Inhibiting the Fibrillation of Serum Albumin Proteins in Presence of

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Inhibiting the Fibrillation of Serum Albumin Proteins in Presence of Surface Active Ionic Liquids (SAILs) at Low pH: A Spectroscopic and Microscopic Study Sangita Kundu, Chiranjib Banerjee, and Nilmoni Sarkar J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b03457 • Publication Date (Web): 19 Jul 2017 Downloaded from http://pubs.acs.org on July 20, 2017

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The Journal of Physical Chemistry

Inhibiting the Fibrillation of Serum Albumin Proteins in Presence of Surface Active Ionic Liquids (SAILs) at low pH: A Spectroscopic and Microscopic Study Sangita Kundu, Chiranjib Banerjee and Nilmoni Sarkar* Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, WB, India E-mail: [email protected] Fax: 91-3222-255303 Abstract One of the key necessary steps to prevent human neurological disorders is the efficient disruption of protein aggregation or amyloid fibril. In this article, we have explored the effect of three amphiphilic surface active ionic liquids (SAILs) namely 1-methyl-3-octylimidazolium chloride ([C8mim]Cl), 1-dodecyl-3-methyllimidazolium chloride ([C12mim]Cl) and 1-hexadecyl-3methyllimidazolium chloride ([C16mim]Cl) having concentration of 5.8 mM, 0.29 mM and 0.08 mM respectively on bovine serum albumin (BSA) and human serum albumin (HSA) fibril. These SAILs have different alkyl chain length attached to the cationic imidazolium headgroups. Interestingly, it is observed that all of the three SAILs exhibit fibril inhibition at room temperature itself as initially evidenced from thioflavin T (ThT) fluorescence assay study. However, C16mimCl is found as the most efficient quencher having highest quenching constant than other two analogues. In addition, circular dichroism (CD) data gives valuable insights into the conformational changes of BSA fibril as a consequence of interaction with SAILs. The field emission scanning electron microscopy (FESEM) and fluorescence lifetime imaging microscopy (FLIM) confirm the inhibitory effect of SAILs. It is evident from fluorescence correlation spectroscopy (FCS) study that 62% fibril is ruptured in presence of C8mimCl while C12mimCl and C16mimCl completely destroy the fibrillar morphology. So the inhibition efficiency is related to the hydrophobicity associated with the long alkyl chain attached with the cationic imidazolium headgroup of SAILs. 1. Introduction A range of human neurological disorders, such as Alzheimer’s, Prion, Parkinson’s, Huntington’s, type 2 diabetes diseases are known to be caused by the self aggregation of proteins and peptides due to the protein misfolding.1-5 The self-assembly of proteins under several in vitro biophysical conditions result the formation of amyloid fibrils.6-11 These amyloid fibrils consist of a characteristic cross beta structure in which the β-strand segments are oriented perpendicular to 1 ACS Paragon Plus Environment


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the fibril’s main axis, stabilized by the intermolecular hydrogen bonds running parallel to it.7,12 The fibril formation can be tuned by optimizing the conditions such as pH, temperature, concentration of protein, ionic strength etc.9,13 Under these conditions, the native state of protein is destabilized and partially unfolded conformers are accumulated in significant population which are not accessible in native structure. Further conformational rearrangements of such nonnative intermediates are facilitated by certain noncovalent intermolecular interactions such as Van der Waals, hydrogen bonding, hydrophobic, electrostatic interactions that can favour to subsequent oligomerization and fibril formation.14 BSA, a mammalian globular protein has been extensively studied as a model α- helical protein because of its structural similarity with human serum albumin, a major transport carrier of drugs and metabolites in blood plasma in addition to the preservation of blood pH.15 At physiological pH (7.4), both the serum proteins, BSA and HSA16 consists of 585 amino acids, ensembled in three homologous domains (I, II, and III), which further made up with two subdomains (A and B), adapting a heart-shaped structure stabilized by 17 intradomain disulphide bonds. These serum proteins have tendency to adopt different conformations depending on the pH of the medium. BSA at pH 7.4, have normal helical heart shaped structure (N form), which transforms to an expanded conformation (E form) at pH below 4, where the protein undergoes denaturation and complete unfolding leading to rise in the hydrodynamic axial ratio from about 4 to 9.17,18 Several investigations have been performed related to feasible conditions of fibril formation, mechanism as well as driving forces of formation, and characterization of the fibrillar aggregates.19,20 Most of the studies have been designed at pH 7.4 as it is normal physiological pH and only few reports are there at low pH regime.6,21,22 However, from the physiological point of view, study at low pH is also important because in human body membrane surface area of few tissues are acidic in nature and the carrier proteins can come in contact with this acidic region.9 It has been pointed out that compared to the fibrils the intermediate oligomers are more toxic to the cells. These can cause the death of cells in several ways, such as by forming reactive oxygen species after complexation with metals, inflammatory response due to accumulation of the misfolded protein, membrane disruption.23 However, it is also reported that the excessive accumulation of fibril, may lead to the organ failure24 and several diseases.25 Therefore, different approaches have been made to inhibit or disrupt this protein aggregation in the initial stages itself 2 ACS Paragon Plus Environment

