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

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Triblock Co-polymer Assisted Mixed-Micelle Formation Results in the Refolding of Unfolded Protein Ramakanta Mondal, Narayani Ghosh, Bijan Kumar Paul, and Saptarshi Mukherjee Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02367 • Publication Date (Web): 25 Aug 2017 Downloaded from http://pubs.acs.org on September 4, 2017

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Triblock Co-polymer Assisted Mixed-Micelle Formation Results in the Refolding of Unfolded Protein

Ramakanta Mondal, Narayani Ghosh, Bijan K. Paul, Saptarshi Mukherjee* Department of Chemistry Indian Institute of Science Education and Research Bhopal Bhopal Bypass Road, Bhauri, Bhopal 426 066, Madhya Pradesh, India *Corresponding author. E-mail: [email protected], Tel.: +917556691301.

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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 the 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 by a step wise manner through three different phases of binding of SDS which is followed by a saturation of interaction. Interestingly, addition of the polymeric surfactant P123 to the unfolded protein results in recovery of ~87% of its α-helical structure which was lost during SDS induced unfolding. This is further corroborated from the recovery of the steady-state and time-resolved fluorescence decay parameters of the intrinsic tryptophan (Trp214) residue of HSA to the initial native-like 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. Based on 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 native-like state.


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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 which 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 which 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) or 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 for its biological functioning.1 Inappropriate folding and improper aggregation of proteins has been documented as the reason for several age-related diseases, including Alzheimer’s and Parkinson’s disease along with other neurodegenerative disorders.6 During refolding, aggregation of protein is a common phenomenon,6 thus preventing proteins from aggregating has always been a topic of growing interest. The in-vitro refolding of a model protein has been established for understanding 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 both at in-vitro and in-vivo conditions and the driving forces involved, we can successfully design small peptides and enzymes.


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Over the past decade or so, many methods of protein refolding were developed to increase the refolding efficiency and yield.8-10 Refolding of a protein into its ordered biologically active state from structure-less 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 multi-domain 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 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 in effective refolding of the denatured proteins.18,19 The extensively used anionic surfactant SDS has been reported to denature proteins20 and the addition of β-cyclodextrin (βCD) has been instrumental in bringing a subsequent refolding.21,22 The mechanism involves the stripping of SDS by βCD due to the formation of inclusion complexes.21,22 In a pioneering work, Gellman and co-worker have used artificial chaperone βCD as stripping agent for 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 to be the major intrinsic fluorophore for HSA and this makes the protein rather unique to be investigated by fluorescence spectroscopy.20,28 The fluorescence monitoring of interaction of SDS with proteins has been reported earlier by several


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researchers.20,21,29-31 In solution, surfactants are found to interact with proteins in monomer, shared-micelle, micelle and vesicle form(s) and the binding isotherm of protein-surfactant interaction is mainly divided into three regions i.e., monomeric, sub-micellar, and post-micellar domains.29 In the monomeric binding region, SDS is found to stabilize the structure of serum proteins thermally, while it denatures the proteins in the submicellar region.29-31 The cationic surfactants like 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 very seminal contribution Pedersen and co-workers have studied the refolding of four SDS-induced denatured proteins; bovine serum albumin, αlactalbumin, 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 native-like structures.37 Our results suggest that although the nonionic triblock copolymer P123 does not have any significant effect on the conformation of HSA in the low concentrations regime, however addition of P123 in the post-miceller concentration region (~1 mM) makes the SDS-induced refolded protein regain a substantial extent of its secondary structure. Pluronics block copolymers have broad range of applications in pharmaceutical industries, targeted drug delivery and food industry due to their low toxicity, high biodegradability and high solubility.36,38-41 Triblock copolymers are comprised of one hydrophobic poly(propylene oxide) (PPO) block and two hydrophilic poly(ethylene oxide) (PEO) blocks having the general formula


