has been examined under various conditions of HSA, such as the native state, the SDSinduced unfolded state, and the nativelike refolded state (upon addition of P123 to the unfolded protein induced by 4 mM SDS). One unit of esterase activity has been defined as the amount of enzyme (HSA) required to liberate 1 μmol of p-nitrophenol from PNPA at 37 °C per minute.45,52 Our results show that in the presence of 4 mM SDS the protein loses its esterase-like activity almost completely and upon addition of P123 to the SDSinduced unfolded protein it recovers up to ∼56% of its esteraselike activity compared to its native state (Figure S5 in the SI). 901

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ACKNOWLEDGMENTS R.M. acknowledges IISER Bhopal for a research fellowship. N.G. acknowledges a research fellowship from the Government of India through CSIR-NET. S.M. sincerely thanks DST, Government of India for financial support. We express our sincere thanks to the Central Instrumentation Facility (CIF) of our Institute for access to ITC measurements.

interpreted on the basis of the classical hydrophobic effect indicating the release of structured water molecules into the bulk aqueous phase during the interaction process.54,55 In both processes, the negative free-energy changes (ΔG1 = −32.31 kJ mol−1 and ΔG2 = −17.18 kJ mol−1) indicate the spontaneity of the processes. It is pertinent to state that the thermodynamics interpreted on the basis of the ITC experiment gives only an averaged depiction of the overall complex equilibrium phenomenon. Consequently, one-to-one mapping of the structural properties with the thermodynamic parameters is not possible here.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b02367. Far-UV CD spectra of HSA in the presence of P123, farUV CD and emission spectra of native HSA and GdHClinduced unfolded HSA in the presence of P123, emission profiles of PSF in the presence of SDS and P123, zeta potential measurement, emission spectra of HSA in the presence of P123, kinetic profiles for the release of pnitrophenol from the different action states of HSA, ITC parameters for the interaction of unfolded HSA with P123, and ITC profile for the titration of HSA with P123 (PDF)

