Evaluation of an Ultrafast Molecular Rotor, Auramine O, as a

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Evaluation of An Ultrafast Molecular Rotor, Auramine O, as Fluorescent Amyloid Marker Niyati H. Mudliar, Biswajit Sadhu, Aafrin M. Pettiwala, and Prabhat K. Singh J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b07807 • Publication Date (Web): 18 Sep 2016 Downloaded from http://pubs.acs.org on September 24, 2016

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The Journal of Physical Chemistry

Evaluation of An Ultrafast Molecular Rotor, Auramine O, as Fluorescent Amyloid Marker Niyati Mudliar,a Biswajit Sadhub Aafrin M. Pettiwala,a and Prabhat K. Singha,* a

b

Radiation & Photochemistry Division, Radiation Safety Systems Division, Bhabha Atomic Research Centre, Mumbai 400 085, INDIA

*Authors for correspondence: Email: [email protected]; [email protected] Tel. 91-22-25590296, Fax: 91-22-5505151

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Abstract

Recently, Auramine O (AuO), has been projected as a fluorescent fibril sensor, and it has been claimed that AuO has an advantage over the most extensively utilized fibril marker, Thioflavin-T (ThT), owing to the presence of an additional large red shifted emission band for AuO, which was observed exclusively for AuO in the presence of fibrillar media, and not in protein or buffer media. Since fibrils are very rich in β-sheet structure, so a fibril sensor should be more specific towards the β-sheet structure so as to produce a large contrast between the fibril form and the native protein form, for efficient detection and invitro mechanistic studies of fibrillation. However, in this report, we show that AuO interacts significantly with the native form of Bovine Serum Albumin (BSA), which is an all α-helical protein, and lack β-sheet structure that are the hallmarks of fibrillar structure. This strong interaction of AuO with the native form of BSA leads to a large emission enhancement of AuO for the native protein itself, and leads to a low contrast between BSA protein and its fibril. More importantly, the large red shifted emission band of AuO reported in the presence of human insulin fibrils, and which was projected as its major advantage over ThT, is not observed in the presence of BSA fibrils as well as fibrils from other proteins such as Lysozyme, HSA and β-Lactoglobulin. Thus, our results caution about the universal applicability of distinctive, and claimed to be advantageous, photophysical features reported for AuO in human insulin fibrils, towards fibrils from other proteins. Time-resolved fluorescence measurements also support the proposition of strong interaction of AuO with the native BSA. Additionally, tryptophan emission of the protein has been explored to further elucidate the binding mechanism of AuO with native BSA. The evaluation of thermodynamic parameters revealed that the binding of AuO with native BSA involved positive enthalpy and entropy changes, suggesting dominant contributions from hydrophobic and electrostatic interactions towards association of AuO with native BSA. Molecular docking calculations have been performed to identify the principal binding location of AuO in native BSA.


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1. Introduction The transformation of normally soluble protein molecules into fibrillar aggregates is a crucial phenomenon encountered in a range of diseases including Parkinson disease, type II diabetes, and Alzheimer’s disease.1-5 These diseases are characterized by insoluble deposition of plaques which are rich in β-sheet containing amyloid fibrils. The crucial step in the fibril formation is the generation of partially folded intermediates, which are formed by destabilization of protein’s native conformation, when subjected to a change in pH, temperature or ionic strength of the medium.6-8 The significant population of these non-native intermediates then undergoes conformational rearrangement assisted by several specific non-covalent intermolecular interactions such as hydrophobic contacts, electrostatic interactions, hydrogen bonding etc., that eventually results into oligomerization and fibril formation. Due to the toxic nature of these fibrils towards cell, understanding the mechanism of fibril formation and detection of these fibrils is very important from the viewpoint of development of therapeutic strategies. However, because of the limited solubility of the amyloid fibrils, many biochemical techniques become unsuitable for their investigation, and therefore amyloid dyes serves as the predominant method of amyloid investigation.9-11 Among the amyloid dyes, the fluorogenic dyes which works through fluorescence turn-on mechanism, i.e., fluorescence is switched on, in the presence of fibrils, are particularly advantageous, because of the convenience and exceptional sensitivity associated with fluorescence spectroscopy. One such dye is Thioflavin-T, which shows several order of magnitude increase in fluorescence intensity upon fibril binding, and therefore serves as one of the most extensively utilized amyloid reporter for fibrillation in solution.1-2, 10-15 Recently, Auramine O (AuO), (see Scheme -1), a molecule belonging to the ultrafast molecular rotor family,16-17 has been projected as an efficient fluorescent fibril sensor and the method was demonstrated for the human insulin fibrils.18-19 It was shown that AuO undergoes a large emission enhancement in the presence of human insulin fibrils, and more importantly, displays an additional large red shifted emission band (~560 nm) apart from the usual emission band of AuO in water or protein ACS Paragon Plus Environment