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because the oligomeric protofibrils are the principal toxic species compared to the mature fibrils. High temperature (above 100oC), ultrasonification, pH change, denaturants can efficiently disfavour the β sheet aggregation and destabilize the fibril.26-28 Polyphenols, β-cyclodextrin, molecular tweezers and cucurbit[7]uril (CB[7]) can arrest the amyloid fibril formation.29-32 A supramolecular approach has been reported by Ruhs et al.33 to disintegrate the β-lactoglobulin fibrils by the complex formation with the sulphonic acid polyethylene glycol (surfactant). Sabate et al.34 have shown that the alkylammonium bromide surfactants prompt fibrillation above critical micelle concentration (cmc) presumably by means of electrostatic interactions whereas below cmc, they retard the amyloid fibril formation. Other anionic surface active agents like surfactin,35 SDS36,37 can affect the fibril aggregation in concentration dependent fashion. Wang and his coworkers38 have studied that the concentration of the cationic gemini surfactant C12C6C12Br2 modulates the process of fibrillogenesis. Hamill et al.39 have investigated the effect of trimethlyammonium bromide, an azobenzene containing photosurfactant on the dynamics of amyloid aggregation. This surfactant can control the inhibition and fibrillization process photoreversibly with light illumination. Room temperature ionic liquids (RTILs) with long alkyl chains have generated an interesting area of research due to their surface-active properties, which make them superior to the conventional ionic surfactants.40-42 These are advantageous to use because of their special physical and chemical properties such as nonflammability, good solvating properties, variable polarity range, low volatility and high thermal stability and recyclability. Such ionic liquids are amphiphilic in nature because of the coexistence of hydrophilic head groups and long hydrophobic tails. The self aggregation properties of the SAILs in water to form various assemblies such as micelles, vesicles, reverse micelles, microemulsions depend on their structures (specially on the long alkyl chain). There are very limited studies on the protein- RTIL interaction have reported so far.43-50 Kim and his coworkers51,52 have studied the effect of ionic liquids to promote amyloid fibrillization. On the other hand, ionic liquids can also suppress the protein aggregation preserving the conformational stability of the native state depending on the experimental conditions. It is possible to dissolve the amyloid fibril of hen egg white lysozyme in protic ionic liquids (pILs) such as triethylammoniummesylate and ethylammonium nitrate with restoration of enzymatic activity where ionic liquids have been employed as solvents only.53 Heydari

and

his

coworkers54

have demonstrated

that

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very low concentration

of


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tetramethylguanidinium based pILs can prevent the fibril formation whereas carboxyl functional group plays an important role in the inhibition. They have proposed that ionic liquid traps the intermediate oligomers and helps to stabilize the protein in its native state. Recently, Debeljuh et al. have shown that amyloid fibrillation rate can be promoted within few seconds using pILs containing traditional kosmotropic anions i.e. phosphate or sulphate, whilst, fibril formation can be suppressed completely using pILs containing the mesylate anion.55 In this regard, mature fibril breakdown with SAILs has not been explored yet which may be an interesting and necessary area of research concerning biological and medical importance. In the present work, we demonstrate the self aggregation of BSA into mature fibrils followed by the disruption of fibrils in presence of a low concentration of SAILs. For this purpose, three different SAILs having different chain length of the alkyl group attached to their cationic part, are used. Furthermore, structural alterations of these fibrils in presence of SAILs have been investigated using FESEM and FLIM techniques. The morphological changes are further corroborated by fluorescence, CD spectroscopic studies and FCS measurements. Experimental results show that all the three SAILs used in this study are able to disrupt the fibrillar structure of BSA at room temperature. However, the extent of disruption is found to depend on the alkyl chain length of the respective SAILs. The effect is higher with the SAIL containing a long alkyl chain; C16mimCl and it is lower for SAIL, with shorter alkyl chain; C8mimCl. 2. Experimental section 2.1. Chemicals: Bovine serum albumin (BSA) and Thioflavin T (ThT) are purchased from Sigma Chemical Co. (St.Louis, USA) and are used as received. Human Serum Albumin (HSA) is purchased from TCI Chemicals

(Japan).

4-(Dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran

(DCM) dye is purchased from Exciton. The SAILs, 1-methyl-3-octyl imidazolium chloride (C8mimCl), 1-dodecyl-3-methyl imidazolium chloride (C12mimCl) and 1-hexadecyl-3-methyl imidazolium chloride (C16mimCl), are obtained from Kanto Chemicals (98% purity) and dried in vacuum for 24 h at 70−80 °C before use. The structures of all the chemicals are shown in Scheme 1. Scheme 1. Chemical structures of DCM, Thioflavin T (ThT) and SAILs


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2.2. Preparation of fibrillar sample and Its Inhibition: The stock solution of BSA is prepared by dissolving the protein in Milli-Q water and stored at 40 C.