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REFERENCES

(1) Lu, D.; Liu, Z.; Liu, Z.; Zhang, M. Molecular Simulation of Surfactant-Assisted Protein Refolding. J. Chem. Phys. 2005, 122, 134902−134911. (2) Anfinsen, C. B. Principles that Govern the Folding of Protein Chains. Science 1973, 181, 223−230. (3) Konermann, L. Protein Unfolding and Denaturants. Encyclopedia of Life Sciences; Nature Publishing Group: New York, 2002. (4) Turro, N. J.; Lei, X. -G; Ananthapadmanabhan, K. P.; Aronson, M. Spectroscopic Probe Analysis of Protein-Surfactant Interactions: The BSA/SDS System. Langmuir 1995, 11, 2525−2533. (5) Gelamo, E. L.; Tabak, M. Spectroscopic Studies on the Interaction of Bovine (BSA) and Human (HSA) Serum Albumins with Ionic Surfactants. Spectrochim. Acta, Part A 2000, 56, 2255−2271. (6) Dobson, C. M. Protein Folding and Misfolding. Nature 2003, 426, 884−890. (7) Yon, J. M. Protein Folding: A Perspective for Biology, Medicine and Biotechnology. Braz. J. Med. Biol. Res. 2001, 34, 419−435. (8) Wang, X.; Lu, D.; Austin, R.; Agarwal, A.; Mueller, L. J.; Liu, Z.; Wu, J.; Feng, P. Protein Refolding Assisted by Periodic Mesoporous Organosilicas. Langmuir 2007, 23, 5735−5739. (9) Martin, N.; Li, M.; Mann, S. Selective Uptake and Refolding of Globular Proteins in Coacervate Microdroplets. Langmuir 2016, 32, 5881−5889. (10) Liu, H.; Dong, X.; Sun, Y. Enhanced Protein Adsorption and Facilitated Refolding of Like-Charged Protein with Highly Charged Silica Nanoparticles Fabricated by Sequential Double Modifications. Langmuir 2015, 31, 655−658. (11) Muzammil, S.; Kumar, Y.; Tayyab, S. Molten Globule-Like State of Human Serum Albumin at Low pH. Eur. J. Biochem. 1999, 266, 26− 32. (12) Wetlaufer, D. B. Nucleation, Rapid Folding and Globular Intrachain Regions in Proteins. Proc. Natl. Acad. Sci. U. S. A. 1973, 70, 697−701. (13) Dahiya, V.; Chaudhuri, T. K. Functional Intermediate in the Refolding Pathway of a Large and Multidomain Protein Malate Synthase G. Biochemistry 2013, 52, 4517−4530. (14) Dill, K. A.; Shortle, D. Denatured States of Proteins. Annu. Rev. Biochem. 1991, 60, 795−825. (15) Yamaguchi, H.; Miyazaki, M. Refolding Techniques for Recovering Biologically Active Recombinant Proteins from Inclusion Bodies. Biomolecules 2014, 4, 235−251. (16) Anand, U.; Jash, C.; Mukherjee, S. Protein Unfolding and Subsequent Refolding: A Spectroscopic Investigation. Phys. Chem. Chem. Phys. 2011, 13, 20418−20426. (17) Otzen, D. E.; Andersen, K. K. Folding of Outer Membrane Proteins. Arch. Biochem. Biophys. 2013, 531, 34−43. (18) Clark, E. D. B. Refolding of Recombinant Proteins. Curr. Opin. Biotechnol. 1998, 9, 157−163. (19) Gupta, P.; Hall, C. K.; Voegler, A. Computer Simulation of the Competition between Protein Folding and Aggregation. Fluid Phase Equilib. 1999, 158-160, 87−93. (20) Anand, U.; Jash, C.; Mukherjee, S. Spectroscopic Probing of the Microenvironment in a Protein-Surfactant Assembly. J. Phys. Chem. B 2010, 114, 15839−15845. (21) Anand, U.; Mukherjee, S. Reversibility in Protein Folding: Effect of β-Cyclodextrin on Bovine Serum Albumin Unfolded by Sodium Dodecyl Sulphate. Phys. Chem. Chem. Phys. 2013, 15, 9375−9383. (22) Matei, I.; Ariciu, A. M.; Neacsu, M. V.; Collauto, A.; Salifoglou, A.; Ionita, G. Cationic Spin Probe Reporting on Thermal Denaturation and

4. CONCLUSIONS We demonstrate the potential application of triblock copolymer P123 in the refolding of SDS-unfolded serum protein HSA. Our CD results establish the considerable efficiency of P123 in refolding as exemplified by an 87% recovery of its α-helical structure. This is further strongly corroborated by the intrinsic fluorescence behavior of the sole Trp residue (Trp214) of HSA as well as its excited-state lifetime measurements. Our steadystate and time-resolved measurements conclusively establish the stepwise unfolding of the protein (induced by the addition of SDS) and the subsequent recovery to the nativelike conformation. The CD results are in excellent harmony with those obtained from spectroscopic measurements. The addition of P123 results in the formation of mixed micelles with SDS, and the interaction of the triblock copolymer with the surfactant molecules embedded in the protein matrix results in the subsequent refolding of the unfolded protein. The ITC data emphatically shows that there is almost no interaction of P123 with the native state of the protein. However, as seen from the ITC enthalpogram, the binding of P123 to SDS present in the protein−surfactant assemblies is indeed quite dramatic as characterized by high binding constants. Thus, on the basis of the results of suitably designed experiments we propose that the formation of mixed micelles between SDS and P123 is the possible mechanism underlying the stripping of SDS molecules from the protein surface leading to its subsequent refolding.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +917556691301. ORCID