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(~500 nm).18-19 On the contrary, the emission maximum of Thioflavin-T does not show any appreciable change, while going from protein to fibril (~490 nm).20-22 This distinguishing feature, of presence of an additional red-shifted band for AuO in human insulin fibril, was claimed to be a significant advantage of AuO over ThT for fibril sensing.18 Interestingly, it has been observed that even an all α-helical protein, without having any predisposition towards β-sheet formation, can form β-sheet structures, in response to the modification of solution conditions, e.g., temperature, pH and ionic strength, suggesting that fibrillation is rather a generic properties of the polypeptides.23 This seminal observation has spurred a lot of interest in this field. Therefore, we wanted to study the fibrillation of a model all α-helical protein, Bovine serum albumin (BSA), using AuO as a probe, due to its recently reported advantages. The most abundant protein of blood plasma is the serum albumin, which serves as a carrier for several endogenous compounds.24-25 Albumins maintain the osmotic pressure of the blood and also contributes towards the maintenance of the pH of the blood.24, 26 Bovine serum albumin is 585 residue protein consisting of three homologous domains linked together by several intradomain disulfide bonds. In contrast to amyloid fibrils which are β-sheet rich structure, BSA is devoid of β-sheet structure. It is well understood that a fibril marker should be specific to β-sheet cross-linked structure, and should have minimum affinity towards non β-sheet structure or native form of the protein, so that a large contrast between the fluorescence properties of the sensor could be achieved between the protein in the native form and the fibril form. However, surprisingly, in this study, we found that, the projected amyloid marker, AuO, interacts significantly with the native form of the protein itself, leading to large changes in the emission intensity of AuO in the native protein solution. Further, the large red shifted band of AuO (~560 nm),18 which was projected as its advantage over ThT, could not be observed in the presence of BSA fibrils. These results question the universal applicability of distinctive and advantageous photophysical features reported for AuO in human insulin fibrils, towards fibrils from other proteins and suggest that care must be taken while using AuO as a probe for fibrillation.


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In this paper, we have employed ground-state absorption, steady-state and time-resolved fluorescence measurements, along with circular dichroism measurements, to obtain information related to the binding mechanisms of the AuO to BSA. Molecular docking calculation has been also performed to identify the principal binding site of AuO in BSA.

Scheme 1: Chemical structure of Auramine O

2. Experimental Auranine O (AuO) was obtained from Sigma-Aldrich as the chloride salt of the dye and was purified by several sublimation steps. Thioflavin-T (ThT) was also obtained from Sigma as the chloride salt and was re-crystallized twice from methanol. Bovine Serum Albumin (essentially globulin free, ≥99%), Human Serum Albumin (≥98%), Lysozyme from chicken egg white, β-Lactoglobulin from Bovine milk, Poly(sodium 4-styrenesulfonate) (Average MW ~70000), and guanidinium chloride was obtained from Sigma-Aldrich. A stock solution of BSA (1mM) was prepared in 5 mM phosphate buffer of pH 7.4, and was stoted at 40 C. In a typical aggregation experiment, the stock solution of BSA was diluted appropriately to a concentration of 100 µM. The protein sample was heated in an oil bath maintained at 65 ± 10 C without agitation for 2 hrs.27 Freshly prepared stock solution of AuO in 5mM Phosphate buffer solution was added to the protein and fibril solution to reach a final desired concentration of AuO, and 20 minutes incubation of the solution at room temperature was maintained before final measurements. The formation of BSA fibrils was confirmed by ThT assay. (Figure S1, supporting information). ACS Paragon Plus Environment

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For the preparation of HSA fibrils, 2 mg/ml of HSA was dissolved in Tris–HCl buffer (pH 7.4) and incubated at 650C for 4 hrs.28 The formation of HSA fibril was confirmed by thioflavin-T fluorescence assay. (Figure S2, supporting information) Lysozyme fibrils were prepared by following the method reported by Pagano et. al.29 1 mg/ml of lysozyme was dissolved in 20 mM phosphate buffer (pH 6.3) containing 3M of Guanidinium hydrochloride, and the solution was incubated at 450C for 16 hrs with continuous stirring. The fibril formation was confirmed by ThT assay (Figure S3, supporting information). For preparation of Bovine β-Lactoglobulin fibril, it was dissolved in pH 2 solution (HCl) at a concentration of 10 mg/ml. The Bovine β-Lactoglobulin sample was then heated at 80 0C for 20 hrs,30 and the fibrillation was checked by ThT assay (Figure S4, supporting information). Ground state absorption measurements were carried out in JASCO model V650 spectrophotometer.

Steady-state

fluorescence

measurements

were

performed

in

a

Hitachi

spectrofluorimeter, model F-4500. For the time-resolved fluorescence measurements, a diode laser based time-correlated singlephoton counting (TCSPC) spectrometer from IBH, U.K was used, the details of which has been described elsewhere.31-32 For the excitation of Auramine O, a 440 nm diode laser (1 MHz repetition rate) was used, whereas for the excitation of the intrinsic fluorophore of BSA, tryptophan, a LED source at 295 nm was used. The emitted photon, after passing through a monochromator, was detected using a photomultiplier tube based detector. The fluorescence transients were collected at magic angle (54.7°) configuration in case of laser source excitation. The magic angle collection avoids the influence of the rotational relaxation of the probe molecule on the observed transient decays. For the measurement of instrument response function (IRF), the scattered excitation light from the suspended TiO2 particles in water was collected. The measured IRF was ∼160 ps. A thermoelectric controller from IBH, UK was used for controlling the temperature of the solution. The decay traces are fitted with a multi-exponential function of the following form,33


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I (t ) = I (0)∑αi exp(−t / τ i )

(1)

The mean fluorescence lifetime is calculated according to the equation,33 < τ >= ∑ Aiτ i

where

Ai = αiτ i / ∑αiτ i

(2)

Time-dependent anisotropy was calculated using the following equation. r (t ) =

I (t ) − GI ⊥ (t ) I (t ) + 2GI ⊥ (t )

(3)

