Ionic Liquid Surfactant Mediated Structural Transitions and Self

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The Journal of Physical Chemistry

Ionic Liquid Surfactant Mediated Structural Transitions and Self-assembly of Bovine Serum Albumin in Aqueous Media: Effect of Functionalization of Ionic Liquid Surfactants Gurbir Singh, Tejwant Singh Kang* Department of Chemistry, UGC-centre for Advance Studies – I, Guru Nanak Dev University, Amritsar, 143005, India.

Abstract The self-assembly of globular protein, bovine serum albumin (BSA) has been investigated in aqueous solutions of ionic liquid surfactants (ILSs), 1-dodecyl-3-methyl imidazolium chloride, [C12mim][Cl] and its amide, [C12Amim][Cl] and ester, [C12Emim][Cl] functionalized counterparts. Dynamic light scattering (DLS) have provided insights into the alterations in hydrodynamic radii (Dh) of BSA as a function of concentration of ILSs establishing the presence of different types BSA-ILS complexes in different concentration regimes of ILSs. Isothermal titration calorimetry (ITC) has been exploited to quantify the ILSs interacting with BSA in dilute concentration regime of ILSs. The zeta-potential measurements shed light on changes in the charged state of BSA. The morphology of various self-assembled structures of BSA in different concentration regimes of ILSs have been explored using confocal laser scanning microscopy (CLSM) and scanning electron microscopy. The structural variations in ILSs have been found to produce remarkable affect on the nature and morphology of self-assembled structures of BSA. The presence of non-functionalized [C12mim][Cl] IL at all investigated concentrations has led to the formation of unordered large self-assembled structures of BSA. On the other hand, in specific concentration regimes, ordered self-assembled structures such as long rods and right-handedly twisted helical amyloid fibers have been observed in the presence of functionalized [C12Amim][Cl] and [C12Emim][Cl] ILSs, respectively. The nature of the formed helical fibers as amyloid ones has been confirmed using FTIR spectroscopy. Steady-state fluorescence and circular dichroism (CD) spectroscopy have provided insights into folding and unfolding of BSA as fashioned by interactions with ILSs in different concentrations regimes supporting the observations made from other studies.

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Keywords: Functionalized Ionic Liquids; Ordered self-assembled structures; long rods; helical amyloid fibers *To whom correspondence should be addressed: e-mail: [email protected]; Tel: +91-183-2258802-Ext-3207


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1. Introduction The self-assembly of proteins is a naturally occurring phenomenon, which give rise to a variety of structural architectures depending upon the prevailing conditions.1,2 Such self-assembly can be controlled as well as tuned by judicious choice of conditions such as addition of suitable additives, utilizing optimum temperature and pH, and the concentration of protein etc.3-9 Bovine serum albumin (BSA), a mammalian globular protein, is widely investigated for its self-assembly owing to its function as major transporter of many biological compounds, and drugs in plasma as well as its structural similarity to human serum albumin (HSA).10 The native structure of BSA in crystal form comprises of three domains i.e. domain I, II and III, forming a heart shape structure, where nine loops comprising 583 amino acid residues are interconnected with 17 disulphide bonds.11 In solution form, structural transformations in BSA are governed by the prevailing conditions of pH, temperature, ionic strength and the presence of additive. For example, BSA remains in its native form (N-form) at physiological pH, whereas it adopts F-form around pH 5.0 leading to decreased solubility along with decrease in α-helical content. At further lower pH (pH < 4.0), BSA adopts expanded form (E-form), where it undergoes unfolding and hence expansion, leading to increase in axial ration from 4 to 9.12-14 The important property of globular proteins such as BSA, HSA, ovalbumin and β-lactoglobulin etc. is their self-assembly into discrete morphological architectures such as amyloid fibrils and non-amyloid fibrillar aggregates having great importance in several scientific areas.3,4,7-9 The study of formation of such fibrils in light of type of the fibrils, mechanism of formation and the nature of forces governing the fibril formation is of great importance from biological and medical point of view due to their unique mechanical properties, relevance in medicinal field and in nanotechnology.2 Moreover, some of the neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and diabetes mellitus type II are closely associated with deposition of amyloid fibrils via protein misfolding disrupting the normal function of concerned body part.15-17 These amyloid fibrils are characterized by their β-sheet rich structure formed by perpendicular orientation of βsheets around axis of fibril interconnected by H-bonds.2 In a broader sense, the fibrillar arrangement of proteins are driven by H-bonding, hydrophobic, electrostatic and weak van der Waals forces of attractions.3



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On the other hand, the use of surfactants in conjunction with globular proteins has been widely investigated from the view point of diverse applications of such colloidal systems in the field of pharmaceuticals, cosmetics, emulsifiers, paints and coatings etc.18-20 The presence of surfactant generally improves the interfacial and bulk properties of aqueous solutions of different proteins. Besides that, a variety of surfactants have been found to induce the structural transitions in different proteins in terms of unfolding or refolding of native structure of proteins, and their solubilization as well as stability which, in turn affects the functional activity of the protein.21-25 Anionic surfactant such as sodium dodecyl sulfate (SDS) has been shown to induce stabilization of secondary structure of BSA in its monomeric concentration regime.21-23 On the other hand, cationic

surfactants

such

as

cetyltrimethylammonium

bromide

(CTAB)

and

dodecyltrimethylammonium bromide (DTAB) have been found to denature the BSA even in their monomeric concentration regime.21,25 Over the past few years, a new class of surfactants called as “ionic liquid surfactants (ILSs)” have attracted a great interest from the scientific community around the globe owing to their better surface active properties as compared to conventional ionic surfactants while possessing unique physico-chemical properties.26-32 There are limited studies, where the effect of ILSs on the structure of BSA in aqueous medium has been investigated. In this regard, our previous report established the destabilizing effect of imidazolium based ILSs, 3-methyl-1-octylimidazolum chloride, [C8mim][Cl] and 1-butyl-3methylimidaozlium Octylsulfate, [C4mim][C8OSO3], on the native structure of BSA.33 On the similar lines, Bharmoria et al., reported biamphiphilic ILSs which induce significant folding alterations in structure of BSA, wherein the native BSA has been stabilized by vesicular structures formed by ILSs.34 Geng et al., have reported the interactional behavior of ILS, 1tetradecyl-3-methyimidazolium bromide, [C14mim][Br] with BSA, where stabilization of secondary structure along with destabilization of tertiary structure of BSA have been reported at low concentration of ILS.35,36 The unfolding of secondary structure of BSA by short chain imidazolium and pyrolidinium based ionic liquids (ILs) have also been reported.37-39 Although, some conventional surfactants have been found to induce or inhibit the fibril formation by BSA,40,41 however no work in connection with formation of different morphological selfassembled architectures of BSA in the presence of ILSs has been reported, specially in context to the β-sheet rich architectures such as amyloid fibers.