Accurate

protein

concentration

is

determined

by

measuring

the

absorbance

spectrophotometrically at 280 nm using molar extinction coefficient of 43824 M-1 cm-1.15 Keeping the protein concentration at 150 µM, the protein samples are adjusted to pH 2 with HCl. The samples are then incubated at a temperature of 70 0C in water bath in presence of 20 mM NaCl and 60% (v/v) ethanol for 24 hours without any agitation. The fibril solutions are finally diluted to a concentration of 50 µM for experimental purpose. We have used three different SAILs (C8minCl, C12mimCl and C16mimCl) having different chain lengths to study the effect of SAILs on fibril formation. This study has been performed below the cmc of the respective SAILs. Mixtures are shaken thoroughly and kept at room temperature for 5-6 days before use. 2.3. Instrumentation: We have performed steady state emission spectroscopy, circular dichroism (CD) spectroscopy, field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), surface tension measurement. The detailed instrumentation is discussed in the Supporting Information. The details of the fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS) are given below. 2.3.1. Fluorescence Lifetime Imaging Microscopy (FLIM) and Fluorescence Correlation Spectroscopy (FCS): FLIM and FCS techniques are used to characterize the formation and disruption of BSA fibril. Both the experiments are carried out using a DCS-120 (complete laser scanning confocal FLIM microscope by Becker & Hickl GmbH) system. For both FLIM and FCS techniques, we have



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used DCM as a fluorescent marker. The FCS traces are measured using picosecond diode lasers with excitation source connected to inverted microscopes of Zeiss, equipped with a 40× waterimmersion objective (NA 1.2). Furthermore, 40× objectives have also been used for FLIM measurements. A diode laser of 488 nm, working at pulse mode (5mW), is used for sample excitations, and long pass filters (498 nm) are used to separate the fluorescence signal from the excitation source. The fluorescence signal is collected using two HMP-100-40 GaAsP hybrid detectors, after adjusting the pinhole diameter at 0.5 mm and maintaining the number of molecules between 5 and 10 in the confocal volume, during each measurement.56 For all the FCS experiments, the final concentration of probe molecules (DCM) is 2 nM and for FILM it is 6 µM. In FCS, laser and confocal microscopy are used to produce a very small observation volume (in the order of femtoliters (fL)) inside a sample. The diffusion of fluorescent molecules in and out of that volume leads to fluctuations in the fluorescence intensity which can be time-correlated to get a normalized autocorrelation function G(τ), which is defined as:57 τ =

〈 + τ〉 1 〈 〉

where, ⟨F(t)⟩ is the average fluorescence intensity, and δF(t) and δF(t + τ) are the amounts of fluctuation in intensity around the mean value at time t and t + τ and are given by = − 〈 〉 2 + τ = + τ − 〈 〉 3

The diffusion time (τD) can be obtained by fitting the correlation function, G(τ), for a singlecomponent system, where diffusion occurs in only three dimensions in the solution phase, using the following equation:

1 τ =

1

α

τ + τ 1 + ω τ τ

4

Where, N denotes the number of particles in the observation volume and the depth-to-diameter ratio of 3D Gaussian volume is represented as, ω = ωz/ωxy. The diffusion coefficient of the molecule can be calculated from the diffusion time (τD) and radius of the observation volume (ωxy) using the following equation.


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!

ω "# = 5 4τ$

Using a sample having known diffusion coefficient [Rhodamine 6G (R6G) in water, Dt = 4.14 × 10−6 cm2 s−1], the structural parameter (ω) of the excitation volume was calibrated employing the given equation.58 (

1 4 !τ τ = &1 + ' ω"#

4

(

!τ &1 + ' 6 ω ω"#

In the fitting analysis, ωxy and ω were kept as free global parameters. Using the obtained ω value, Veff is calculated from equation 7. .

*+,, = - ω/"# ω 7 The observation volume, as estimated from the global analysis of the fluorescence correlation function of R6G of varying concentration, is 1.7 fL, with a transverse radius of 283 nm. 3. Results and discussion In this article, our aim is to elucidate the effect of SAILs on BSA fibril. Hence, initially the experimental evidences for the aggregation and fibrillation propensity of BSA have been provided, followed by inhibition process of fibril. The formation of BSA fibril at pH 2 and in the presence of 20 mM NaCl (details in experimental section) has been investigated by observing the changes in the thioflavin T (ThT) fluorescence intensity. Different research groups have used thioflavin T (ThT) as an amyloid marker in the study of protein aggregation including amyloid-β peptide, lysozyme, insulin, amylin, transthyretin, α- synuclein, β-2 microglobulin etc.59-61 ThT molecules insert itself into the ‘channels’ formed between the side chains of the extended β sheet present in amyloid fibrils with its long axis parallel to that of the fibrils.62,63 Thus ThT acts as an amyloid sensor by showing enhanced fluorescence with a broad emission peak having maximum at 482 nm when bound to amyloid fibrils.9 The fluorescence spectrum of ThT in fibril solution is shown in Figure S1. Furthermore, to monitor secondary structural changes of protein upon formation of the fibrillar network, we have performed CD measurements. At pH 2 and in presence of 20 mM NaCl, the far-UV CD spectrum of BSA shows characteristic minima at 209 and 222 nm of an α-helix rich protein, which may arise because of the π-π*and n-π* transition of the peptide backbone of α-helicity.46 Interestingly, a pronounced peak in the CD spectrum around the wavelength value of 217 nm is observed which may imply the formation of both β7 ACS Paragon Plus Environment