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PEOx-PPOy-PEOx and they interact with ionic surfactants.38,42-44 Earlier studies on the interaction of block copolymer with ionic surfactants in aqueous media have shown to result in the formation of either mixed-micelles or 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 the 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 as a 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 which has been otherwise unfolded by SDS. Thus, the current work demonstrates that combination of two surfactants (SDS and P123) of varying structures can serve as refolding agents for a 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. 2. Experimental Section 2.1. Materials Human Serum Albumin (HSA), Tris buffer, guanidine hydrochloride (GdHCl), pnitrophenyl acetate (PNPA) and P123 were used as purchased from Sigma-Aldrich Chemical Co., USA. 0.01 M Tris-HCl buffer of pH 7.4 was prepared in deionized triply distilled Milli pore water. The protein concentration was kept at 2.5 µM throughout all the experiments. All experiments were carried out at 25±0.5 °C unless specified otherwise.


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2.2. Instrumentation and Methods 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 performed with appropriate baseline correction and was the average of four successive scans collected at a scan rate 20 nm min-1. 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 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 IBH-NanoLED-295 light source (FWHM ~810 ps) and the signals were collected using a Hamamatsu MCP Photomultiplier (Model R-3809U-50) at the magic angle polarization of 54.7°.45 The decays were deconvoluted on DAS-6 decay analysis software. Zeta Potential Measurements: Zeta potential for the various micellar systems were determined using Delsa Nano-C (Beckman Coulter Instruments), using a flow cell at 25 °C in 0.01 M Tris-HCl buffer of pH 7.4. Isothermal Titration Calorimetry (ITC) Measurements: The ITC experiments were carried out on a Nano ITC, 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


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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 P123-HSA interaction was evaluated by the titration of 10 mM P123 with 2.5 µM HSA under the same experimental condition mention 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, TA instrument, the exothermic and endothermic heat burst curves are 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 ∆H as obtained from fitting of the experimental data provides the genuine thermodynamic signature of the concerned process and we have interpreted our data accordingly.38,45,47 Esterase-Like Activity: The esterase-like activity of HSA has been investigated by the action of HSA on the substrate p-nitrophenyl acetate (PNPA). Here, the absorbance of the released product, p-nitrophenol has been monitored on a Cary-100 UV-vis spectrophotometer (λabs = 400 nm, molar absorption coefficient, ε = 17700 M-1 cm-1).45 The reaction conditions for these experiments were kept as: [HSA] = 2.5 µM, [PNPA] = 30 µM, pH = 7.4, temperature was kept constant at 37±0.5 °C. 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


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protein.21,48 The Mean Residue Ellipticity (MRE) was estimated from the recorded ellipticity (θobs in mdeg at 222 nm) value using the following equation:21 ‫݃݁݀(ܧܴܯ‬. ܿ݉ଶ . ݀݉‫ି ݈݋‬ଵ ) =

ఏ

(1)

௖೛ ௡௟×ଵ଴

where, Cp is the molar concentration of HSA, n is the number of amino acid residues present in protein (for HSA, n is 585), l is the path-length of the cuvette (here 0.1 cm).21,45 For the 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 and consequently the random coil content increases. The α-helicity of HSA decreases by ~25% and correspondingly the random coil content increases by ~48% in the presence of 4 mM SDS (Table 1). With further addition of SDS to HSA the decrement of CD signal becomes insignificant which indicates a saturation of interaction of SDS with HSA (please see Section 3.2. for details).


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A

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B

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: −□− −; unfolded HSA+1 mM P123: −◊−. (B) Variation of percentage α-helix: −●− and random coil: −○− in the unfolded state of HSA (in the presence of 4 mM SDS) as a function of [P123].

Interestingly, a complete reverse trend of the CD signal of HSA was obtained when we added block copolymer P123 to the partially denatured HSA (Figure 1). In order to monitor the refolding process by P123, we did not use a concentration of SDS beyond 4 mM (please refer to Figure 2B for estimation of the saturation point) in order to avoid the presence of excess micellar SDS which 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 in structural compactness and the refolding process results in an 87% recovery of α-helicity and correspondingly decreases of 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 the labile SDS molecules which induced the unfolding of HSA by forming the protein-surfactant assembly are removed from the protein scaffolds and form mixed-micelle with P123.