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In the present study, we report the self-assembly of BSA into a variety of structures such as micro-particles, micro-rods, and long helical fibers etc., mediated by different imidazolium based ILSs shown in Scheme 1. The alterations in native structure of BSA and formation of selfassembled structures have been followed by dynamic light scattering, zeta potential, fluorescence, and circular dichroism spectroscopic measurements. The morphological aspects of the formed self-assembled structures have been explored using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The results obtained from different technique supports each other very well. The functionalization of alkyl chain of ILSs via amide (ILS-2) or ester (ILS-3) moiety have shown contrasting behavior with each other as well as with non-functionalized ILS-1 (Scheme 1). Although there are reports pertaining to affect of ILSs on the structure of BSA,33-39,40 however, there exists no report on the effect of modification in alkyl chain of imidazolium based cationic ILSs via amide or ester group on their interactions with BSA. Further a detailed investigation into the self-assembling behavior of BSA in the presence of ILSs is much lacking. Here, for the very first time, the functionalization of alkyl chain of ILSs has been found to govern the formation of diverse structures at physiological pH and room temperature without the need of harsh conditions such as very low pH, high temperature or long incubation time. Further, looking at the nano-technological and biological importance of β-sheet rich structures such as amyloid fibrils, the work reported here presents the novelty in terms of induction of amyloid fibril formation along with other morphological architectures via self-assembly of globular protein BSA, induced by varying content of structurally different ILSs in aqueous solutions. 2. Experimental: The aqueous solutions of BSA (0.1% w/v) were prepared in phosphate buffer having an ionic strength of 7 mmol L-1 using Millipore water. As the BSA undergoes degradation with time, aqueous solutions of BSA were freshly prepared before every measurement. All the measurements were performed via titration method at 298.15 K, if not mentioned otherwise. An appropriate amount of concentrated solution of respective ILSs was added in adequate amount of solution of BSA, followed by stirring for complete solubilization of ILSs. Dynamic light scattering measurements were performed using a light scattering apparatus (Zeta-sizer, nanoseries, nano-ZS) Malvern Instruments, equipped with a built-in temperature controller having an



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accuracy of ± 0.1 K at an scattering angle of 1730. The samples were thermally equilibrated for 5 minutes before each measurement and an average of 30 measurement runs were considered as data. The data was analyzed using the standard algorithms and is reported with an uncertainty of less than 8%. Zeta potential (ξ-potential) measurements were performed using the same instrument that was used for DLS measurements. For ξ-potential measurements, electrophoretic mobility was measured using a gold electrode. Isothermal titration calorimetry (ITC) was performed using a MicroCal ITC200 microcalorimeter, equipped with an instrument controlled Hamilton syringe with a volume capacity of 40 µL. The titration was performed automatically by adding 2 µL aliquots of prepared concentrated stock solution of ILSs into the sample cell containing 200 µL of aqueous solution of BSA with continuous stirring of 700 rpm. Confocal laser scanning microscopy (CLSM) was performed using a Carl Zeiss LSM 510 confocal microscope. A drop of solution of FITC-labeled BSA having respective ILS at desired concentration was placed on glass bottomed dishes (Matsunami Glass Ind., Ltd.). Green fluorescence images of FITC-labeled BSA in the presence of ILSs were obtained through a BP505-530 band-pass filter using Ar laser at λex = 488 nm. Scanning electron microscopy (SEM) measurements were performed using Zeiss Ultra 55-Limited edition scanning electron microscope. Different solutions of BSA having ILSs were placed on a thoroughly cleaned glass surface and dried for 24 hours in air before imaging. Silver coating was done on samples before measurement. Transmission electron microscopy (TEM) measurements were performed on JEM2100 transmission electron microscope (TEM) at a working voltage of 200 kV without staining the sample. A drop of freshly prepared aqueous solution of BSA having ILSs was placed on a carbon coated copper grid (300 mesh) and the residual solution was blotted off. The samples for TEM measurements were dried in air at room temperature for 24 hours before measurements. Fourier transformed infrared (FTIR) spectra were recorded on NICOLET 6700 FTIR spectrometer. For recording spectra, a cell with BaF2 windows and a Teflon spacer was used: the optical path length was 0.02mm. As sometimes it is difficult to observe the band of interest i.e. Amide I band of BSA centered around 1650 cm-1 in aqueous solutions of BSA, we have tried different resolutions for measurement and the amide I band was observed at a resolution of 2 cm1

. For each spectrum 132 scans were made. Steady-state fluorescence spectra were recorded on a

Perkin Elmer LS55 spectrophotometer using a quartz cuvette of path-length 10 mm at excitation wavelength (λex.) of 280 nm. The spectra were recorded in the wavelength range of 300 to 440


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nm using an excitation and emission slit width of 3.0 nm, each. Far-UV Circular dichroism (CD) spectroscopy measurements were made on JASCO J-815 spectrometer in the wavelength range 200-250 nm in a 1 mm path length quartz cuvette at a response time of 2 s and band width of 0.2 nm. 3. Results and Discussions: 3.1. Complexation of BSA and ILSs from dynamic light scattering measurements: The molecular structure of ILSs under investigation is provided in Scheme 1. The presence of solution structures of BSA and transformations in size of such structures have been monitored by dynamic light scattering (DLS), which provides real time information about the changes occurring in the solution.33,34 Figure 1 shows the variation of count rate of scattered light from the aqueous solutions of BSA as a function of concentration of respective ILSs. All the ILSs have shown almost identical behavior, where variation is observed only in terms of concentrations corresponding to abrupt changes in count rate. In general, the count rate increases marginally up to certain lower concentrations, marked as C1, of respective ILSs followed by a steep increase in count rate in middle concentration regime up to C2. Afterwards the count rate remains constant in a small concentration regime between C2 and C3, followed by an exponential decrease in count rate before reaching a plateau region after C4. The concentrations corresponding to various transitions observed in the presence of different ILSs are provided in Table 1. As the count rate in DLS represents the number of photons detected per second scattered from the sample, therefore a change in count rate can be viewed as a consequence of change in number or size of the species present in solution.42 The variation in C1, which indicates the onset of observable interactions between BSA and ILSs using DLS follows the order: ILS-1 > ILS-2 > ILS-3. This indicates that ester functionalized ILSs interacts with BSA at relatively low concentration as compared to other ILSs in terms of producing visible affects in alterations in solution structures of BSA. Figure 2 (A-C) shows the intensity weighed size distribution profiles corresponding to various transitions as indicated by C1 to C4 for different ILSs. As can be seen from Figure 2A, the hydrodynamic diameter (Dh) of solution structures increases from ≈ 8.0 nm (native BSA) to ≈ 11 nm up to C1 (1.0 mmol L-1) in case of ILS-1. The results are quite similar to that observed in the case of relatively short chain ILS, 3-methyl-1-octylimidazolium chloride, [C8mim][Cl] and biamphiphilic ILSs.33,34 The ILSs can interact with the BSA in monomeric, hemi-micellar or micellar form depending on the concentration.33,34 All the ILSs i.e.



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ILS-1, ILS-2 and ILS-3 remains in monomeric form in the concentration range at least below C1 as indicated by their higher critical micellization (cmc) values.32,43,44 It is important to mention that the cmc of ILSs decreases in the presence of BSA. However, the average concentrations corresponding to C1 for all the ILSs are much lower (≈3 to 10 times) than respective cmc values in the presence of BSA as can be seen from Table 1, which supports the assumption that at concentrations below C1, ILSs remain in monomeric form. Further, the quantification of ILSs interacting with BSA has been deduced from isothermal titration calorimetry (ITC) measurements at varying concentrations of BSA using the following equation.34 = +