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sheet and unfolded conformation confirming the amyloid nature of fibrils as substantiated by increase in ThT fluorescence (Figure S2).20. The aggregation of the BSA fibril is further ensured by FESEM and FLIM images. The FESEM images of BSA fibril are shown in Figure 1a,b at two different resolutions. The FESEM images show fairly rigid mature long (of the order of micrometers) fibrillar network having characteristic branched long-rod-shaped structure. The fluorescence intensity and lifetime images of these fibrillar aggregates are shown in Figure 2(ad) and Figure S3(a-d).These images collected at different time interval confirm fibril formation. Apart from this, unlike intensity images, lifetime images additionally supply valuable informations like microheterogeneity and the excited state lifetime of the fluorophores. In a recent report, we have successfully employed FLIM technique for monitoring fibril formation from L-Phenylalanine and its inhibition by crown ethers.64 In addition, BSA fibrils are further characterized by AFM images (shown in Figure S4).

Figure 1: FESEM images of BSA fibrils at (a) 400 µm, (b) 200 µm resolution.


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Figure 2: (a),(b) Lifetime images of BSA fibril after 7 days and (c), (d) after 15 days. The effect of SAILs on BSA fibril and the subsequent disruption of fibril leading to the formation of different aggregates have also been established by the above techniques along with FCS measurements. As we have mentioned earlier, the effect of SAILs on fibrillation process has not been studied extensively, and to the best of our knowledge, this is the first report which reveals that SAILs can be used to arrest the fibril formation. Here, three SAILs, namely, C8mimCl, C12mimCl and C16mimCl, have been employed for the entire inhibition processes. All the three SAILs mentioned above contain chloride ion as anion and imadazolium ion as the cationic moiety. However, the chain lengths of the alkyl group attached to the cationic part differ in each case. C16mimCl (long chain) contains a hexadecyl, C12mimCl (intermediate chain) contains a dodecyl and C8mimCl (short chain) contains an octyl chain attached to their respective imidazolium cations (shown in Scheme 1). The emission intensity of ThT decreases with increasing concentration of the SAILs; however, the extent of the decrement is different for each SAIL (shown in Figure 3). The decrease in emission intensity of ThT in presence of SAILs indicating strong interaction between BSA fibril and SAILs and could be the reason for fibril inhibition which is further confirmed by other techniques. The following Stern–Volmer equation has been employed for the quantitative estimation of the quenching in terms of the quenching constant (123 .

4 = 1 + 123 567 8

Where, F0 and F represents fluorescence intensity of ThT in absence and presence of quencher with quencher concentration [Q] and KSV is the Stern–Volmer (S–V) quenching constant which can be obtained from the slope of plot of F0/F versus [Q]. The calculated quenching constants



provide a preliminary idea regarding the efficiency of inhibition of the fibrillar structure. It should be mentioned here that, this study has been performed below the cmc of the respective SAILs as at cmc, the fibrils are precipitated out of the solution. The extent of interaction between the fibrils and the SAILs depend on the alkyl chain of the respective SAIL. It is clear from the quenching constants that the effect has been found to be greater in case of long chain (C16mimCl) SAIL compared to the shorter ones (C12mimCl and C8mimCl). The obtained quenching constants are 26.63 × 103, 6.18 × 103, and 0.23 × 103 M-1 for C16mimCl, C12mimCl, and C8mimCl, respectively, that justifies the above statement. 1.5

(a)

1.6

1.8

1.0 0

0.6

1

2

Concentration (mM)

0.3 0.0

480 540 Wavelength (nm)

600

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F 0/F

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Fl. Intensity (a.u.)

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1.2

2.0 1.5 1.0

0.9

0.000

0.025

0.050

0.075

Concentration (mM)

0.6 0.3 0.0

480

540 Wavelength (nm)

600

Figure 3: Fluorescence spectra of ThT ((λex= 450 nm) in BSA fibril in presence of different SAILs (a) C8mimCl, (b) C12mimCl, (c) C16mimCl, whereas the corresponding Stern-Volmer plots are shown in the inset of the figures. The inhibition process is further corroborated by FESEM and FLIM techniques. It should be mentioned earlier that we have studied imaging and lateral diffusion with the help of another amyloid fibril binder dye DCM65 because ThT is weak fluorescent molecule until it bounds to fibril. In addition, there would be no control as free ThT does not really have any fluorescence, so ThT is not expected to be a good dye for the FCS experiments. From the FESEM images (Figure 4 and S5), there is a clear indication of arresting the fibrillation process in presence of the SAILs. The concentrations of C8mimCl, C12mimCl and C16mimCl are maintained at 5.8 mM, 0.29 mM, and 0.08 mM respectively throughout the experimental work. The corresponding FLIM images are shown in Figure 5 and S6. Figure 4a and 5a clearly shows that the fibril structure is disassembled in presence of 5.8 mM C8mimCl but it is unable to rupture the morphology of fibrils completely. Compared to the neat fibril, in presence of C8mimCl, the fibers are looking thin and seem to begin disrupted, this may be because of its shorter chain length. In presence of C12mimCl and C16mimCl, there is no trace of fibril and some 10 ACS Paragon Plus Environment