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Table 1: Variation of Secondary Structure of HSA with Increasing Concentrations of SDS and P123

HSA 2.5 µM

α-helix# (%) 65.5

Anti-parallel β-sheet# (%) 3.3

Parallel βsheet# (%) 3.4

β-turn# (%) 12.4

Random coil# (%) 16.0

+4 mM SDS

48.9

5.2

5.6

14.2

23.6

0.007

49.0

5.2

5.6

14.5

23.6

0.04

49.6

5.2

5.5

14.4

23.3

0.11

50.8

5.0

5.3

14.3

22.7

0.15

53.6

4.6

4.9

13.9

21.4

0.18

55.1

4.5

4.7

13.7

20.7

0.25

55.4

4.4

4.6

13.6

20.5

0.40

56.1

4.4

4.6

13.6

19.9

0.81

56.8

4.3

4.4

13.5

19.9

1.00

56.7

4.3

4.4

13.5

19.9

1.35

56.9

4.3

4.5

13.5

20.2

System

+[P123] (mM)

#

±0.5% 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 indicate that P123 have almost insignificant effect on the protein conformation at 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 the protein induced by removal of SDS as a result of mixed-micelle formation between P123 and SDS molecule. 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 11 ACS Paragon Plus Environment

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the Trp214 amino acid residue by exciting HSA at 295 nm.20,45,50 As tryptophan fluorescence is highly sensitive towards the small change in its micro-environment, 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 step-wise 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 due to the presence of hydrophobic alkyl chains of the surfactant molecules as well as internalization of Trp residue into the more hydrophobic protein scaffolds due to structural loss of the protein.20,21 Further, when 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 into 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 specific binding of SDS with HSA.20,21 Further addition of SDS results in 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 of Figure 2B) is termed as non-cooperative binding. In the third region (up to 3.6 mM, up to C3 of Figure 2B), an extreme diminution in fluorescence intensity was observed and beyond that there was almost no change, which are identified as the co-operative binding region and saturation phase, respectively.20,21,45 Thus, the unfolding of HSA induced by SDS proceeds


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through a step-wise mechanism characterized by three distinct regions: specific, non-cooperative, and co-operative binding regions which is followed by a saturation phase.

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 illustrates the effect of increasing P123 on the fluorescence properties of the unfolded HSA in the presence of 4 mM of SDS. As seen in the figure, increasing concentration of P123 results in a significant increase in fluorescence intensity of the protein; a completely opposite pattern of modulation of the fluorescence intensity of HSA was observed as compared to the unfolding process, Figure 2A. However, the emission maximum of Trp214 does not show any considerable shift with increasing concentration of P123. A 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 intrinsic fluorescence intensity of unfolded 13 ACS Paragon Plus Environment

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HSA was regained. Similar observations were reported previously by using bovine serum albumin (BSA, an analogue of HSA), where about ~75% of fluorescence intensity had been recovered by using βCD as a refolding agent. The mechanism of 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 the 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 GdHCl-denatured HSA which confirms that P123 does not recover the secondary structure of 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 substantiates the mechanism of refolding initiated and implemented through the formation of mixed-micelles between P123 and SDS.


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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, 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 refolding process.

In the present investigation, we have also tried to establish the 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 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 (λ௠௔௫ ௘௠ ∼582 nm in aqueous buffer to ∼567 nm in 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 SI exemplifies a notable enhancement in emission intensity followed by an additional 3 nm blue-shift of 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 mixedmicelles 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 mixed-micelle between P123 and SDS we have also performed zeta potential measurements. As expected, SDS being an anionic surfactant having negative surface charge exhibits a negative value of zeta potential, Table S1 in Supporting Information (SI). As seen from Table S1, SI, the values of zeta potential becomes more positive (less negative) as we add the non-ionic pluronic P123 to 4 mM SDS in a titrimetric manner. This significant change in 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 transfer of the 15 ACS Paragon Plus Environment