(1)

where is the concentration of ILS at C1, is the concentration of free ILS, [P] is the concentration of BSA and N is the number of molecules of ILS bound to BSA at C1. Figure S2A and S2B (supporting information) shows the representative enthalpograms in case of ILS-1 and variation of as a function of [P] for investigated ILSs, respectively. From Figure S2B (supporting information), it has been found that N for different ILSs is 57 (ILS-1), 76 (ILS2) and 86 (ILS-3), which indicates the comparatively greater binding capacity of ILS-3 with BSA. Considering the relatively smaller size imidazolium head group of ILSs as compared to that of BSA along with ability of ILSs to unfold the BSA (discussed later) which leads to increase in number of available binding sites, it is natural to assume that the given number (N) of ILSs binds with BSA in monomeric form. Therefore the formed complexes between BSA and ILSs up to C1 is termed as BSA-ILS (monomer) complexes, where ILSs interacts with BSA in monomeric form. Henceforth, BSA-ILS (monomer) complexes will be simply abbreviated as MCs. The smaller increase in Dh from ≈ 8.0 nm to ≈ 11.0 nm in case of BSA-ILS-1 system is assigned to the monomeric interaction of ILS with BSA via electrostatic and hydrophobic interactions leading to partial unfolding of its secondary structure forming ILS-1-MCs. Such marginal increase in Dh due to unfolding in monomeric concentration regime has also been reported for other imidazolium based ILSs bearing simple alkyl chain as well as for conventional surfactants.18,33,34 However, in case of BSA-ILS-2 and BSA-ILS-3 systems, a substantial increase in Dh from ≈8 nm to 126 nm, and to ≈207 nm, respectively via intermediate structures having Dh ≈40 nm up to C1 is observed. This indicates the greater extent of unfolding of BSA caused by interactions with ILS-2 and ILS-3 as compared to that by ILS-1 as also supported by circular


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dichroism (CD) spectroscopic results (discussed later). With further increase in concentration from C1 to C2, MCs increases in size for all the ILSs under investigation. The increase in Dh is from ≈11 nm to ≈ 207 nm in case of ILS-1 (Figure 2A), from ≈126 nm to ≈ 850 nm in case of ILS-2 (Figure 2B) and ≈ 207 to 1250 nm in case of ILS-3 (Figure 2C). It is interesting to mention that the increases in Dh of BSA in case of ILS-1 is much higher (≈20 fold) as compared to that produced by relatively small chain [C8mim][Cl]33, suggesting the possible role of enhanced hydrophobic interactions. The large increase in Dh between C1 and C2 can be accounted by the ILS mediated self-assembly of BSA-ILS-MC to form BSA-ILS (aggregate) complexes. Henceforth, the BSA-ILS (aggregate) complexes will be abbreviated as ACs. The unfolding of BSA in MCs drives the self-assembly of BSA stabilized by weaker H-bonding interactions between polypeptide chains, where electrostatic repulsions are counterbalanced by ILSs mediated electrostatic interactions and hydrophobic interactions. As the change in Dh is different in case of different ILSs, it seems that along with electrostatic and hydrophobic interactions, the presence of H-bonding prone amide (ILS-2) and ester (ILS-3) moiety are also playing an important role in the formation and stability of ACs. Between C2 and C3, the count rate and Dh remains almost constant for all the investigated systems indicating the dimensional stability of formed ACs. Here, the added ILSs gets adsorbed on the ACs via interacting hydrophobically with already adsorbed ILSs on to the surface of ACs, triggering the formation of hemi-micelle type of structures onto the surface of ACs. After C3, a decrease in count rate and Dh is due to breakdown of some of the ACs by the forming hemi-micelles until the appearance of free micelles of ILSs at C4. Such type of behavior has been reported in case of other imidazolium based ILSs.33,34 The increase in hydrophobic interactions between alkyl chains of ILSs forming hemi-micelles lead to decrease in interactions between BSA and ILSs stabilizing the ACs between C2 and C3. This destabilizes the ACs leading to structural changes. Further, the nature of correlation coefficient (Figure S1, supporting information) between C3 and C4 as compared to that before C2 or after C4, specifically in case of ILS-3 indicates the formation of large sized selfassembled architectures. The relative decrease in count rate between C3 and C4 follows the order: ILS-1 < ILS-2 < ILS-3. This suggests the maximum change in size or shape of the ACs in case of ILS-3 between C3 and C4 as compared to other systems. From the DLS measurements, it has been established that the ILS-1 is least affective in bringing the formation and change in solution structures of BSA as compared to ILS-2 and ILS-3.



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3.2. Interactional forces operating in BSA-ILSs systems from zeta-potential measurements: At the investigated pH, BSA bears an overall negative charge as confiremd from zeta potential (ξ-potential) measurements (Figure 3). Owing to negative charge on BSA, positively charged monomers of ILSs interact with BSA via electrostatic interactions along with hydrophobic and hydrogen bonding (H-bonding) interactions. The magnitude of negative ξ-potential at similar concentrations below C1 for all the ILSs follows the order: BSA-ILS-1 > BSA-ILS-2 > BSAILS-3, which suggests that ILS-3 binds more strongly with BSA as compared to other two ILSs corroborating the results obtained from DLS measurements. The presence of H-bonding amide (ILS-2) and ester (ILS-3) functionality lead to formation of H-bonding between ILS-2 and ILS-3 and polypeptide backbone. This results into enhancement of already existing electrostatic forces of interactions between ILSs and BSA. In the concentration regime between C1 and C2, the ξpotential decreases in magnitude. This decrease can be accounted by the cooperative adsorption of added monomers of ILSs onto MCs formed at C1. The increased unfolding of BSA (discussed later) exposes more binding sites of BSA for interaction with ILSs leading to cooperative adsorption. This large decrease in surface charge accompanied by higher degree of unfolding (discussed later) in this concentration regime results in formation of ILS mediated self-assembly of MCs to form ACs as indicated by DLS measurements. As can be seen from Figure 3, the concentrations corresponding to complete neutrality of charge of BSA by monomers of ILSs i.e. zero ξ-potential values matches well with the C2 observed from DLS measurements (Table 1). Between C2 and C3, ξ-potential don’t vary much for ILS-1 and ILS-2, supporting the electrical stability of ACs. However, in case of BSA-ILS-3 system, there is relatively larger increase in ξpotential values towards positive end indicating that even between C2 and C3, the interactions between BSA and monomers of ILS-3 are operative. Further, the ξ-potential increases but slowly between C3 and C4 in case of ILS-1 and ILS-2, however it increases sharply in case of ILS-3 (Figure 3) suggesting the enhanced interactions of BSA with ILS-3. The presence of high positive charge on ILS-3-ACs results into electrostatic repulsions between the self-assembled ILS-3-ACs, which may leads to either partial or full breakdown of ILS-3-ACs in this concentration regime as compared to other systems. This could lead to formation of discrete selfassembled structure of BSA in this concentration regime. The variation of ξ-potential between C3 and C4 follows the order: ILS-1 < ILS-2 < ILS-3, which supports the above aspects. After C4, ξpotential increase in a small concentration range before attaining constancy upon the formation