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smaller aggregates are formed. In the FESEM image, in presence of 0.29 mM C12mimCl, there exists a new type of aggregated morphology that may be formed after the complete disintegration of fibrillar structure (Figure 4b). In comparison to former, C16mimCl has the better efficiency for inhibition which is further confirmed in the FESEM and FLIM images. However, it should be mentioned that C16mimCl arrest the fibrillation at much lower concentration compared to other two SAILs. Recently, we have shown the formation of different types of aggregates like flakes, flower or long rod shaped structures in presence of crown ethers and after disassembly of L-Phe fibril.64 Lanthanides can also disrupt the fibrillar morphology of Phe fibril and forms flakes and “Palmyra leaf” type aggregates.66 In our present work, SAILs have been employed to inhibit amyloid fibril which subsequently forms new types of smaller aggregates after disintegration of the fibrils.

Figure 4: FESEM images of BSA fibril inhibition with the addition of (a) 5.8 mM C8mimCl (scale bar, 100 µm), (b) 0.29 mM C12mimCl (scale bar, 10 µm) and (c) 0.08 mM C16mimCl (scale bar, 10 µm). Images are taken 5-6 after the addition of SAILs.

Figure 5: Lifetime images of BSA fibril inhibition by the addition of different SAILs, (a) 5.8 mM C8mimCl, (b) 0.29 mM C12mimCl, (c) 0.08 mM C16mimCl. 11 ACS Paragon Plus Environment


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To explain the inhibition process, we have plotted the lifetime distribution histograms of DCM in BSA fibril and in presence of different SAILs in Figure S7 and 6 respectively. The lifetime distribution of DCM is in the range of 2200– 2500 ps with a maximum around 2350 ps inside the neat fibril. With increasing the chain length of SAILs, the lifetime distribution of DCM is decreased which is supported by our previous reports.64,

66

The fluorescence lifetime of dye

obtained from imaging technique depends on the local environment surrounding the probe. The interaction between the fibrils and SAILs may monitor the localized changes of the probe reflecting the changes in fluorescence lifetime. The sensitivity of fluorescence lifetime towards any environmental change is utilized as an intrinsic molecular property in a variety of application to discriminate the fluorescent species on the basis of the lifetime. This is an exclusive and novel technique to analyse the microheterogeneity of different kinds of aggregates. On investigating the distribution plots (Figure 6) in detail, it is comprehended that in presence of SAILs, the lifetime distribution is broadened compared to neat fibril because the disruption of fibrils leading to form various aggregates with structural heterogeneities. Here, we have monitored the structural changes of BSA fibril with the addition of SAILs and it is obvious that the disrupted assemblies are different because the interaction is solely dependent on the alkyl chain length of the SAILs. So, the heterogeneities are there in the aggregated structures which are due to the different sizes of the aggregates. Because of the heterogeneous population inside the aggregates, the dye molecules are in a physically inhomogeneous environment. The heterogeneity parameter gives us the information regarding the local heterogeneity in the sample and is closely related to the width of the lifetime distribution which suggests whether the lifetime distribution will be narrow or broad.67 This helps us to find out the mechanisms or factors responsible to broaden the lifetime distribution. Furthermore, it is very difficult to obtain a particular lifetime value from the FLIM images as the lifetime values vary in different regions of the aggregates.68,69 The broader lifetime distribution also implies that the DCM faces different heterogeneous milieu in different regions of formed aggregates70 which are confirmed by FLIM and FESEM techniques. It is because of the fact that the SAILs are structurally different (different chain lengths for C16mimCl, C12mimCl, and C8mimCl) as well as their extent of interaction with fibrils (different binding constants) resulting the differences in the heterogeneity of their aggregated structure. Our earlier reports64,66 show similar type of broad lifetime distribution of DCM inside the formed aggregates after disruption of L-Phenylalanine fibril. From a close inspection of the plot of 12 ACS Paragon Plus Environment

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lifetime distribution in case of C8mimCl, it is observed that the lifetime of dye is shifted to lower lifetime region having maximum at 2020 ps in lifetime distribution histogram whereas it is 2350 ps in neat fibril. Furthermore 2200-2400 ps lifetime region (Figure 6(a)) may contain a significant contribution of fibrillar morphology. In presence of C8mimCl, though inhibition of fibril is started but complete disruption is not occurred and thus the result implies that there should be some fibrous structure along with the disrupted fibril. In contrary, no trace of fibril is found in presence of C12miml and C16mimCl which is confirmed from FLIM images. Here, the broader lifetime distribution results due to the presence of heterogeneous population of different aggregates in completely disintegrated fibrils. In these cases, absence of any distinct band leads to indistinguishable aggregated structure. On analyzing the distribution plots of the same in presence of the C12mimCl and C16mimCl, we can observe that the maxima are shifted to lower lifetimes around 1920 ps and 1500 ps respectively. So, it is obvious that the structures of SAILs are playing important roles which cause the inhomogeneous interaction with the fibrils. As a result, the fluorescence intensity as well as the lifetime of the dye is decreased in presence of SAILs and rate of quenching increases with the increase in chain length, which further confirms our steady state measurements. From these observations it is confirmed that the molecular state and the environment of the fluorophore is changed from C8mimCl to C16mimCl which is the probable reason of change in fluorescence lifetime of the dye. So, we can conclude that chain length of the SAILs has a profound effect on the lifetime histograms of the dye.