Where, Iǁ (t) and I⊥(t) represent the emission transient decay for the parallel and perpendicular polarizations, respectively. The polarization of the excitation beam was vertical. G-factor accounts for the polarization sensitivity of the detector, and was measured independently. CD measurements: Circular dichroism measurements were carried out with a Biologic MOS 450 spectropolarimeter over a wavelength range of 190− 300 nm under constant nitrogen flow at room temperature, using 1 mm path length cell. A scan rate of 50 nm /min was set while recording the spectra, and each spectrum is an average of 3 scans. All the spectra were baseline substracted with the spectra of only buffer solution, serving as a baseline, under the same condition. CD spectra were recorded as ellipticity (θ) in mdeg. Molecular Docking: Docking simulations were performed using Auto Dock 4.2 software34 to elucidate the optimum binding location of Auramine-O (AuO) in bovine serum albumin (BSA) protein. Crystal structure of the protein was taken from protein data bank (PDB: 4F5S).35 Before the docking procedure, charges on ligand atoms were derived using natural population analysis (NPA)36 at B3LYP/TZVP level.37-39 Partial Kollman charges are assigned to BSA upon removal of crystallographic water molecules from the structure. For blind docking, Grid size was set to 80, 80, 80 along X, Y and Z axis with 0.8 Å grid spacing. The grid maps for the docking studies were computed by using autogrid module. Finally docking simulations were performed using Lamarckian genetic algorithm (LGA). For the simulations, GA population size 150 was taken and 2500000 steps of maximum number of energy evaluations were considered for each run. Total 50 conformations were generated in the docking analysis. Finally, the most stable conformation was chosen after the cluster analysis on the generated ACS Paragon Plus Environment

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docked conformations with a root-mean-square deviation (rmsd) tolerance of 2.0 Å. Discovery Studio Visualizer v16.1 software from Accelrys Software Inc. was used for visualization and schematic presentations of the docked conformations.

3. Results and Discussion Steady–state emission and absorption measurements: The emission spectrum of AuO at different concentrations of native BSA has been measured, and is presented in figure 1A. It is observed from figure 1A that the emission intensity of AuO progressively increases with gradual addition of native BSA in solution, and leads to a large emission enhancement of ~ 80 times (at 140 µM concentration of BSA), as compared to the AuO in only buffer solution.

Figure 1. (A) Steady-state emission spectra (λex = 440 nm) of AuO (16 µM) at different native BSA concentrations: (1) 3 (2) 6 (3) 9(4) 12 (5) 15 (6) 20 (7) 28 (8) 41.5 (9) 54 (10)78 (11) 99 (12) 137 µM. (B) Fluorescence titration curve for the AuO-native BSA. The blue circles are the data points (at λem = 505 nm) and the solid red line represents the fitting according to the 1:1 binding model.


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AuO displays very weak emission in the only buffer solution with a maximum at ~505 nm. The weak emission yield of AuO in aqueous solution, or in low viscosity solvents, is reported to be due to a very efficient non-radiative conformational relaxation process operative in the excited state of AuO.40-42 This conformational relaxation, identified as phenyl group twisting motion in the excited state, leads to the population of a non-fluorescent state of charge transfer character (TICT) in the solution, from an initially formed locally excited state (LE) state, thus causing a very low emission yield.40-42 This phenyl group twisting motion, when impeded in a confined environment or in high viscosity solvents, leads to an increase in the emission yield of the AuO.43-48 Thus, such a large increase in the emission intensity of AuO in the presence of native BSA can be ascribed to the complexation of AuO with the native BSA. Due to the complexation of AuO within the binding pockets of BSA, the available free volume for the excited state conformational change in AuO decreases, which leads to the reduction of the non-radiative phenyl group twisting process in the excited state. This leads to an immense enhancement in the emission yield of AuO, in the protein bound form. Additionally, the lower polarity at the hydrophobic binding sites of BSA may also reduce the propensity of AuO to form a polar non-emissive TICT state, which will further add to the increase in the emission yield of AuO in the presence of BSA protein. The interaction of AuO with native BSA is also supported by ground state absorption measurements. For this, we have measured absorption spectra of AuO in the presence and absence of BSA, and observed a red shift of λmax of the dye by about 5 nm. (Figure S5, Supporting information). The observed red shift in the absorption spectra can be assigned to the hydrophobic interaction at the binding location of protein pocket with AuO. Since at pH 7.4, the overall charge of BSA protein is negative, thus, besides hydrophobic effect, the observed red shift may be also caused by electrostatic interaction between cationic AuO and negatively charged amino acid of BSA at the binding location, as also supported by docking simulation (see later). Similar bathochromic shift has been observed for the AuO in polyanionic DNA solution,49 Sulfobutyl ether β-cyclodextrin,48 and SDS micelle45 which offers potential negatively charged binding sites for electrostatic interaction with the cationic AuO.


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In an attempt to evaluate the strength of binding and also the stoichiometric compositions of the complex formed between AuO and BSA, the fluorescence titration data for AuO with the gradually increasing BSA concentration was analyzed using the 1:1 complexation model48,

50-52

which gave

satisfactory fitting to the experimental results. During the experiment, total dye concentration [AuO]0 was kept constant. The observed fluorescence intensity, If, at any concentration of the BSA protein can be expressed as,50, 52 ∞

0

If = I A u O

[A uO ] eq [A uO ] 0

[A uO • B S A ] eq + I A uO • B S A

[A uO ] 0

(4) ∞

where,

0

I A uO

is the fluorescence intensity of AuO in absence of BSA,

I

AuO•BSA

is the extrapolate

fluorescence intensity when all the AuO will be converted to AuO●BSA complex, [AuO]0 represents the total concentration of AuO, and [AuO]eq represents equilibrium concentration of the free AuO in the solution. For the purpose of fitting of fluorescence titration curves, the changes in fluorescence intensity ( ∆ ) were estimated at its emission maximum (λem=505 nm). ∆

is then plotted against the total protein

If

concentration [BSA]0 used. Following eq. 6,

∆

If

If

is expressed as,

∞

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 [ A u O ] eq  0 ∆ If =  1 - I A uO )  (I [A u O ]0  A uO • BSA 