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of free micelles. It has been observed that how the introduction of functional group in the alkyl chain of ILS leads to dimensional and other peculiar changes in the surface charge of BSA in different concentration regimes. 3.3. Morphological investigations on complexes of BSA and ILSs: The size and shape of selfassembled structures of BSA in aqueous systems comprising BSA and ILSs, have been explored employing confocal laser scanning microscopy (CLSM) for real time observations, results from which have been further corroborated by scanning electron microscopy (SEM) measurements. Figure 4 (A-F) shows the CLSM (A-C) and SEM (D-F) images for the complexes formed between BSA and ILS-3, in the concentration range C1 and C2, C3 and C4 and beyond C4, respectively. Similar images for ILS-1 and ILS-2 are shown in Figure S3 and S4, (supporting information), respectively. It is important to mention that due to technique limitations, it was impossible to observe BSA-ILS complexes below C1 using CLSM measurements. The images for all the systems in the concentration range C2, and C3 does not show much change as compared to that observed between C1 and C2, and hence are not provided. In case of BSA-ILS1, elongated structures with dimensions of ≈ 250 nm in width and ≈ 250 nm to ≈1 µm in length have been observed between C1 and C2 (Figure S3A and D). The observed sizes are in good agreement with Dh obtained from DLS measurements with some discrepancy, which is due to the fact that DLS provides the Dh considering the morphology of the dispersed particles as spherical one. SEM images confirmed that these elongated structures are formed by assembly of few spherical structures having diameter of ≈ 250 nm. Between C1 and C2, the ILSs monomer mediated self-assembly of relatively smaller ILS-1-MCs leads to formation of larger ILS-1-ACs. A careful look at corresponding CLSM image also establishes that the elongated structures observed from SEM imaging are also present in real time situation and is not an artifact of sample drying before SEM measurements. With increase in concentration of ILS-1, well dispersed near spherical (≈ 250 nm) particles with few elongated particles emerges out between C3 and C4, which is in good agreement with the results obtained from DLS measurements (Figure S3B and E). It is natural to assume that these near spherical structures are formed by breakdown of elongated structures formed between C1 and C2. The onset of formation of hemi-micelle like aggregates decorating the ILS-1-ACs destabilizes the ACs leading to its breakdown. With further increase in content of ILS-1 beyond C4, elongated ILS-1-ACs reappear again with almost similar morphology as observed in concentration regime between C1 and C2 (Figure S3C and F). These



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ILS-1-ACs, however, seems to possess higher degree of aggregation where BSA could wrap around forming micelles of ILS-1. In case of BSA-ILS-2, between C1 and C2, near spherical structures with dimensions of ≈ 700 nm to ≈ 2 µm in line with the DLS measurements have been observed from SEM measurements, (Figure S4A and D). The relatively more unfolding of BSA (Section 3.4.2) by monomers of ILS2 as compared to ILS-1 due to its stronger interactions with BSA followed by ILS mediated selfassembly of ILS-2 MCs complex leads to such large sized aggregates. These structures transforms to elongated rods with dimensions of width ≈ 800 nm and length ≈ 2 µm between C3 and C4 (Figure S4B and E). There are structural differences between the elongated structures observed between C1 and C2 for ILS-1 (Figure S3D) and between C3 and C4 for ILS-2 (Figure S4E). The elongated structures observed in the case of ILS-1 (Figure S3D) seems to be originated by loose self-assembly of formed ILS-1-MCs at lower concentrations, whereas the formed rod like structures in case of ILS-2 (Figure S4E) seems to be more compact. The relative amount of structural contents of BSA i.e. α-helical and β-sheet content along with the extent of interactions between BSA and ILSs are expected to govern the nature of formed architectures. The balancing set of interactions such as electrostatic, hydrophobic and H-bonding interactions leads to formation of compact ILS-2 mediated self-assembly as compared to that of ILS-1, where there is almost complete absence of H-bonding. With further increase in concentration, relatively smaller ILS-2-ACs with dimensions of ≈ 100 nm to in size appears due to breakdown of elongated structures via forming micelles of ILS-2, as can be seen from Figure S4C and F. The breakdown of ILS-2-ACs after C4 in contrast to that observed in case of ILS-1-ACs seems to be governed by enhanced interactions of ILS-2 with BSA owing to presence of H-bonding prone amide moiety. In case of ILS-3, unique behavior has been observed in terms of dimensions as well as morphology of the formed BSA-ILS-3 complexes in different concentration regimes. Smaller spherical structures (≈ 100 nm) along with a few relatively larger structures (≈ 500-1000 nm) have been observed between C1 and C2 as can be seen from Figure 4 (A and D). A careful examination of SEM images shows that the large sized near spherical particles are formed by self-assembly of smaller particles which are assumed to be ILS-3-MCs as shown in inset of Figure 4D. The driving force for the formation of ILS-3-ACs complexes is the ILS mediated self-assembly of smaller ILS-3-MCs complexes, where BSA adopts highly extended from due to


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greater extent of unfolding. Very interestingly, at concentrations between C3 and C4, ILS-3-ACs as long helical fibers (≈ 1 µm in length and ≈ 40 µm in length) have been observed both from CLSM and SEM images (Figure 4B and E). A magnified CLSM image corresponding to Figure 4B is shown in Figure 4G, which supports the right handedly twisted helical nature of elongated fibers in real time measurements. After C4, the formed helical structures disappear at the cost of somewhat elongated ILS-3-ACswith dimensions of ≈ 700 nm - 1.5 µm in width and ≈ 1 µm in length. To confirm the contrasting observations made from CLSM and SEM measurements, transmission electron microscopy (TEM) imaging has been performed on aqueous systems comprising BSA and ILS-3 at different concentrations. Figure 5 (A-F) shows the various structures observed from TEM measurements. Figure 5A shows the TEM images corresponding to SEM and CLSM images shown in Figure 4A and D at concentrations between C1 and C2. TEM image clearly indicates that the large ILS-3-ACs is comprised of small and spherical selfassembled ILS-3-MCs of size ≈50-70 nm. Figure 5B-E shows the TEM images of various structures observed between concentration of interest i.e. C3 and C4, which also helped in putting forward a mechanism for the formation of helical fibers in this concentration range. To provide a clear understanding of the process, a schematic is shown in Scheme 2. Generally, near the isoelectric point, a protein self-assembles into amorphous unordered aggregates. However, away from isoelectric point, where the protein is charged and partially unfolded, it self-assembles into ordered aggregates such as amyloid fibers. In the present study, the pH of the solutions is above the isoelectric point of BSA, where BSA is negatively charged. At lower content of ILS-3, BSA gets unfolded, where it adopt a large size of ≈ 100 nm in the expanded form as ILS-3-MC. The ILS-3 decorated ILS-3-MCs complex self-assembles to form spherical ILS-3-ACs (Figure 5A). As can be seen from Figure 5B, between C3 and C4 thin fibers (≈100 nm in width and 2.5 µm in length) further self-assemble to form a sheet like structure, which twists around its short axis providing the helical nature to the growing fibers as shown in Scheme 2. Near to completion of folding of the thin sheet into helical fiber, the thin fibers points towards the growing axis of helical fiber as can be seen from Figure 5D, which finally twists to give rise to complete fiber (Figure 5E). The width of thin fibers (≈ 100 nm) suggests that the BSA molecules in its expanded from arranges themselves to form long thin fiber (≈100 nm in width and 2.5 µm in length), which self-assembles hierarchically to form long helical fibers via sheet like