Intensity (a.u.)

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Figure 6: Lifetime distribution histograms of DCM after destruction of BSA fibril with addition of different SAILs; (a) C8mimCl, (b) C12mimCl, (c) C16mimCl. FCS is a useful technique and successfully employed in cell biology to study the molecular diffusion, mobility, molecular interaction, binding processes, transition in conformational dynamics of the biomolecules in single molecular level. This technique is based on the measurement of fluctuations in fluorescence intensity of the fluorophores ranging in the nM concentration inside a small observation volume (in the order of fL). The fluctuations in the intensity may originate as a result of change in fluorescence properties of the molecule due to chemical reaction or conformational fluctuation, or due to change in the number of fluorescent particles in the small volume because of diffusion. The use of FCS technique to monitor the conformational dynamics of proteins covalently labelled with a fluorescent dye, has been shown in the recent literature.71,72 It is reported that the behaviour of labelled and unlabelled proteins are different and only limited concentration of proteins can be studied by FCS. Therefore, in our present study, the probe molecules are not covalently labelled with the protein, as the formation of fibril is a concentration dependent process and requires high protein concentration (µM range) and above 1µM concentration, the FCS signal is very low.73 We have mentioned earlier that DCM is used as a fluorescence marker for the FCS study because of the weak fluorescence of ThT. The fluorescence auto-correlation trace (FCS trace) of DCM in BSA fibril at pH 2 is measured first, followed by the measurement of the FCS traces in presence of each of the three SAILs. The autocorrelation traces are shown in Figure S8 and 7. After analyzing the FCS data, the diffusion times of the probe molecule are determined, which are further employed to calculate the values of diffusion coefficient in each case. The diffusion coefficients of the fluorophore in these systems, calculated from an average of three data sets, are tabulated in Table 1. The results are quite interesting for all the cases. With addition of SAILs to fibrillar solution, the diffusion time of DCM changes in different ways. After fitting the autocorrelation trace with single component diffusion model (equation 4), the value of diffusion coefficient (Dt) of DCM in fibrillar solution is found to be 64.67 µm2s-1, which is slower compared to that in the neat water (300 µm2s-1).74 This supports that the dye molecules bind with the fibril. Abelian et al.75 have also reported similar translational diffusion coefficient of Aβ40. A similar type of environment throughout the fibrillar system might have resulted in the single diffusion coefficient of DCM in


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fibrillar solution. However, in presence of C8mimCl, the autocorrelation curve is fitted by twocomponent diffusion model, as the fits considering single-component diffusion model is not satisfactory (as judged by residuals and R2 values). It is thus evident that there exist two different ensembles which have distinctly different diffusion coefficients. In this case, a new faster diffusion component (123.35 µm2s-1) arises, with a relative contribution of 62%. This new diffusion coefficient could probably be due to the movement of DCM in the comparatively smaller aggregates, generated after the disassembly of fibril. The data of 0.29 mM C12mimCl implies complete disruption as the slower diffusion component becomes zero and faster component is the only contributing one. Diffusion coefficient in case of C12mimCl is found to be 137.23 µm2s-1. In addition, the same value is further enhanced to 155.89 µm2s-1 in presence of C16mimCl. The presence of single component (in between 123 to 155 µm2 s-1) in FCS traces of DCM in longer alkyl chain SAILs containing fibrillar solution confirm the complete inhibition of fibrils that supports our previous results. Therefore, the extent of changes in the lateral diffusion of probe in different aggregates relies on the difference of the hydrophobicity of the SAILs. The data suggests that in presence of SAILs, a new type of smaller aggregates may be formed after disintegration of fibril structure. Close inspection of the diffusion coefficient data (Table 1) one can enable the comparison of the extent of disruption of fibrils using each of the three SAILs. The highest diffusion coefficient is obtained when C16mimCl is used as a SAIL and the value is lowest in presence of C8mimCl. However, intriguingly, by calculating the magnitude of the diffusion coefficient we can evaluate the percentage of the disassembled fibril and this is almost 62% in presence of C8mimCl whereas other two are able to disrupt the fibril completely but discrepancy in diffusion component arises due to the difference in the aggregated structure. In a nutshell, we have successfully monitored the fibril arresting process by SAILs using FCS and the result completely correlates with the FLIM and FESEM findings.



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Table 1. Diffusion parameters of DCM inside different aggregates.

