Tyrosine fluorescence probing of conformational changes in tryptophan-lacking domain of albumins

Accepted Manuscript Tyrosine fluorescence probing of conformational changes in tryptophan-lacking domain of albumins

N.G. Zhdanova, E.G. Maksimov, A.M. Arutyunyan, V.V. Fadeev, E.A. Shirshin PII: DOI: Reference:

S1386-1425(16)30700-4 doi: 10.1016/j.saa.2016.11.038 SAA 14798

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date: Revised date: Accepted date:

2 August 2016 17 November 2016 22 November 2016

Please cite this article as: N.G. Zhdanova, E.G. Maksimov, A.M. Arutyunyan, V.V. Fadeev, E.A. Shirshin , Tyrosine fluorescence probing of conformational changes in tryptophanlacking domain of albumins. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Saa(2016), doi: 10.1016/ j.saa.2016.11.038

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ACCEPTED MANUSCRIPT Tyrosine fluorescence probing of conformational changes in tryptophan-lacking domain of albumins

N.G. Zhdanova,a† E.G. Maksimov,b A.M. Arutyunyan,c V.V. Fadeev a, E.A. Shirshina†† a

b

c

Department of Physics, M.V. Lomonosov Moscow State University, 119991, Russia

Department of Biology, M.V. Lomonosov Moscow State University, 119991, Russia

A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State

[email protected], †† [email protected]

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†

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University, 119991, Russia

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Abstract

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We addressed the possibility of using tyrosine (Tyr) fluorescence for monitoring conformational changes of proteins which are undetectable via tryptophan (Trp) fluorescence. The model

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objects, human (HSA) and bovine (BSA) serum albumins, contain one and two Trp residues, respectively, while Tyr is more uniformly distributed over their structure. The results of the

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investigation of albumins interaction with ethanol using intrinsic Trp and Tyr steady-state and

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time-resolved picosecond fluorescence indicated the presense of an intermediate at 10% (v/v) of ethanol in solution, that was supported by the results of extrinsic fluorescence measurements

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with the Nile Red dye. Based on the comparison of HSA and BSA Trp and Tyr fluorescence, it was suggested that conformational changes at low ethanol concentration are located in the

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domain III of albumins, which lacks tryptophan residues. The sensitivity of Tyr fluorescence to domain III alterations was further verified by studying albumins interaction with GdnHCl.

1. Introduction Conformational changes of protein molecules are known to cause pathological processes in the human organism, and the assessment of proteins conformation in vivo is important for medical diagnostics. Serum albumin is one of the most prospective candidates for this purpose, as it is the most abundant protein in blood plasma and is responsible for binding 1

ACCEPTED MANUSCRIPT and transport of a wide variety of molecules (drugs, metabolites, fatty acids (FAs), etc.) [1-3]. Serum albumin is a globular protein, which adopts a heart-like shape formed by three homologous domains in its native conformation [1]. The ligand binding activity of serum albumin is determined by the presence of different binding sites in its structure [4-6]. For

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instance, in the albumin structure there are 6-9 binding sites for FAs, which are insoluble in water and are present in blood plasma majorly in complexes with albumin [7-8]. In

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normal conditions there are 0.1 – 2 molecules of FAs per a single albumin molecule,

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while in pathological cases this ratio increases up to 6 [9-11].

Binding of ligands and environmental conditions may induce conformational changes of

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albumin, which could influence on its transport functions. However, the studies of protein

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conformation changes are usually performed in vitro in model solutions, and an extensive investigation of albumin conformational changes in vivo is almost lacking. One of the rare

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examples is the application of electron spin resonance (ESR) spectroscopy of human

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blood plasma for diagnosis of cancer and sepsis [12-13]. This technique is based on the determination of alterations in the binding modes of spin-labelled FA when the protein structure is perturbed by addition of ethanol (EtOH) [13]. At low EtOH concentrations

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([EtOH]<2% (v/v)) conformational changes of human serum albumin (HSA) are driven

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by an alteration of the hydrogen bonds network in the first hydration sphere of the protein molecule [14-15], as well as by binding of EtOH molecules [14-17]. Namely, EtOH molecules, which displace water in the first hydration sphere, may form hydrogen bonds with amino acid side-chains (e.g. with that of tyrosine (Tyr)) [15]. As EtOH concentration increases ([EtOH] = 2–30% (v/v)), unfolding of protein molecules occurs that leads to formation of intermediates [14]. Finally, at high EtOH concentrations ([EtOH] = 30-60% (v/v)) protein aggregation starts due to the formation of intermolecular beta-structures [14]. As a result, changes in rearrangements of albumin structure induced by EtOH as 2