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intermediates (Scheme 2, Figure 5B-E). The process described here can be termed as fibrillation of fibrils which have not been observed before at least in case of BSA. During this self-assembly, it is be possible that some of the ILS-3 remains intact with the self-assembling ILS-3-MCs complex, where the surface of forming ILS-3-ACs complex is decorated with hemi-micelles of ILSs. It is expected that β-sheet runs perpendicular to the long axis of the thin fiber as shown in Scheme 2 similar to that exhibited by amyloid fibers. Further, from SEM images, it is clear that the helical fibers possess a periodicity of around 100 nm (Figure 4E), which matches well with the width of the parent fiber giving rise to hierarchical self-assembled fibrous structure. The right-handed helical fibers observed here are much larger (≈ 1 µm in length and ≈ 40 µm in length) in dimension than those reported earlier (≈ 10 nm to ≈ 30 nm in width and 500 nm to 16 µm in length).3,4,7 The nature of the formed fibers as amyloid fibers has been confirmed by FTIR investigations, which is an invasive tool to investigate the conformational changes in protein structure owing to the presence of amide I band appearing at 1600-1700 cm-1. Different conformational structures of proteins absorbs at different wavenumber such as 1610-1630 (intermolecular β-sheet structure), 1630-1643 cm-1 (native β-sheet), 1638-1648 cm-1 (random coil), 1650-1660 cm-1 (α-helix), 1660-1680 cm-1 (turn), and 1680-1692 cm-1 (intermolecular βsheet structure).39,45,46 Out of all these conformational variants, the presence of intermolecular βsheet structure also termed as anti-parallel β-sheet structure is a unique feature of amyloid structures. Figure 6 shows the second derivative FTIR spectral change of BSA in the presence of different amounts of ILS-3, where contrastingly different self-assembled long and helically twisted fibers have been observed. Pure BSA exhibits absorption ≈ 1648 cm-1 characteristic of αhelical structure along with absorption at ≈1640, ≈1655 and ≈1663 cm-1 corresponding to βsheet, coil and turn, respectively. With the addition of ILS-3 up to C2, the absorption of bands corresponding to α-helix, coil and turn decreases at the cost of increasing β-sheet structure. As expected, band corresponding to anti-parallel β-sheet structure ≈ 1618 and 1680 cm-1 appears at C2 which exists even up to C4 establishing the formed helical fibers as amyloid fibers. Interestingly, in case of ILS-1 and ILS-2, no anti-parallel β-sheet structure has been observed (Figure S5A,B, supporting information). The driving force for the transformation of near spherical self-assembled ILS-3ACs formed between C1 and C2 (Figure 4B and E, Figure 5A) into thin elongated fibers, which further self-assemble hierarchically to form long helical fibers (Figure 4E and 5E) seems important to


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discuss. Based on the DLS and ξ-potential measurements, it is natural to assume that the ILS-3AC formed between C1 and C2 does not change much in size and morphology between C2 and C3, however, as observed from CD measurements (Section 3.4.2), there is refolding of BSA between C2 and C4. The refolding of BSA results from decreased interactions between BSA and adsorbed ILSs-3 at the cost of enhanced interactions between adsorbed ILSs-3 and the coming ILSs-3 forming a hemi-micelle type of structure appended with BSA. Between C3 and C4, the forming hemi-micelles on to surface of BSA increase the overall positive charge on the complex. Here, the dominance of electrostatic repulsions (large positive ξ-potential) between selfassembled ILS-3-MCs forming ILS-3-ACs and hydrophobic interactions between ILSs over weak H-bonding and electrostatic interactions stabilizing the ILS-3-AC complex is expected to result in rearrangement of BSA in ILS-3-ACs to from elongated thin fibers (Figure 5B, Scheme 2). Elongated helical fibers of BSA observed in the concentration range of C3 to C4 in case of ILS-3 have never been reported in the presence of conventional surfactants or ILSs. 3.4. Alterations in secondary structure of BSA: 3.4.1.

Fluorescence measurements: The intrinsic fluorescence offered by BSA due to

the presence of three amino acid residues i.e. Tryptophan (Trp), Tyrosine (Tyr) and Phenylalanine (Phe) is an intensive tool to investigate the conformation, dynamics and interactional behavior of proteins. In most of the cases, Phe is not excited owing to its rather low quantum yield, hence fluorescence from Phe can be ignored. Therefore, the fluorescence of BSA is mainly offered by Trp and Tyr as established using 3D fluorescence spectroscopy.38 By using an excitation wavelength (λex) of 280 nm, these amino acid residues absorbs in ultraviolet (UV) region due to aromatic n-π* transitions and fluorescence at emission wavelength (λem) around 340 nm is observed. BSA has two Trp residues located in sub-domain IC of domain I (Trp134) and in sub-domain IIA of domain II (Trp 214) of BSA, respectively, whereas 19 Tyr residues are located in different structural domains of BSA.11 However, Trp contributes the maximum towards fluorescence of BSA, therefore any change in fluorescence of BSA at λex = 280 nm in the presence of different ILSs can be viewed in relation to the change mainly in microenvironment of Trp residues, fashioned by interactions of BSA with ILSs. Figure 7A shows the representative fluorescence spectra of BSA in the presence of ILS-3 at different concentrations, whereas, Figure 7B and 7C shows the variation of emission intensity (Iem) and emission wavelength (λem) as a function of concentration of different ILSs, respectively,



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at λex = 280 nm. For all the investigated ILSs, with an increase in concentration of ILSs, quenching of fluorescence accompanying blue shift has been observed. In the profiles of Iem and λem (Figure 7C), different transitions in various concentration regimes has been observed (Table 1), which matches well with that obtained from DLS and ξ-potential measurements. With the addition of ILSs, the Iem decreases linearly up to C1 in all the cases with almost equal slope. With further addition of ILSs, the Iem decreases very sharply after C1 till C2 following an equal slope for all the investigated ILSs. The transition corresponding to C2 is not observed in case of ILS-1. The decrease in Iem between C2 and C3 follows a relatively lower slope in case of ILS-2 and ILS3 as compared to that observed between C1 and C2 (Figure 7B) in accordance with stability of ACs between C2 and C3, as indicated by DLS and ξ-potential measurements. We could observe a break corresponding to C4 in profiles of Iem, after which it decreases exponentially in all the investigated systems. As can be seen from Figure 7C, a blue shift in λem till C1 is observed for all the ILSs which follows the order: ILS-1 (7 nm) < ILS-2 (9 nm) < ILS-3 (10 nm). Between C1 and C2, relatively smaller blue shift (2 nm) has been observed for all the investigated systems as compared to that up to C1, which remains almost same between C2 and C3 in case of ILS-2 and ILS-3. After C4, no blue shift in λem has been observed. The observations mentioned above can be explained on the basis of interactional behavior of ILSs with BSA, nature of ILSs and changes that ILSs bring in structure of BSA as discussed below. In general, below C1, a decrease in Iem accompanied by blue shift has been observed. The exposure of the fluorophore to the aqueous media may lead to enhanced solvent relaxation around the fluorophore resulting in fluorescence quenching. On the other hand, a blue shift indicates that Trp is experiencing more hydrophobic environment in the presence of ILSs as compared to that in aqueous media. Up to C1, fluorescence quenching follows a similar slope in all the investigated systems, whereas at similar concentrations below C1, a blue shift in λem follows the order: ILS-1 < ILS-2 < ILS-3. In this concentration regime, there is partial unfolding of BSA following the order: ILS-1 < ILS-2 < ILS-3 as observed from CD measurements (Section 3.4.2). This unfolding of BSA accompanied by formation of ILS-MCs exposes the Trp214 towards relatively polar medium along with change in its microenvironment in terms of change in solvation and crowding by ILSs. A similar change in microenvironment of Trp134 is also expected. The fluorescence quenching following a similar slope in all the investigated ILSs suggests that the nature of formed complex between ILSs and amino acid residues neighboring