Systems

α1

α2

DCM in BSA fibril

D1

D2

(µm2/s)

(µm2/s)

1 0.38

123.35

R2

64.67

0.95

68.11

0.98

+ C8mimCl

0.62

+C12mimCl

1

137.23

0.97

+C16mimCl

1

155.89

0.97

Figure 7: Fluorescence correlation curves of DCM in BSA fibril in presence of different SAILs ;(a) C8mimCl, (b) C12mimCl, (c) C16mimCl. The points are the experimental data, and the lines represent best fits data To further explore the secondary structural changes of amyloid fibril in presence of SAILs, far UV CD analyses have been performed. It is very interesting to observe that in presence of C8mimCl, the band of fibril at 217 nm is red shifted by 4 nm (Figure 8).This red shift in CD band may be attributed to the hydrogen bond formation involving imidazolium protons and –CO and – NH groups of different amino acids or polypepdide bond.43 But this observation is absent in cases of longer alkyl chain containing imidazolium chloride. Furthermore, in presence of C12mimCl and C16mimCl, the CD spectra show two negative peaks at 208 nm and 223 nm confirming the destabilization of amyloid structure of BSA. But the nature of the spectra is quite distinct from the spectrum of native BSA (Figure S2) as the peak near 223 nm becomes broad in 16 ACS Paragon Plus Environment

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presence of SAILs. It is confirmed from the spectra, that the fibrils are disrupted but not refold back to the native state rather forms some inhomogeneous amorphous aggregates in presence of SAILs. We are also speculating that the hydrogen bonding interaction is not the reason for disruption of the fibril in cases of C12mimCl and C16mimCl as the hydrogen bonding interaction is solely governed by the distance as well as the angle between hydrogen bond donor and acceptor groups,76 and because of longer alkyl chains attached to the imidazolium headgroups, this type of interaction is not preferred. Rather, we believe that the hydrophobic and electrostatic interaction may be responsible for the disruption of fibrils that we will discuss later.

fibril fibril+ c8mimCl

0

fibril+ c12mimCl

CD (mdeg)

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fibril+ c16mimCl

-50 -100 -150 -200 -250 200

210 220 230 240 Wavelength (nm)

250

Figure 8: Circular Dichroism (CD) spectra of BSA fibril and the same in presence of three SAILs. To observe the effect of SAILs on fibril inhibition in other proteins, HSA has also been studied as it has 76% sequence homology with BSA. At the similar conditions, HSA also forms fibril (Figure S9 and S11). In the similar fashion, imidazoliumn based SAILs are effective for fibril inhibition and inhibition efficiency follows the order C16mimCl> C12mimCl> C8mimCl. The corresponding FESEM and FLIM images are shown in Figure S10 and S12. In this context, we have proposed some mechanisms of SAILs induced inhibition of fibrils. There are several interaction forces involved in the binding of biomacromolecules with small 17 ACS Paragon Plus Environment


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molecules which generally comprise hydrophobic, electrostatic, Van der Waals and hydrogen bonding interactions. The most important feature of SAILs in aqueous environment is the hydrophobic moiety in the cationic part of the structures. The length of alkyl side chains increase the hydrophobicity of the cationic imidazolium group, thus the hydrophobicity of C16mimCl is larger than that of the C8mimCl. It is well reported that the hydrophobicity of any molecule increases with its aliphatic chain length due to non-polar nature of aliphatic chain.77 Furthermore, for better clarification, we have also investigated the micellization property of SAILs78 in water by using a model hydrophobic dye, pyrene79 because the aggregation of amphiphilic molecules is primarily caused by the hydrophobic effect.80 As shown in Figure S13, a remarkable decrease in the cmc values going from C8mimCl to C16mimCl implies that with increase in chain length of SAILs, stronger hydrophobic interactions occur between the long hydrocarbon chains. Hence, they can easily form micelles and the cmc values decrease. Again for a particular concentration of CnmimCl, surface tension of the solution containing CnmimCl decreases with increasing the value of ‘n’ (Table S1). This also implies the presence of larger hydrophobic domain in the structure of C16mimCl.81,82 On the other hand, the BSA in the lower pH region becomes more unfolded causing weakening of the intermolecular repulsion and make more hydrophobic groups exposed to the solution to favour the aggregation propensity. These larger aggregates are well corroborated with the FESEM and FLIM images. In order to disrupt these aggregates, strong interaction between fibril inhibiting agent and BSA fibril is necessary. Indeed, interaction may be possible by the insertion of hydrophobic part of fibril inhibiting agent inside the hydrophobic groups of proteins. Therefore to inhibit protein fibril, the key requirement is the possession of long hydrophobic chain in fibril inhibiting agent. Thus, aliphatic chain of SAILs could penetrate easily in hydrophobic part of protein aggregates and the amount of penetration or in other words disruption of proteins fibril by SAILs will increase with the aliphatic chain length of the respective SAIL. In our proposed design, disruption of BSA fibril by the three different SAILs is also notified from the FESEM images and the difference is arisen only in the extent of disruption. So, we may concern in the difference in the extent of binding of SAILs with fibril that depends on the headgroup and alkyl chain length of SAILs. Therefore, the binding sites for ([Cnmim]) to BSA are suggested as the negatively charged amino acids Glutamic Acid (Glu), Aspartic Acid (Asp) and the hydrophobic residues i.e., Tyrosine (Tyr) Alanine (Ala), Phenylalanine (Phe), Valine (Val), Leucine (Leu), Serine (Ser) and Trytophan (Trp).43, 46 As in 18 ACS Paragon Plus Environment