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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-micelle is more negative compared to pure P123 micelles which indicate that the property of mixed-micellar system is different from 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 SI. As seen from 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 the non-ionic block copolymer P123 does not interact prominently with HSA at 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 subsequent addition of P123 to SDS-denatured HSA are shown in Figure 4A and 4B, respectively. The bi-exponential 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 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


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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 of 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 average lifetime follows the same stepwise mechanism of unfolding as encountered in steady-state 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 decrement of structural compactness which provides the freedom of interconvertion of the rotameric forms of Trp214 discussed elsewhere.20,21 In the refolding process (Figure 4B, Table 3), the amplitude of the faster lifetime component (α1) decreases gradually from about 49% to about 21% whereas, the amplitude of the slower lifetime component (α2) progressively increases from about 51% to about 79%, and show no significant variation thereafter. The average lifetime of HSA increases up to 4.62 ns due to addition of 0.8 mM P123. These results indicate the partial refolding of HSA as 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 to those obtained from steadystate emission and CD measurements (please refer to similar nature of the plots as seen from the insets of Figure 4A and 4B and Figures 2B and 3B).


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Figure 4. Fluorescence lifetime decay transients of (A) native HSA in presence of various concentrations of SDS: 1 → 6 correspond to [SDS] = 0.0, 0.006, 0.08, 2.8, 3.2, 4 mM, (B) unfolded HSA (HSA+4 mM SDS) in presence of various concentrations of P123: 1 → 5 correspond to [P123] = 0, 0.015, 0.075, 0.12, 0.8 mM. Insets show the variation of average lifetime of (A) native HSA with increasing concentrations of SDS and (B) unfolded HSA with increasing concentrations of P123. Table 2: Time-Resolved Fluorescence Decay Parameters of HSA with Increasing Concentrations of SDS

†

χ2

SDS

τ1

α1

τ2

α2

>†

(mM)

(ns)

(%)

(ns)

(%)

(ns)

0

2.68

17.12

7.13

82.88

6.37

1.04

0.006

2.31

24.39

6.62

75.61

5.57

1.00

0.01

2.25

26.91

6.37

73.09

5.26

1.06

0.08

2.10

30.83

5.77

69.17

4.64

1.04

0.2

1.91

30.78

5.73

69.22

4.55

1.03

0.6

1.83

30.23

5.73

69.77

4.55

1.17

1.2

1.69

31.67

5.33

68.33

4.18

1.10

2.4

1.31

35.95

4.45

64.05

3.32

1.14

2.8

1.28

37.93

4.19

62.07

3.09

1.13

3.2

1.25

43.89

4.15

56.11

2.88

1.16

3.6

1.18

46.41

4.15

53.59

2.77

1.15

4

1.18

48.72

4.32

51.28

2.80

1.18

±5% 18 ACS Paragon Plus Environment

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Table 3: Time-Resolved Fluorescence Decay Parameters of Unfolded HSA (in the Presence of 4 mM SDS) with Increasing Concentrations of P123

††

χ2

P123

τ1

α1

τ2

α2

>††

(mM)

(ns)

(%)

(ns)

(%)

(ns)

0

1.18

48.72

4.32

51.28

2.80

1.18

0.01

1.41

41.48

4.57

58.52

3.26

1.14

0.015

1.48

37.32

4.66

62.68

3.47

1.14

0.075

2.17

18.28

4.95

81.72

4.44

1.12

0.12

2.35

19.68

5.02

80.32

4.49

1.02

0.3

2.29

18.96

5.10

81.04

4.57

1.03

0.4

2.31

19.16

5.11

80.84

4.58

1.08

0.5

2.48

21.61

5.19

78.39

4.61

1.06

0.6

2.42

22.22

5.24

77.78

4.61

1.02

0.8

2.31

21.01

5.21

78.99

4.62

1.05

± 5%

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 substrate.45,52 The esterase-like activity of HSA has been examined under various conditions of HSA, such as native state, SDS induced unfolded state and native-like 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 µM 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 SDS induced unfolded protein, it recovers up to ~56% esterase-like activity compared to its native state (Figure S5 in SI).