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fluorophores is almost identical in nature. The blue shift can be accounted due to following reasons: (a) enhanced hydrophobicity of the fluorophore, which can either be due to internalization of fluorophore due to folding of protein; (b) displacement of more polar water molecules by relatively less polar ILSs surrounding the fluorophore; (c) due to hydrophobic interactions between the long alkyl chains of ILSs with fluorophore in case of unfolded protein. In this concentration regime up to C1, the BSA gets unfolded as indicated by CD measurements, which rules out the internalization of fluorophore. The full displacement of water molecules solvating the fluorophores is ruled out considering the hydrophilic nature of imidazolium head group, however, the partial removal or structural rearrangement of H2O molecules around the hydrophilic imidazolium head group can provide relatively non-polar environment to fluorescent residues. Further, the possibility of change in microenvironment of the fluorophores via interaction with hydrophobic alkyl chains of ILSs in vicinity of fluorophores creating a hydrophobic environment also cannot be ruled out. The other possible reasons for the observed variations in fluorescence of BSA are the formation of variety of solution structures of BSA in presence of different ILSs. A sharp decrease in Iem along with a blue shift in λem between C1 and C2 can be assigned to the self-assembly of ILS-MCs complexes to form ILS-ACs. The fluorescent residues at surface (Trp134) or in hydrophobic pocket of BSA (Trp214) got buried towards

more

hydrophobic

environment

inside

the

complexes

with

alterations

in

microenvironment of fluorophores. For ILS-2 and ILS-3, between C2 and C3, relatively lesser change in Iem along with almost constant value of λem indicates the marginal change in physiological environment of formed complex between fluorophores of BSA and ILSs on molecular level. This information corroborates well with negligible change in Dh and ξ-potential values between C2 and C3. While going from C3 to C4, a decrease in Iem and a blue shift in λem has been observed which follows the order: ILS-1 (1 nm) < ILS-2 ≈ ILS-3 (3 nm). After C4, Iem decreases exponentially, whereas λem attains a constant value. These observations support the assumption that there is breakdown of ILS-ACs in case of ILS-3 induced by the forming micelles where the immediate surroundings of the fluorophores don’t change much. The marked affect of structural variation of ILSs on the produced blue shift can be accounted by the varying extent of hydrophobic interactions between alkyl chains and hydrophobic residues of BSA near the fluorophores. The presence of amide (ILS-2) or ester (ILS-3) moiety provides the sites for H-bonding between ILSs and polypeptide chain



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neighboring the fluorophores. This strengthens the hydrophobic interactions between alkyl chains and hydrophobic amino acid residues on polypeptide chain along with electrostatic interactions. Although the ester moiety is relatively poor H-bond acceptor as compared to amide group, however, the conformational flexibility of ester moiety (ILS-3) is assumed to provide enhanced set of interactions between BSA and ILS-3, where ILS-3 can easily interacts via electrostatic, hydrophobic and H-bonding interactions even with closely spaced, otherwise unapproachable interactional sites on BSA leading to a larger blue shift in case of ILS-3. In case of ILS-1, the observed behavior is quite different as compared to short chain analogous [C8mim][Cl], where comparatively a lesser blue shift has been observed up to similar concentration, amounting to the affect of lesser hydrophobicity of smaller alkyl chain.33 Further, the ILSs investigated here produced contrasting results in terms of fluorescence quenching in lower concentration regime below C1 as compared to earlier reported biamphiphilic ILs, where an increase in Iem has been observed following quenching.34 The presence of enhanced electrostatic and hydrophobic interactions owing to presence of amphiphilic [C12OSO3] anion and [C8mim] cation, is supposed to stabilize the BSA in a different manner as compared to that observed in present study.34 Although it is not possible to provide exact information about the binding sites of BSA, however, the relative positions of various fluorescent residues along with their neighboring environment can provide useful information in this regard. Various binding sites has been proposed for interactions of fatty acids with structurally similar HSA.47,48 In some studies, the total number of cationic residues have been considered as possible binding sites for anionic surfactant.18 As mentioned earlier, the two Trp residues fluorescent at λexc = 280 nm have different location in BSA. Trp134 is located at the protein surface, whereas Trp214 is present in hydrophobic binding pocket of protein.11 Trp134 is surrounded by a number of negatively charged hydrophilic aspartic (Asp) and gutamic acid (Glu) residues, whereas Trp214 is surrounded by hydrophobic and positively charged Lys and Val residues.11 Therefore, positively charged monomers of ILSs can interact with Trp134 via electrostatic interactions while retaining some of the solvating water molecules due to their hydrophilic nature. On other side, Trp214 can be surrounded by ILSs, interacting mainly via hydrophobic interactions with no or little solvent associated with it. From the observations, it is inferred the ILS-1 interacts mainly via electrostatic and hydrophobic interactions with Trp134 and Trp214, whereas the additional ability of H-bonding by ILS-2 and ILS-3 leads to their greater interactions with the amino acid


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residues neighboring the fluorescent residues. It is proposed that with increase in concentration of ILSs up to C1, the micropolairty around the fluorescent residues goes on changing whereas, it don’t exert any affect on the nature formed ILS-MCs, thereby exerting no effect in Iem while modifying the λem. 3.4.2. Folding and defolding of BSA: As fluorescence spectroscopy cannot be solely used to predict the folding or defolding transitions in BSA, therefore the changes in secondary structure of BSA as a consequence of interactions with different ILSs have been probed by using the farUV circular dichroism (CD) spectroscopy. BSA is a globular protein possessing both α-helical and β-sheet content in its native sate. The far-UV CD spectra of BSA exhibit two characteristic negative bands situated at -θ212 and -θ223 representing the α-helical nature of the BSA. The relative content of α-helical and β-sheet structure of BSA as a function of concentration of ILSs can be valuably used to get information about unfolding and refolding of BSA.33,34,37,38 Figure 8(A) shows the representative far-UV CD spectrum of BSA in presence of ILS-3 at various concentrations. The presence of particles affects the photons detected by the detector, therefore the intensity of CD band at -θ223 can have some artifacts at higher concentrations of ILSs above C2. Therefore, the variation in intensity of band at -θ223 can only be used to get qualitative information about the change in secondary structure of BSA. The variation of intensity at -θ223 as a function of concentration of ILSs is provided in Figure S6 (supporting information). As can be seen from Figure S6, intensity at -θ223 decreases linearly up to C1, which further decreases with relatively lower slope up to C4 before attaining a constant value after C4, in case of ILS-1. This indicates the unfolding of BSA at lower concentration of ILS-1 without accompanying any refolding of BSA in the investigated concentration range of ILS-1. On the other hand, the intensity of band at -θ223 first decreases in magnitude up to C2 followed by an increase up to C4, which again decreases after C4 in case of ILS-2 and ILS-3. Initial decrease in intensity indicates the unfolding of BSA up to C2, after which refolding of BSA takes place up to C4. After C4, again unfolding of BSA takes place. To get the precise idea about changes in secondary structure of BSA quantitatively, the relative content of α-helical and β-sheet structures in the presence of different ILSs at varying concentrations have been calculated using K2D3 secondary structural analysis software.49 As the α-helical and β-sheet contents are relative to each other, therefore there content is not expected to change substantially with intensity even at higher concentrations of ILSs. Figure 8B, Figure S7 (supporting information) and Table S1 (supporting information)