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the range of pH 4.0 to 2.0 BSA bears a positive charge, so the electrostatic interaction is not facilitated in our study but the cation and anions of the SAILs can interact electrostatically with the oppositely charged amino acid residues of BSA. This suggests that hydrophobic force is mainly responsible for the interaction of imidazolium SAILs to BSA and our results may be explained in terms of higher hydrophobicity of the long hydrocarbon chain in the [C16mim] cation as compared to the [C8mim] cation. In the cases of longer alkyl chain containing SAILs, their self aggregation tendency increases which further increases their interaction with the hydrophobic moieties of the peptide backbones. We have already discussed earlier the role of hydrogen bonding interaction in C8mimCl while the efficiency gets diminished with increase in hydrophobicity. Moreover, this facts are also in well supported by the better spatial interaction in cases of long alkyl chains with the hydrophobic patches of the BSA fibril making them more efficient for the fibrillar disassembly.83 Shu et al.46 have reported that the electrostatic attraction between the positively charged ILs and negatively charged BSA favours the binding of imidazolium ionic liquids with BSA. However, another type of noncovalent binding may stabilize the native protein structure would be through the formation of cation-π interactions.54,84 This type of interaction involves the aromatic side chains in BSA i.e., Phe, Tyr, Trp and the imidazolium cation (C3N2H5+).85,86 Among these, the indole ring of Trp is the most prominent site for cation binding. Tyr provides comparatively larger electrostatic potential than Phe because of the presence of oxygen in the structure.87 The interaction between the charged species and the aromatic rings is governed by the electrostatic and induction effects involving the quadruple moment of the aromatic and the polarizing nature of the cations and polarizability of the aromatic ring are two important factors determining the strength of interactions.88 The imidazolium cation is stacked over the polarisable electron cloud of aromatic ring of the amino acids. The nitrogen atom of imidazolium cation may form the hydrogen bonds with the carboxylic/amino group of the amino acid (CH⋯O and NH⋯N) establishing cation-π interaction with the aromatic residues.89 However, these aromatic rings become more exposed while undergoing partial unfolding at acidic pH, therefore they can bind to the cationic headgroups of SAILs through a predominately electrostatic interaction ultimately destabilize the fibril.84 But based on our experimental evidences, hydrogen bonding interaction is not predominant in cases of long alkyl chain containing SAILs. Although these aromatic residues are hydrophobic in nature, they may capable to bind with SAILs through the interaction with the hydrophobic 19 ACS Paragon Plus Environment


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aliphatic chain of SAILs. Considering all the facts, we speculate that the hydrophobic force is the main driving force along with the noncovalent binding for the disintegration of fibrils by SAILs which is supported by the several microscopic and spectroscopic evidences. For the treatment of disorders associated with fibril formation, one of the plausible remedies is to identify molecules which can either prevent fibril formation or disintegrate the formed fibrils. Though the mechanism of formation of oligomeric intermediates from the native protein is not well understood till now, but it has been reported that oligomeric prefibrillar species are the primary toxic species compare to polymeric fibrils. It is also reported that the cytotoxicity in neuronal cell depends on the different fibrillar morphologies and the surface properties of the peptide.25,90 Therefore, inhibition of fibril formation is very essential and interestingly SAILs could be employed to disrupt fibrillation of proteins. 4. Conclusion: In conclusion, we have investigated the role of three SAILs; C8mimCl, C12mimCl, C16mimCl to inhibit the formation of protein fibril using various spectroscopic and microscopic techniques. It is well understood from this study that long chain imidazolium based ionic liquids are potent to disrupt the fibrillar morphology of protein. However, the inhibitory efficacy of C16mimCl is comparatively higher than C8mimCl. The extent of disruption depends on the hydrocarbon chain length of respective SAILs. From FESEM images, it is clearly observed that the SAILs destroy the morphology of fibril formed. Furthermore, C8mimCl is able to inhibit the fibril formation by nearly 62%, whereas complete inhibition occurs in presence of C12mimCl and C16mimCl. The disruption process, although monitored initially by ThT fluorescence experiments is further confirmed through FLIM and FCS techniques, making the present study more unique. The CD analyses also corroborate our observation. We have discussed probable mechanisms for disintegration of fibrils. Considering all the forces involved, the hydrophobic interaction is main governing force for SAILs promoted inhibition of fibril. This study sheds light on the inhibitory property of SAILs towards fibrillogenesis which may help to future development of antiamyloid drugs. Supporting Information Steady state emission spectra, CD spectra, Fluorescence intensity images of neat BSA fibril and disrupted fibrils, AFM image of fibril, Lifetime distribution of DCM in BSA fibril, fitted


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correlation curve of DCM in BSA fibril, Fluorescence intensity and FESEM images of HSA fibril and in presence of SAILs are given in supporting information. Acknowledgement: N.S. is thankful to SERB, Department of Science and Technology (DST), Government of India, for generous research grants. S.K. is thankful to CSIR for the research fellowship.

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Inhibiting the Fibrillation of Serum Albumin Proteins in Presence of

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