(5)

where [AuO]eq is expressed in terms of the total protein concentration used, [BSA]0, and the equilibrium constant Keq for the AuO●BSA complex by the following equation. [AuO]eq =

1 2 Keq

{(K

eq

[AuO]0 - K eq [BSA]0 - 1) +

(K eq [AuO]0 + K eq [BSA]0 +1) 2 - 4(K eq ) 2 [AuO]0 [BSA]0

}

(6)

The binding constant (Keq) for AuO with BSA protein as obtained from the nonlinear fitting of the titration curve in figure 1B using the equation 6 was thus estimated to be 3.5±0.2 x104 M-1. The high value of Keq indicates a very strong interaction of the AuO with the native form of the BSA. Thus, such a strong binding of AuO with the native form of the BSA is responsible for large changes in emission yield of the molecule, where the torsional motion of the phenyl group in the excited state is hindered to a significant extent upon binding with the protein pocket. ACS Paragon Plus Environment

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Time-resolved measurements: Fluorescence lifetime serves as an important and sensitive parameter for investigating the local environment around a fluorophore, and it is very sensitive to excited-state interactions. Thus, to understand the effect of protein binding on the fluorescence lifetime of AuO, we have performed time-resolved fluorescence measurements using time-correlated single photon counting (TCSPC) technique. The transient emission decay traces for AuO has been measured at their emission maximum (λem = 505 nm) in buffer solution and in native BSA solution, and has been presented in figure 2. AuO decays very rapidly in the only buffer solution and cannot be measured with the limited time-resolution of our current TCSPC setup (~160 ps). The lifetime of AuO in the aqueous solution

Figure 2. The transient emission decay (λex = 440 nm, λem = 505 nm) of AuO in 5mM phosphate buffer solution (dotted red) and 100 µM native BSA solution. The black solid line represents the instrument response function (IRF). is reported to be ~1 ps.44-45 The ultrafast decay of the excited state of AuO in the buffer solution arise from the relaxation of AuO on the barrier-less excited state potential energy surface.44-45 However, in the presence of native BSA protein, the emission decay trace becomes significantly slower, extending upto nanoseconds. The decay trace for AuO in BSA was found to be non-single exponential in nature which is typical for AuO excited state dynamics, as well as for similar molecular rotors which display significant conformational flexibility.42,

44-45, 53-56

The average excited state lifetime (equation 2) for

AuO in BSA protein has been calculated to be 0.94 ns. Such a large value of the fluorescence lifetime of


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AuO upon binding to native BSA, in comparison to the aqueous buffer solution, suggests a severe restriction of the phenyl group twisting motion of AuO in the protein bound state. Time-resolved anisotropy measurements: Time-resolved fluorescence anisotropy of a fluorescent probe is often used to investigate the rotational dynamics of the probe while associated to the biomacromolecule of interest, which can provide important insights into the rigidity of the probe in its bound state. Thus, to understand the rotational dynamics of AuO in the native BSA system, timeresolved fluorescence depolarization studies have been carried out. The temporal behavior of the fluorescence anisotropy for AuO in native BSA is shown in figure 3. It is evident that the fluorescence anisotropy of AuO shows a very insignificant decay within

Figure 3. Fluorescence anisotropy decay traces for AuO in 100 µM native BSA solution. (λex = 440 nm, λem = 505 nm) The filled circles are the data points and the solid blue line is the fitting according to the exponential decay model our experimental time window. Such a slow decay of the fluorescence anisotropy indicates strong immobilization of AuO in the protein bound state. A single exponential fitting of the anisotropy decay trace yields a correlation time of > 1 µs, which is suggestive of the rotation of the whole AuO-BSA complex. It should be noted that the fluorescence anisotropy of the AuO in the only buffer media could not be measured because of the very fast lifetime of the AuO molecule in the buffer media. However, considering the molecular dimension of the AuO molecule, rotational correlation time of AuO in the aqueous buffer medium would be much faster (< 50 ps) than the time-resolution of our experimental set


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up. Thus, such a strong immobilization of AuO at the binding location in the protein pocket is consistent with the large increase in emission yield and a significant slowdown of the excited state dynamics of the AuO molecules in the protein bound state. AuO in the BSA fibril system: As stated earlier, AuO has been recently projected as a potential fibril sensing probe, with the concept being demonstrated for the human insulin fibrils. Fibrils are known to be rich in β-sheet structure. Thus, a fibril sensing probe is assumed to have a high specificity and strong interaction with the β-sheet structure. However, on the contrary, we have discovered that AuO interacts significantly with the native form of the BSA protein itself, which is an all α-helical protein, and lack any β-sheet structure in its native form. Thus, to investigate whether AuO displays any distinguishing feature for the fibrils of BSA as compared to the native form of the protein, we have carried out steadystate emission measurements of AuO in the BSA fibrils. Figure 4A displays the emission spectra of AuO with increasing concentration of the BSA fibrils.