Saptarshi Mukherjee: 0000-0001-8280-0754 Notes

The authors declare no competing financial interest. 902

DOI: 10.1021/acs.langmuir.7b02367 Langmuir 2018, 34, 896−903

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and Ionic Surfactants: Equilibrium and Structure. Langmuir 2002, 18, 9267−9275. (43) Ganguly, R.; Aswal, V. K.; Hassan, P. A.; Gopalakrishnan, I. K.; Kulshreshtha, S. K. Effect of SDS on the Self-Assembly Behavior of the PEO-PPO-PEO Triblock Copolymer (EO)20(PO)70(EO)20. J. Phys. Chem. B 2006, 110, 9843−9849. (44) Jansson, J.; Schillén, K.; Olofsson, G.; da Silva, R. C.; Loh, W. The Interaction between PEO-PPO-PEO Triblock Copolymers and Ionic Surfactants in Aqueous Solution Studied using Light Scattering and Calorimetry. J. Phys. Chem. B 2004, 108, 82−92. (45) Ghosh, N.; Mondal, R.; Mukherjee, S. Hydrophobicity Is the Governing Factor in the Interaction of Human Serum Albumin with Bile Salts. Langmuir 2015, 31, 1095−1104. (46) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer, 2006. (47) Roman-Guerrero, A.; Vernon-Carter, E. J.; Demarse, N. A. Thermodynamics of Micelle Formation. TA Instruments Application Note, MCPAN-2010-05; TA Instruments-Waters LLC, 2010. (48) Moriyama, Y.; Takeda, K. Re-formation of the Helical Structure of Human Serum Albumin by the Addition of Small Amounts of Sodium Dodecyl Sulfate after the Disruption of the Structure by Urea. A Comparison with Bovine Serum Albumin. Langmuir 1999, 15, 2003− 2008. (49) Bohlooli, M.; Moosavi-Movahedi, A. A.; Taghavi, F.; HabibiRezaei, M.; Seyedarabi, A.; Saboury, A. A.; Ahmad, F. Thermodynamics of a Molten Globule State of Human Serum Albumin by 3-βHydroxybutyrate as a Ketone Body. Int. J. Biol. Macromol. 2013, 54, 258−263. (50) Sharma, A. S.; Ilanchelian, M. Comprehensive Multispectroscopic Analysis on the Interaction and Corona Formation of Human Serum Albumin with Gold/Silver Alloy Nanoparticles. J. Phys. Chem. B 2015, 119, 9461−9476. (51) Pertich, J. W.; Chang, M. C.; McDonald, D. B.; Fleming, G. R. On the Origin of Nonexponential Fluorescence Decay in Tryptophan and its Derivatives. J. Am. Chem. Soc. 1983, 105, 3824−3832. (52) Ostdal, H.; Andersen, H. Non-Enzymatic Protein Induced Hydrolysis of p-Nitrophenyl Acyl Esters in Relation to Lipase/Esterase Assays. Food Chem. 1996, 55, 55−61. (53) da Silva, R. C.; Olofsson, G.; Schillén, K.; Loh, W. Influence of Ionic Surfactants on the Aggregation of Poly(Ethylene Oxide)Poly(Propylene Oxide)-Poly(Ethylene Oxide) Block Copolymers Studied by Differential Scanning and Isothermal Titration Calorimetry. J. Phys. Chem. B 2002, 106, 1239−1246. (54) Paul, B. K.; Ghosh, N.; Mukherjee, S. Interplay of Multiple Interaction Forces: Binding of Norfloxacin to Human Serum Albumin. J. Phys. Chem. B 2015, 119, 13093−13102. (55) Ross, P. D.; Subramanian, S. Thermodynamics of Protein Association Reactions: Forces Contributing to Stability. Biochemistry 1981, 20, 3096−3102.