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3.5. Isothermal Titration Calorimetry (ITC) Having established that the SDS unfolded HSA can be refolded by the SDS/P123 mixedmicellar system; we were interested in estimating the energetics of the concerned processes in terms of the associated thermodynamic parameters. Figure 5 displays the calorimetric titration data (after subtraction for heat of dilution) and the thermodynamic parameters as-obtained from fitting of the integrated heat data to a multiple-sites binding model are summarized in Table S2 in SI. It has been reported earlier20 that the binding of SDS to the native state of HSA occurs in a sequential manner, characterized by a three-stage binding (with very high binding affinity especially for the cooperative stage). Herein, we try to establish the fact the binding of P123 to the SDS molecules within the protein scaffolds results in a mixed-micelle formation which in turn subsequently refolds the protein to its native-like conformation. To establish this, we have carried out the ITC experiments using varying concentrations of P123 and HSA in the native state (please refer to Figure S6, SI). As seen, the heat of interaction is almost negligible and hence we can rationally conclude that the block co-polymer does not interact with the native state of the protein. Hence, our ITC results are in good agreement to the steady-state emission and CD data. However, when P123 was added to the unfolded state of the protein (resulted by 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 mixedmicelles that results 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 the anionic surfactant SDS and the tri block copolymer P123 is mainly controlled by the balance of two binding forces. The initial exothermic heat change (∆H0, Table S2 in SI) with high positive entropy change signifies the change in hydration from hydrated SDS monomers which 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 itself from the solvent environment to the hydrophobic aggregate of the non-polar core of the micelle38 with accompanying release of water molecules (water of hydration). It is to be noted that such entropy driven process is usually interpreted on the basis of the classical hydrophobic effect indicating the release of structured water molecules to the bulk aqueous phase during the interaction process.54,55 In both the process the negative free energy change (∆G1 = -32.31 kJ mol-1 and ∆G2 = -17.18 kJ mol-1) indicates the spontaneity of the processes. It is pertinent to state that the thermodynamics interpreted based on 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.

B

A


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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) The raw data for the integrated heat after 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 Instrument and Method section (Section 2.2) for power sign convention.

4. Conclusion We demonstrate the potential application of a triblock copolymer P123 in refolding of SDS-unfolded serum protein, HSA. Our CD results establish a considerable efficiency of P123 in refolding as exemplified by an 87% recovery of its α-helical structure. This is further strongly corroborated from the intrinsic fluorescence behavior of the sole Trp residue (Trp214) of HSA as well as its excited-state lifetime measurements. Our steady-state and time-resolved measurements conclusively establish the step-wise unfolding of the protein (induced by the addition of SDS) and the subsequent recovery to the native-like conformation. The CD results are in excellent harmony to 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 unforlded protein. The ITC data emphatically concludes 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 in indeed quite dramatic characterized by high binding constants. Thus, based on the results of suitably designed experiments we propose that 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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Acknowledgments RM acknowledges IISER Bhopal for a research fellowship. NG acknowledges a research fellowship from the Govt. of India through CSIR-NET. SM sincerely thanks DST, Govt. of India for financial support. We express our sincere thanks to the Central Instrumentation Facility (CIF) of our Institute for access to ITC measurements.

Supporting Information: Far-UV CD spectra of HSA in presence of P123, Far-UV CD and emission spectra of native HSA and GdHCl induced unfolded HSA in presence of P123, emission profiles of PSF in the presence of SDS and P123, zeta potential measurement, emission spectra of HSA in presence of P123, kinetic profiles for the release of p-nitrophenol from the action different state of HSA, ITC parameters for the interaction of unfolded HSA with P123, ITC profile for the titration of HSA with P123. This information is available free of charge via the Internet at http://pubs.acs.org.

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

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