Figure 4. (A) Steady-state fluorescence spectra (λex = 440 nm) of AuO (16 µM) at different BSA concentrations in the fibril form: (1) 0.7 (2) 2 (3) 4 (4) 5.7 (5) 7 (6) 8.4 (7) 11 (8) 13.5 (9) 16 (10) 19


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(11) 25 (12) 34 (13) 50 (14) 75 µM. (B)Fluorescence titration curve for the AuO- BSA fibril. The blue triangles are the data points (at λem = 505 nm) and the solid red line is the fitting according to the 1:1 binding model. It is evident that similar to the case of AuO in the native BSA, the emission intensity of AuO gradually increases with increase in the concentration of BSA fibrils. , a slightly higher emission enhancement (a factor of ~4) has been obtained at the saturation condition of the BSA fibril as compared to native BSA. But, most importantly, the emission peak maximum for the AuO in the BSA fibril appears at ~505 nm, which is in sharp contrast to reported emission peak position of AuO in human insulin fibrils at ~560 nm.18 This additional largely red shifted emission peak position of AuO reported for human insulin fibril, as compared to AuO in buffer or protein, was projected as one of the major advantage for AuO in fibril sensing18 in comparison to the most routinely used fibril sensor, Thioflavin-T, because ThioflavinT also leads to the emission enhancement in the presence of fibrils but the emission peak position largely remains invariant in comparison to that of the buffer or protein media.18, 20-22 It should be noted that, to further check the generality of the red-shifted new emission band of AuO in the fibrillar media, we carried out measurements for AuO in fibrillar media of few other proteins which include Lysozyme, Human serum albumin, and β-Lactoglobulin. Figure 5 shows the emission spectra of AuO in these fibrillar media, where it is evident that the emission intensity of AuO increases in the presence of these fibrillar media, but the emission maximum remains centered at its normal emission band (~500 nm), and we do not observe the new red-shifted emission band (~560 nm), in any of the cases, that was reported for AuO in Human insulin fibrils. Thus, we observe that the presence of a large red shifted emission band for AuO, as reported in human insulin fibrils, is not a general phenomenon, and may be specific for the human insulin fibrils.


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Figure 5. Steady-state emission spectra(λex = 440 nm)of Auramine-O in different fibrillar system: (A) Lysozyme fibril, (B) β-Lactoglobulin fibril and (C) Human Serum Albumin (HSA). To evaluate the strength of binding of AuO with the BSA fibrils, we analyzed the fluorescence titration data according to 1:1 binding model as described above for the native BSA case, which provided a satisfactory fit to the data (Figure 4B). The binding constant (Keq) for AuO with BSA fibril was evaluated to be 5.2±0.3 x 104 M-1. A slightly higher value of binding constant for AuO in BSA fibril as compared to the native BSA is consistent with a slightly higher emission yield of AuO in the BSA fibril as compared to the native BSA. To evaluate the effect of BSA fibril binding on the excited state dynamics of AuO, we have also performed the time- resolved fluorescence measurements for AuO in the presence of BSA fibrils. Figure 6 presents the transient decay trace for AuO, monitored at its emission maximum, for both BSA protein in its native form and in fibrillar form. It is evident that the emission decay trace for AuO is slightly more slower in BSA fibril as compared to native BSA protein. Similar to the case of AuO in native BSA, the AuO in fibril also displays a non-single exponential kinetics. The average lifetime for AuO in BSA fibril is calculated to be ~1.25 ns which is slightly higher than AuO in the native BSA (~ 0.94 ns).


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This slightly higher average lifetime for AuO in fibril is consistent with the higher emission yield of AuO in the BSA fibril as compared to that of protein.

Figure 6. The transient fluorescence decay (λex = 440 nm, λem = 505 nm) of AuO in (A) 5 mM phosphate buffer solution (dotted red) (B) 100 µM native BSA solution prepared in 5mM phosphate buffer solution, pH 7.4. (C) 100 µM BSA fibril solution prepared in 5 mM phosphate buffer solution, pH 7.4. The solid black line represents the instrument response function (IRF). In this regard, it is important to note that the AuO displays a similar long excited-state lifetime in the human insulin fibrillar media.18 This long excited state lifetime of AuO upon association with human insulin fibrils was argued to be the reason for the observation of the new band of AuO, which was attributed to a species formed upon a fast proton dissociation from the excited AuO.18 On the contrary, it was argued that, owing to the very short (picoseconds) lifetime for AuO in water or native protein, this proposed faster proton dissociation could not be observed in water or native protein medium, leading to only normal emission band centred at ~500 nm. However, in the present case of both BSA native protein and fibrils, AuO displays a long excited state lifetime, but the new emission band could not be observed. Further, AuO also displays long lifetime in Lysozyme (Figure 7, τavg=1.3 ns) and HSA (Figure S6, supporting information), (τavg= 1.6 ns) fibrils but the new red-shifted emission band remains absent. This greatly limits the validity of the proposition of proton transfer theory for the observation of the new emission for AuO.


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Figure 7. The transient emission decay (λex = 440 nm, λem = 505 nm) of AuO in 20mM Phosphate (pH 6.7) buffer (dotted red) and in Lysozyme Fibril solution (solid blue line). The black solid line represents the instrument response function (IRF). Interestingly, very recently, ThT has been reported to display a similar large-red shifted emission band in the presence of a supramolecular host, which has been assigned to ThT aggregates.57 Infact, we could observe this low energy emission band for AuO on the surface of a negatively charged polyelectrolyte, polystyrene sulphonate (Figure S7, supporting information), which is very conducive for the formation of dye aggregates.58 Thus, it is possible that AuO dimers/aggregates may form in the presence of Human insulin fibrils, which might be responsible for the observation of new emission band for AuO in the presence of Human insulin fibrils. To evaluate the effect of fibrillar environment on the rotational dynanics of AuO, and also to investigate whether it displays any difference from the BSA in its native form, we have performed timeresolved fluorescence anisotropy measurements for AuO in BSA fibril. Figure 8 represents the temporal profile for the fluorescence anisotropy, and similar to the case of AuO in native BSA, here also the anisotropy was found to display an insignificant decrease within our experimental time window. This observation is completely consistent with the immobilization of AuO within the BSA fibril, leading to large emission enhancement and slow excited state dynamics in the presence of BSA fibril. However, because of the limited lifetime (~1 ns) of the probe, any significant difference between the temporal ACS Paragon Plus Environment