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shows the variation of α-helical and β-sheet content in solutions of BSA as a function of concentration of ILSs. The native BSA has been shown to possess around 66% α-helical content, whereas some other reports established the 49% α-helical and 9.8% β-sheet content.50,51 The discrepancy may be assigned to the concentration of BSA used, the ionic strength of the medium or the method used for calculaiton. In the present study, BSA in its native state possesses 56% αhelical and 11% of β-sheet content. In case of ILS-1, the content of α-helices decreases sharply from 56% to 34 % up to C1, which further decreases to 6% up to C4 accompanying an increase in content of β-sheet from 10% to 15% up to C1, which remains almost constant, thereafter. This indicates the unfolding of helical structure of BSA, where conformations other than β-sheet, such as turns and random coils dominate as also indicated by differential FTIR spectra (Figure S5A, supporting information). With further increase in content of ILS-1, content of α-helices decreases dramatically up to C4 with no change in content of β-sheet structures. Such type of unfolding is expected to give rise to undefined self-assembled structures of BSA in different concentration regimes of ILS-1 as observed from spectroscopic techniques. On the other hand, the content of αhelices decreases sharply from 56% to 30% and 56% to 27 % in case of ILS-2 and ILS-3, respectively, while going up to C2 (Figure 8B), whereas β-sheet content rises from 10% to 19% and 10% to 22% (Figure S7) in case of ILS-2 and ILS-3, respectively, which is qualitatively supported by differential FRIT spectra (Figure 6 and Figure S5B, supporting information). This establishes the unfolding of BSA by ILS-2 and ILS-3 up to C2 supporting the hypothesis made during discussion in earlier sections and corroborates well with the fluorescence results. Unfolding of BSA exposes more binding sites of BSA, where ILSs interact with BSA cooperatively up to C1. The unfolding of BSA, where the adsorbed ILS in the form of ILS-MCs counterbalances the electrostatic forces of repulsion between different unfolded BSA molecules leads to self-assembly of ILS-MCs rich in β-sheet structure between C1 and C2. Interestingly, despite possessing the high content of β-sheet structure in its unfolded form, ILS-MCs selfassembles into undefined spherical or near spherical architectures in this concentration regime as observed from CLSM and SEM microscopy. Further, up to C2, although there is an increase in βsheet content, however, no cross-β arrangement specific of amyloid structures has been observed from FTIR measurements as discussed earlier. This indicates that the aggregates formed by BSA-ILSs up to C2 are loosely bound aggregates with no degree of ordering. There can be two reasons behind such phenomenon: (i) The amount of ILSs which is assumed to be lower to fully


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counterbalance the electrostatic forces of repulsion between the self-assembling ILS-MCs; (ii) the lesser hydrophobicity of ILS-MCs than that required for ordered self-assembly. After C2, which is assigned as the saturation concentration of BSA, there occurs refolding up to C4, where the content of α-helix increases (Figure 8B) from 30% to 39% and 27% to 42% and β-helical content decrease (Figure S7) from 19% to 14% and 22% to 18% in case of ILS-2 and ILS-3, respectively, (Figure S7, Table S1). As mentioned earlier, the onset of formation of hemi-micelle like structures of ILSs leads to decrease in extent of interactions between ILSs and BSA between C2 and C3. As a consequence, refolding of ILSs decorated BSA takes place, which is more in the presence of ILS-3 as compared to ILS-2. Such stabilization of BSA in lower concentration range of ILSs have been reported, where positively charged ILSs cross-links between different polypeptide chains via electrostatic interactions with one chain and via hydrophobic interactions with other polypeptide chain.34 Further refolding of BSA between C3 and C4 is due to destabilization of ILS-ACs via forming hemi-micelles, which interacts with BSA hydrophobically. The dissolution of BSA results in increased number of binding sites for ILSs, where adsorption of ILSs result in some structural transition as observed from CLSM and SEM measurements. With further increase in concentration after C4, again defolding of BSA takes place, which is due to interaction of ACs with the forming micelles of ILSs where micelles of ILSs could wrap around the BSA leading to destabilization of its secondary structure. It has been observed that the nature of functionalization of alkyl chain of ILSs has a significant affect in their interactions with BSA leading to formation of a variety of structurally distinct complexes between ILSs and BSA in different concentration regimes. Further, the modulation of the secondary structure of BSA, which fashions the self-assembly of BSA is also found to be very much dependent on the nature of ILSs. ILS-1, ILS-2 and ILS-3 have structural differences in terms of absence of functionality (ILS-1) and presence of amide (ILS-2) and ester (ILS-3) moiety in the alkyl chain. The presence of amide or ester group is expected to govern the interactions of ILSs with BSA via H-bonding along with other set of interactions, which is lacking in case of ILS-1. The amide functionality in ILS-2 is very much prone to H-bonding, which can form H-bond with water solvating BSA and with amino acid residues of peptide backbone via both C=O and N-H group. If we compare the BSA-ILS-2 and -ILS-3 systems, it is expected that the symmetry and rigidity of amide group leads to formation of H-bond by C=O and N-H of amide group of ILS-2 with N-H group of one peptide chain and with C=O of amino



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acid present on neighboring polypeptide chain leading to formation of more compact structures. On the other hand, flexible nature of ester group along with H-bond forming ability of C=O group may make it more available to the same peptide back bone, where the alkyl chain can interact with other neighboring peptide back bone via hydrophobic interactions. This leads to formation of loosely packed and large amyloid structures. It is expected that the results presented here will open up a new avenue of research in the field of ILSs mediated self-assembly of a variety of proteins where diverse self-assembled structures depending on the nature of protein and constituent ions of ILSs will appear. Conclusions The functionalization of alkyl chain of imidazolium based ionic liquid surfactant (ILS) with hydrogen bonding prone moieties i.e. amide (ILS-2) and ester (ILS-3) have produced remarkable effect on the size and shape of self-assembled structures of BSA as compared to nonfunctionalized ILS-1. The ILSs interacts with BSA as monomers in lower concentration range to form

BSA-ILS

(monomer)

complex,

which

self-assembles

hierarchically

to

from

morphologically distinct BSA-ILS (aggregate) complex at higher concentration of ILSs. Unordered near spherical structures are produced by ILS-1 in low concentrations below critical micelle concentration (cmc), which transforms into fused elongated architectures at concentrations near cmc. The presence of ILS-2 led to formation of compact elongated structures rich in β-sheet content without showing any crossed β-structure in a specific concentration range below cmc, whereas exceptionally long and wide helical fibers possessing crossed β-structure have been observed in the presence of ILS-3. The difference in nature of prevailing interactions between BSA and different ILSs in different concentration regimes of ILSs have produced such contrasting effect, which is well supported by variation in size, surface charge and unfolding or refolding on BSA as a function of concentration of ILS-3. The refolding of BSA along with distinct set of interactions with ILS-3 functionalized with flexible H-bonding prone ester moiety as compared to other systems produced longer right handedly twisted amyloid fibers of BSA in specific concentration range of ester functionalized ILS-3. Acknowledgement: The authors are thankful to CSIR, Govt. of India, for financial assistance wide Project Scheme No. 01(2774)/14/EMR-II. The authors are also thankful to Prof. Nobuo Kimizuka, Kyushu University, Japan for assistance in CLSM measurements. Authors are very


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thankful to the reviewers for their valuable suggestions which improved the quality of manuscript. Supporting Information: Table S1 and Figures S1 to S7. This information is available free of charge via the Internet at http://pubs.acs.org.

References: 1. Dobson, C. M. Protein Folding and Unfolding. Nature 2003, 426, 884-890. 2. Knowles, T. P. J.; Buehler, M. J. Nanomechanics of Functional and Pathological Amyloid Materials. Nat. Nanotechnol. 2011, 6, 469-479. 3. Usov, I.; Adamcik, J.; Mezzenga, R. Polymorphism Complexity and Handedness Inversion in Serum Albumin Amyloid Fibrils. ACS Nano 2013, 7, 10465-10474. 4. Lara, C.; Gourdin-Bertin, S.; Adamcik, J.; Bolisetty, S.; Mezzenga, R. Self-Assembly of Ovalbumin into Amyloid and Non-Amyloid Fibrils. Biomacromolecules 2012, 13, 42134221. 5. Koseki, T.; Kitabatake, N.; Doi, E. Irreversible Thermal Denaturation and Formation of Linear Aggregate of Ovalbumin. Food Hydrocolloids 1989, 3, 123-134. 6. Loveday, S. M.; Wang, X. L.; Rao, M. A.; Anema, S. G.; Creamer, L. K.; Singh, H. Tunning the Properties of β-Lactoglobulin Nanofibers with pH, NaCl and CaCl2. Int. Dairy J. 2010, 20, 571-579. 7. Bhattacharya, M.; Jain, N.; Mukhopadhyay, S. Insights into the Mechanism of Aggregation and Fibril Formation from Bovine Serum Albumin. J. Phys. Chem. B 2011, 115, 4195-4205. 8. Pearce, G.; Mackintosh, S. H.; Gerrard, J. A. Formation of Amyloid-Like Fibrils by Ovalbumin and Related Proteins under Conditions Relevant to Food Processing. J. Agric. Food Chem. 2007, 55, 318-322. 9. Kavanagh, G. M.; Clark, A. H.; Ross-Murphy, S. B. Heat-Induced Gelation of Globular Proteins: Part 3. Molecular Studies on Low pH Beta-Lactoglobulin Gels. Int. J. Biol. Macromol. 2000, 28, 41-50. 10. Liu, J.; Tian, J.; Tian, X.; Hu, Z.; Chen, X. Interaction of Isofraxidin with Human Serum Albumin. Bioorg. Med. Chem. 2004, 12, 469-474.