Complexation-Decomplexation of BSA with SDS. Potential Applications in Protein Purification Processes. J. Phys. Chem. B 2014, 118, 11238−11252. (23) Rozema, D.; Gellman, S. H. Artificial Chaperones: Protein Refolding via Sequential Use of Detergent and Cyclodextrin. J. Am. Chem. Soc. 1995, 117, 2373−2374. (24) Rozema, D.; Gellman, S. H. Artificial Chaperone-assisted Refolding of Carbonic Anhydrase B. J. Biol. Chem. 1996, 271, 3478− 3487. (25) He, X. M.; Carter, D. C. Atomic Structure and Chemistry of Human Serum Albumin. Nature 1992, 358, 209−215. (26) Campbell, R. A.; Ang, J. C.; Sebastiani, F.; Tummino, A.; White, J. W. Spread Films of Human Serum Albumin at the Air-Water Interface: Optimization, Morphology, and Durability. Langmuir 2015, 31, 13535− 13542. (27) Peters, T., Jr. All about Albumin: Biochemistry, Genetics, and Medical Applications; Academic Press: San Diego, CA, 1996. (28) Bolattin, M. B.; Nandibewoor, S. T.; Joshi, S. D.; Dixit, S. R.; Chimatadar, S. A. Interaction of Hydralazine with Human Serum Albumin and Effect of β Cyclodextrin on Binding: Insights from Spectroscopic and Molecular Docking Techniques. Ind. Eng. Chem. Res. 2016, 55, 5454−5464. (29) Bharmoria, P.; Rao, K. S.; Trivedi, T. J.; Kumar, A. Biamphiphilic Ionic Liquid Induced Folding Alterations in the Structure of Bovine Serum Albumin in Aqueous Medium. J. Phys. Chem. B 2014, 118, 115− 124. (30) Chen, S. H.; Teixeiria, J. Structure and Fractal Dimension of Protein-Detergent Complexes. Phys. Rev. Lett. 1986, 57, 2583−2586. (31) Kelley, D.; McClements, D. J. Interactions of Bovine Serum Albumin with Ionic Surfactants in Aqueous Solutions. Food Hydrocolloids 2003, 17, 73−85. (32) Gelamo, E. L.; Itri, R.; Alonso, A.; da Silva, J. V.; Tabak, M. SmallAngle X-ray Scattering and Electron Paramagnetic Resonance Study of the Interaction of Bovine Serum Albumin with Ionic Surfactants. J. Colloid Interface Sci. 2004, 277, 471−482. (33) Zhang, X.; Poniewierski, A.; Hou, S.; Sozanski, K.; Wisniewska, A.; Wieczorek, S. A.; Kalwarczyk, T.; Sun, L.; Hołyst, R. Tracking Structural Transitions of Bovine Serum Albumin in Surfactant Solutions by Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Analysis. Soft Matter 2015, 11, 2512−2518. (34) Wu, D.; Xu, G.; Sun, Y.; Zhang, H.; Mao, H.; Feng, Y. Interaction between Protein and Cationic Gemini Surfactant. Biomacromolecules 2007, 8, 708−712. (35) Otzen, D. Protein-Surfactant Interactions: A Tale of Many States. Biochim. Biophys. Acta, Proteins Proteomics 2011, 1814, 562−591. (36) Neacsu, M. V.; Matei, I.; Micutz, M.; Staicu, T.; Precupas, A.; Popa, V. T.; Salifoglou, A.; Ionita, G. Interaction between Albumin and Pluronic F127 Block Copolymer Revealed by Global and Local Physicochemical Profiling. J. Phys. Chem. B 2016, 120, 4258−4267. (37) Kaspersen, J. D.; Søndergaard, A.; Madsen, D. J.; Otzen, D. E.; Pedersen, J. S. Refolding of SDS-Unfolded Proteins by Nonionic Surfactants. Biophys. J. 2017, 112, 1609−1620. (38) Mondal, R.; Ghosh, N.; Mukherjee, S. Enhanced Binding of Phenosafranin to Triblock Copolymer F127 Induced by Sodium Dodecyl Sulfate: A Mixed Micellar System as an Efficient Drug Delivery Vehicle. J. Phys. Chem. B 2016, 120, 2968−2976. (39) Jeong, B.; Bae, Y. H.; Lee, D. S.; Kim, S. W. Biodegradable Block Copolymers as Injectable Drug-Delivery Systems. Nature 1997, 388, 860−862. (40) Rösler, A.; Vandermeulen, G. W. M.; Klok, H. A. Advanced Drug Delivery Devices via Self-Assembly of Amphiphilic Block Copolymers. Adv. Drug Delivery Rev. 2012, 64, 270−279. (41) Anand, U.; Mukherjee, S. Microheterogeneity and Microviscosity of F127 Micelle: The Counter Effects of Urea and Temperature. Langmuir 2014, 30, 1012−1021. (42) Thurn, T.; Couderc, S.; Sidhu, J.; Bloor, D. M.; Penfold, J.; Holzwarth, J. F.; Wyn-Jones, E. Study of Mixed Micelles and Interaction Parameters for ABA Triblock Copolymers of the Type EOm-POn-EOm 903

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