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decay of anisotropy in native BSA and fibrillar BSA could not be observed, and qualitatively, they display a very similar trend. Thus, Although AuO has been projected as a superior fibril sensor, as compared to the most routinely used probe, Thioflavin-T, in this report, we have convincingly shown that the emission features reported for AuO in human insulin fibrils are not universal for all the fibrillar system. Futher, despite the fact that fibrils are structures with rich β-sheet content, AuO interacts very significantly with the native form of BSA protein which is predominantly an α-helical protein. Thus, our result suggests that caution needs to be exercised while using AuO as a fibril marker because it may lead to erroneous conclusion on the estimation of fibril.

Figure 8. Fluorescence anisotropy decay traces for AuO in 100 µM BSA solution in the fibril form. (λex = 440 nm, λem = 505 nm) The filled circles are the data points and the solid blue line is the fitting according to the exponential decay model. After establishing that AuO interacts very significantly with the native form of the BSA protein, we wanted to investigate, in detail, the interaction between AuO and native BSA, so as to understand what guides such a strong interaction between AuO and native BSA. For this, we have employed circular dichroism (CD) measurements, tryptophan emission quenching and the molecular docking calculation for the AuO-native BSA system Circular dichroism measurements: To investigate whether binding of AuO causes major structural changes in native BSA structure, circular dichroism (CD) measurements were performed on BSA and AuO-BSA complex. CD spectroscopy is a widely used technique to detect sensititve conformational ACS Paragon Plus Environment

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changes in the structure of protein upon interaction with the ligand. Figure 9 displays the far-UV CD spectra of BSA in the presence of increasing concentration of AuO. The CD spectra of BSA display two negative bands at ~ 208 nm and 222 nm, which represent the characteristic of the α-helical structure of the protein. The CD spectra of BSA display similar shape both in absence and presence of AuO, which indicates that BSA maintains its α-helical structure even after dye association.

Figure 9.Circular Dichroism spectra of native BSA solution (5 µM) prepared in 5mM phosphate buffer at different concentration of AuO (1) 0 (2) 5 (3) 10 (4) 20 µM. The addition of AuO slightly decreased the intensity of both the negative bands, indicating minor alterations in the secondary structure of BSA. Similar minor alterations in the α-helical structure of BSA, on binding of ligands, have been reported earlier.59-60 Tryptophan quenching of native BSA: Fluorescence-quenching of intrinsic fluorophores of proteins have been widely used to study the interactions of ligands with proteins.61-66 This method can reveal accessibility of quenchers to protein fluorophores, and can help in understanding ligand binding mechanism of proteins. Among the intrinsic fluorophores of protein, monitoring tryptophan associated fluorescent changes is the most common practice for deriving information regarding the structural changes in protein as well as its dynamics.67-69 BSA consists of two tryptophan residues: The eighth helix of D129- R144, in the domain I, contains Trp-134, which is well exposed, whereas Trp-212 is buried inside the protein structure and is present on the second helix of E206-F221 of domain II.70 Upon excitation at 295nm, BSA shows emission maximum at 345 nm. The BSA solution was excited at 295 ACS Paragon Plus Environment

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nm to escape any emission contribution from tyrosine residues.71 It should be noted that only AuO contributes insignificantly towards emission under the same experimental conditions. Figure 10 displays the steady-state emission spectrum of BSA at a varying concentrations of AuO (0-50 µM) in an aqueous buffer solution. It is evident that the increasing concentrations of AuO resulted in a gradual decrease in fluorescence intensity associated with a decrease in the emission wavelength maximum (a blue shift from 343 to 338 nm) in the BSA spectrum. This suggests that the Tryptophan residue of BSA gets relatively more buried into the hydrophobic domain, on formation of AuO-BSA complex.

Figure 10. Steady-state emission spectra of Tryptophan in native BSA solution (10 µM) (λex = 295 nm) at different concentration of Auramine O (1) 0 (2) 8.4 (3) 17 (4) 25 (5) 33 (6) 41 (7) 50 µM. The quenching of tryptophan fluorescence can be analysed using Stern- Volmer equation which is given by,33 F0 = 1 + KSV [Q] F

(7)

Here, F and F0 are the fluorescence intensities in the presence and absence of quencher, respectively, and [Q] represnts the quencher concentration. KSV is the Stern-Volmer quenching constant, which measures the efficiency of quenching. Fluorescence quenching can be either dynamic or static. The dynamic quenching results from collisional encounters between the quencher and the fluorophore, whereas static quenching arises when there is a ground state complex formation between the quencher and the fluorophore.33 Type of fluorescence quenching can provide important information about the changes taking place in the vicinity ACS Paragon Plus Environment

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of chromophore molecules. The distinction between static and dynamic quenching can be made by investigating the temperature dependence of Stern-Volmer quenching constant. Higher temperature causes faster diffusion and hence results in larger amount of dynamic quenching. On the contrary, higher temperature usually causes dissociation of the weakly bound complexes and, hence, reduces the extent of static quenching. Thus, to evaluate the mechanism of quenching, AuO concentration dependent quenching of tryptophan fluorescence was performed at three temperatures (295, 302, and 309 K), at which BSA does not undergo chemical degradation.60,

72

Consequently, the Stern-Volmer

analysis was performed, and the results are presented in Figure 11. The result shows that the SternVolmer quenching constant (KSV) increases with increase in temperature, which indicates higher efficiency of quenching at higher temperature. This clearly indicates that the probable quenching mechanism of AuO-BSA binding reaction is initiated by dynamic collision. It should be noted that the efficiency of quenching of BSA fluorescence is relatively weaker which indicates that the probable binding location of AuO in BSA is domain II, where Trp residue is quite buried inside the hydrophobic pocket. This is further supported by molecular docking results (see later).