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Scheme 1. Molecular structure of ILSs Scheme 2. A schematic view showing various self-assembled structures of and model structure of BSA. BSA formed by mediation of different ILSs in different concentration regimes. SA refers to self-assembly.



ILS-1 ILS-2 ILS-3

5000

C3

C2

C2

Count rate / kCps

4000

C3

C2 C3

3000

2000

C4 1000

C1

C1

C1

C4

1

C / mmol L

C4

-1

Figure 1: The variation of observed count rate in aqueous solutions of BSA as a function of concentration of ILSs.

220

A

200 -1

7.7 mmol L

-1

7.7 mmol L

180

180 4.1 mmol L

-1

120 100

-1

2.3 mmol L

80

-1

2.0 mmol L

60

Intensity %

-1

4.4 mmol L

120 -1

100

3.2 mmol L

80 -1

2.3 mmol L

60

-1

-1

1.2 mmol L

120

-1

0.6 mmol L 80

-1

0.4 mmol L -1

1.0 mmol L

40

1.0 mmol L

40

2.0 mmol L

160

-1

2.7 mmol L

-1

6.2 mmol L

5.0 mmol L

140

140

C

200

-1

160

-1

160

B

Intensity %

200

Intensity %

-1

0.2 mmol L

40

20

20

-1

-1

0 mmol L

0 10

100

0 mmol L

0

1000

10

Dh / nm

100

1

1000

Dh / nm

-1

0 mmol L

0 10

100

1000

Dh / nm

Figure 2 (A-C): The size distribution profiles of BSA-ILS systems at different selected concentrations of ILSs for (A) ILS1; (B) ILS-2 and (C) ILS-3 measured from dynamic light scattering at 298.15 K. 20

ILS-1 ILS-2 ILS-3

15

C4

10 5

ζ / mV

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 32

C2

C3 C2

C4

C3

0

C2 C3

-5

C4

-10 -15 -20 0

2

4

-1

C / mmol L

6

8

Figure 3: The variation of zeta-potential in different BSA-ILS systems as a function of concentration of ILSs at 298.15 K.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

20 µm

2 µm

C

B

GG

10 µm

E

D

F

Figure 4 (A-G): CLSM images (A-C) and SEM images (D-F) of complexes formed via interactions of BSA and ILS-3 at various concentrations between C1 and C2 (A and D), C3 and C4 (B and E) and beyond C4 (C and F) at 298.15 K. Figure 4G shows the magnified phase contrast CLSM image corresponding to Figure 4B. Inset of Figure 4D and 4E shows the magnified SEM images of spherical and right handedly twisted helical fibers, respectively.

A

B

C

A

B

C

D

E

F

Figure 5 (A-F): TEM images of various solution structures in case of BSA-ILS-3 system at a concentration of (A) 1.0 mmol L-1 (C2-C3) showing self-assembled spherical BSA-ILS-3-ACs (B-D) different stages of fiber formation observed at 1.5 mmol L-1 (C3-C4) as (B) self-assembled thin fibers in a sheet-like structure, (C) twisting of sheet like structure around its long axis, (D) thin fibers at the growing axis of helical fiber, (E) formed helical fiber and (F) at 3.0 mmol L-1 (beyond C4) of ILS-3.



Second Derivative of Absorbance

0.003

Intermolecular α-helix β - sheet β - sheet

e 0.002

Intermolecular β - sheet

turn

d 0.001

c 0.000

b -0.001

a -0.002 -0.003 1600

1620

1640

1660

1680

wavenumber / cm

1700

-1

Figure 6: FTIR differential spectra of BSA in the presence of ILS-3 at different concentrations corresponding to (a) C1; (b) C2; (c) C3; (d) C4 and (e) after C4.

0.8

ILS-1 ILS-2 ILS-3

600

C1

500

344

ILS-1 ILS-2 ILS-3

340

400 300 200

0.6

C1

C1

C1

C2

C3

100 0 300

C4

B

A 320

340

360

380

400

420

440

C4

C2

C1

336

C3

C2

0.4

λmax / nm

Intensity / a.u.

Intensity / a.u.

C2

C3

C1 C2

332

C3

C4

C

0.2 0.1

1

0.1

-1

C / mmol L

C3 C4 C4

C4

328

wavelength / nm

1

-1

C / mmol L

Figure 7 (A-C): (A) A representative emission spectra of BSA (λex = 280 nm) in aqueous solutions of ILS-3 at varying concentrations; (B) variation of emission intensity and (C) variation of emission maxima of BSA at λex = 280 nm for different ILSs as a function of concentration of ILSs at 298.15 K.

10

60

A

-2

C/mmol L

-5

-1

0 0.25 0.51 0.75 1.00 1.35 1.70 2.00 2.50 3.00 3.75 6.00

6

-10 -15 -20

210

220

230

240

250

α-helical content / %

-1

50

0

200

ILS-1 ILS-2 ILS-3

B

5

[θ] /10 xdeg.cm .dmol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 32

40

C4

C1 C3

30

C2

C3

C2

20

C2

10

0

1

C4 2

3

4

5

C / mmol L

λ / nm

-1

6

7

8

9

Figure 8 (A-B): (A) A representative far-UV CD spectrum of BSA at different concentrations of ILS-3; and (B) the variation of α-helical content of BSA as function of concentration of various ILSs.


Page 31 of 32

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Table 1. The concentrations corresponding to various structural transitions of BSA fashioned by interactions with ILSs as observed from dynamic light scattering (DLS), fluorescence spectroscopy (emission intensity-Iem and emission maxima-λem), and far-UV circular dichroism (CD) spectroscopy at 298.15 K.

C / mmol L-1 C3 [C12mim][Cl]

Technique C1

C2

DLS Flr. (Iem) Flr. (λmax) CD

1.01 1.30 1.10 1.03

2.04 − − 1.73

DLS Flr. (Iem) Flr(λmax) CD

0.40 0.38 0.74 −

1.78 0.71 1.07 1.70

C4 (cmc) 2.40 2.36 2.21 −

3.14 3.67 3.67 −

2.50 1.75 2.06 2.53

3.74 2.93 4.05 −

0.98 2.10 1.95 0.99

2.71 3.27 4.30 2.51

[C12Amim][Cl]

[C12Emim][Cl] DLS Flr. (Iem) Flr. (λmax) CD

0.20 0.40 0.50 −

0.60 0.71 1.08 0.74



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Content:


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Ionic Liquid Surfactant Mediated Structural Transitions and Self

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