Figure 11: Stern-Volmer plots for quenching of BSA by AuO at three different temperatures. 295K (), 302K (), and 309 K (). Fluorescence lifetime decay of BSA in the presence of AuO: Fluorescence lifetime of tryptophan residues in proteins serves as an important parameter for understanding the interaction between the probes and proteins.33 The fluorescence lifetime decay profiles of the native BSA only, and in presence


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of various concentrations of AuO has been measured and results are presented are presented in Figure S8 (supporting information). BSA in only aqueous buffer solution displays a bi-exponential decay traces which is typical for tryptophan in protein.33, 71 BSA shows two emission decay times of 6.3 ns and 2 ns in only buffer, which are in good agreement with the literature reports.71 The emission decay traces for BSA gradually becomes faster with increasing concentration of AuO. Thus, the mean lifetime decreases from 5.85 ns in absence of AuO to 5.15 ns at AuO to BSA ratio of 5:1. Thus, the decrease in the mean lifetime of Tryptophan fluorescence is definitely a consequence of interaction of AuO with BSA, and supports the involvement of dynamic nature of quenching of tryptophan fluorescence by AuO. Further, the minute nature of change in lifetime values of Tryptophan further corroborates the weaker alteration of protein structure by addition of AuO, as also suggested by CD spectra. Thermodynamic parameters for AuO in native BSA: Small molecules binds macromolecules by several type of non-covalent interaction modes, mainly including hydrogen bonding, electrostatic force, the vander Waals force, and hydrophobic interaction force. The thermodynamic parameters including enthalpy change (∆H0) and entropy change (∆S0), provide important clues regarding the binding mode of the ligand to the macromolecule. Hence, the effect of temperature on the binding constant was studied. For this purpose, the quenching data of Tryptophan fluorescence was utilized. The association constant (KA) and the number of binding sites (n) was determined using modified Hill’s equation assuming that BSA has the same independent binding sites,60

log

F0 − F = n log K A + n log{[Q] − (1 − F / F 0 )[ P]} F

(8)

where F0 and F represent the fluorescence intensity without and with the quencher, [Q] represents the total added quencher concentration and [P] is the total protein concentration This modified Hill’s equation takes into account the fact that when ligands bind partially to sites of protein molecules, the total ligand concentration does not represent the free ligand concentration ([Q]free) in solution, which can be defined as,60 [Q] free = [Q] − n(1 − F / F0 )[ P]

(9)


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Thus, binding constant and the number of binding sites can be calculated from the intercept and slope of linear fit of plots of log[(F0-F)/F] versus log {[Q]-(1-F/F0)[P]}] as shown in Figure 12. The obtained value of n is very close to 1, suggesting that the probe is located in one binding site. This is in very good agreement with a previous report where the number of binding sites for AuO in serum albumins was also found to be 1.73 The values of calculated binding constant at different temperatures are presented in table 1. Table 1: The binding parameters of Auramine O with BSA at different temperatures Temp (K)

KSV (x 104 M-1)

KA (x 104 M-1)

295

0.96

1.33

302

1.29

1.53

309

1.68

1.90

For evaluating the thermodynamic parameters, a plot of ln (K) vs 1/T was fitted with a straight line according to the Van’t Hoff equation (Inset of Figure 12): ln K = −

∆H 0 ∆S 0 + RT R

(10)

Where, ∆H0 and ∆S0 are enthalpy change and entropy change, respectively. T is the temperature and R is the universal gas constant. K represents the binding constant at corresponding temperature. If the ∆H0 is assumed to remain constant over the studied temperature range, then ∆H0 and ∆S0 can be evaluated from the Van’t Hoff equation (equation 10). The free energy of the reaction (∆G0) can be evaluated using the following equation ∆G 0 = − RT ln K

(11)


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Figure 12: The plots of log[(Fo - F)/F] vs {[Q]-(1-F/F0)[P]}] for BSA at three different temperatures 295 K (), 302 K (), and 309 K(). Inset shows the plot of ln K vs 1/T and its linear fitting As shown in table 2, the signs for ∆H0 and ∆S0 of the binding reaction were observed to be positive. The negative sign for ∆G0 means that the binding process is spontaneous. Thus, the binding process was endothermic and spontaneous. According to the characterization by Ross and Subramanian,74 based on the sign and magnitude of thermodynamic parameters, positive value of ∆H0 and ∆S0 is typically associated with hydrophobic interaction. Therefore, on our case, hydrophobic interaction seems to play an important role in the binding reaction between AuO and BSA. For ligand–protein interaction, positive entropy is often considered as an evidence of hydrophobic interaction, however, it has been also shown that positive entropy may also be associated with electrostatic interaction.75 Since BSA is negatively charged at physiological pH,76 whereas AuO is positively charged under the same condition, hence, the binding of AuO to BSA, apart from hydrophobic interaction, might also involve electrostatic interaction which is also supported by our molecular docking results shown in the next section. Table 2: Thermodynamic parameters of AuO-BSA interaction Temp (K)

∆G0 (kJ/mol)

∆H0 (kJ/mol)

∆S0 (J/mol K)

295

-23.3

19.2

144

302

-24.2

309

-25.3


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Molecular Docking: Docking of the ligand with macromolecules furnishes important clues about the preferred binding location, and is often used significantly to corroborate experimental observations. Thus, molecular docking study was performed to identify the principal binding site of AuO with bovine serum albumin and to aid in understanding of the interaction of AuO with BSA. The crystal structure of BSA contains three different domains (I, II and III) which can be further categorized in sub-domains "A" and "B". Previously, it has been observed that the hydrophobic pockets present in these domains acts as the probable binding site of ligand.77 Our docking analysis, based on the best energy ranking and the reoccuurence rate, suggests that, for the cationic AuO, the most probable binding location lies at sub-domain IIA of BSA, with a binding free energy of 4.53 kcal mol-1. The docking analysis also revealed another energetically probable conformation, where AuO binds in sub-domain IB, (Figure S9, supporting information) however, the reocurrence rate of this alternative conformation was significantly lower (8 out of 50) as compared to the former (23 out of 50). The binding site and acting interactions for the most probable docked conformation between the AuO and BSA are shown in Figure 13 (lower panel).


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Figure 13: Upper panel: Binding Site of Auramine O in BSA. Auramine O is shown in ball and stick model. The residues at close vicinity of dye molecule are shown in stick model. Lower panel: 2D Schematic diagram representing interactions between AuO and neighbouring residues of BSA. Salt bridge interactions are presented using orange colour while existing hydrogen bonds with the terminal amino group of the dye is represented by green colour. Pink colour corresponds to represent T-shaped ππ interaction. solvent-accessible surface surrounding the residue/atom are shown in light blue circle Our docking results suggest that the cationic AuO undergoes electrostatic interaction with the acidic residue GLU100 due to its proximal presence (OGlu100-HNH2(AuO): 1.86 Å). In addition, the proximal presence of carbonyl groups of LYS242 and SER104 near the N-methyl groups of AuO also indicates the involvement of hydrogen bonding interaction between them (see Fig. 13, lower panel). Further stability at this site is provided by the T-shaped π-π interaction between the aromatic ring of AuO and imidazole moiety of HIS246 residue. Upon binding, loss of residue solvent accessibility (see supporting information, Table S1) were noted for all aforementioned interacting amino acid residues which further confirms the direct involvement of these residues in ligand binding. Thus, these molecular docking are in very good agreement with our experimental data on thermodynamic parameters, which suggested the predominant involvement of hydrophobic and electrostatic interaction in the AuO-BSA binding. Table 3: Result of docking analysis AuO bound to BSA. Energies are in kcal mol-1

a

Binding free energy

Electrostatic energy

Intermolecular energya

-4.53

-1.14

-5.72

Intermolecular Energy correspond to the sum of electrostatic, hydrogen bonding, van der Waal and

desolvation Energy

4. Conclusions In summary, we have shown that despite the absence of β-sheet structure in the native form of Bovine serum albumin, a recently projected fibril sensor, AuO, interacts quite significantly with the native form ACS Paragon Plus Environment

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of the bovine serum albumin (K ~ 104 M-1), leading to large changes in the emission intensity of AuO in the presence of native form of the protein itself. More importantly, the distinctive and claimed to be advantageous, photophysical features of AuO, in the form of an additional large red shifted emission band, reported earlier in the case of AuO in the human insulin fibril, was not observed in the case of BSA fibrils, as well as fibrils from other proteins such as Lysozyme, HSA and β-Lactoglobulin. Excited state lifetime and time-resolved fluorescence anisotropy data also suggested a strong confinement of AuO in the native structure of the protein, and leads to only minor changes in the photophysical features of AuO, when going from protein to fibril. CD measurements suggested only minimal influence on the secondary structure of BSA upon binding of AuO. The evaluation of thermodynamic parameters suggests dominant contributions from hydrophobic and electrostatic interactions towards association of AuO with BSA. Molecular docking calculation suggested that AuO preferentially locates itself at domain II, and suggest the involvement of polar amino acid residues in stabilizing AuO at the binding location of native BSA. Thus, our results suggest that AuO interacts quite significantly with the native form of the protein thereby leading to only minor contrast between protein and fibril. More importantly, our results alarms about the universal applicability of distinctive and advantageous photophysical features reported for AuO in human insulin fibrils, towards fibrils from other proteins, such as BSA lysozyme, HSA, and β-lactoglobulin, and suggest that care must be taken while using AuO as a probe for fibrillation as it may lead to erroneous conclusion on the estimation of fibrils.

ASSOCIATED CONTENT Supporting Information. The ThT assay of fibrils; the ground state absorption spectra for AuO in buffer and in native BSA; the transient decay trace for AuO in HSA fibrils; the emission spectra of AuO in Poly(sodium 4-styrene sulphonate), the transient decay trace for tryptophan of BSA in absence and presence of varying concentration of AuO; loss of residue solvent accessibility of the amino acids of native BSA upon binding to AuO, calculated from molecular docking calculations, has been presented


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in the supporting information. This information is available free of charge via the Internet at http://pubs.acs.org AUTHOR INFORMATION Corresponding Author *Email: [email protected]; [email protected]

Acknowledgments: We acknowledge Dr. H. Pal, Dr. D. K. Palit and Dr. B. N. Jagtap for their constant support and encouragement during this work. NM and AMP thanks Homi Bhabha National Institute, Departement of Atomic energy for approving the project internship. BS also acknowledges the encouragement and support from Dr. Anil Kumar, Dr. Tusar Bandyopadhyay, Shri Rajvir Singh and Dr. K.S. Pradeepkumar.

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Graphical Abstract 55x40mm (300 x 300 DPI)


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Evaluation of an Ultrafast Molecular Rotor, Auramine O, as a

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