ACCEPTED MANUSCRIPT determined by ESR spectroscopy may serve as an indicator of pathologies in the human organism [18]. Among numerous methods for the assessment of protein conformation, fluorescence technique is extensively used because of its sensitivity and simplicity. Intrinsic fluorescence of proteins caused by the presence of tryptophan (Trp) residues is a good

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reporter of changes in molecule structure due to a high sensitivity of Trp photophysical parameters (fluorescence lifetime and quantum yield, position of fluorescence emission

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maximum) to its local microenvironment [19]. Though intrinsic fluorescence

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spectroscopy is a mainstream technique in case of model protein solutions, it can’t be simply extrapolated to analysis of biomaterials and biofluids. For instance, Trp

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fluorescence of human blood plasma could be not indicative of pathological changes in

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the organism [20]. The possible reasons are the presence of a complex mixture of fluorophores or the insufficient sensitivity of Trp to protein conformational changes in

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vivo, which could be less pronounced than in model systems.

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Another aromatic amino acid that could be responsible for proteins intrinsic fluorescence is Tyr. Tyr fluorescence is usually quenched in Trp-containing proteins [21], making it a significantly less used fluorophore in protein structure research compared to Trp.

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However, Tyr residues are generally presented in proteins in a greater number than Trp –

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e.g. in HSA and bovine serum albumin (BSA) the ratio of Tyr/Trp is 18/1 and 20/2, respectively [22-23]. Protein unfolding may result in partial removal of Tyr quenching [21, 24-27], that together with a more uniform distribution of Tyr residues in protein structure make it a prospective indicator of conformational changes. For instance, it was shown in [26] that Tyr is sensitive to specific binding of surfactant molecules (sodium dodecyl sulphate, SDS) to HSA (this system simulate the conformational changes in albumin by specific binding of FAs [28]),, while Trp fluorescence remained unchanged. Intriguingly, it was also demonstrated that in human blood plasma HSA was solely 3

ACCEPTED MANUSCRIPT responsible for the enhancement of Tyr fluorescence upon titration with SDS [27], thus suggesting that Tyr fluorescence may serve as a powerful indicator of albumin conformational changes in human blood plasma. In this work we studied the sensitivity of Tyr fluorescence to conformational changes of BSA and HSA in water-EtOH mixtures aimed at a further elucidation of structural

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determinants underlying Tyr fluorescence sensitivity to conformational changes of albumin. Here by using EtOH as a denaturing agent for serum albumins we verified the

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suggested method for Tyr lifetime assessment [26], which provides additional

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information about conformational changes in protein structure far from Trp residues. We detected the formation of an intermediate state by plotting phase diagrams based on the

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intrinsic fluorescence from Tyr and Trp residues that was supported by extrinsic

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fluorescence measurements of the Nile Red (NR) hydrophobic probe and circular dichroism (CD) measurements. The obtained results allow us to suggest that Tyr

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fluorescence could provide information about conformational changes in the domain III

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of albumins far from Trp residues. This statement was additionally verified by using a chaotropic agent (guanidine hydrochloride, GdnHCl) to direct disruption of this domain

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in the albumin structure.

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2. Experimental 2.1. Materials

Bovine serum albumin (BSA, fatty acid free, Sigma-Aldrich, USA), human serum albumin (HSA, fatty acid free, Sigma-Aldrich, USA), ethanol (EtOH, gradient grade for liquid chromatography, Merck, Germany), Nile Red (NR, technical grade, SigmaAldrich, USA), dimethyl sulphoxide (DMSO, Panreac, Spain), tris-(hydroxymethyl)aminomethane (Tris, Dia-M, Russia), PBS Tablets (Phosphate Buffered Saline Tablets, Dulbecco's Formula, MP Biomedicals, USA) were used as obtained without further 4

ACCEPTED MANUSCRIPT purification. All solutions were prepared using bidistilled water except the NR stock solution that was prepared in DMSO. Tris-HCl buffer (50 mM) was used to maintain pH at 7.40±0.05. In the case of circular dichroism measurements PBS from PBS Tablets was prepared (10 mM, pH 7.40±0.05) to minimize the absorption in the far-UV range. All measurements were performed at room temperature ((25±1)°C) without temperature

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stabilization unless otherwise mentioned. The concentration of proteins and NR were determined spectrophotometrically using the

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known extinction coefficients at the appropriate wavelengths, which are listed in Table 1.

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In the experiments with the NR dye an aliquot of its stock solution in DMSO was added to protein sample. The ratio of molar concentrations of NR to that of BSA was equal to

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1:3. Then an appropriate amount of water-EtOH mixture was added. In the experiments

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with free NR an aliquot of its stock solution was added to water-EtOH mixture directly prior to measurements to minimize the adsorption of the dye onto cuvette walls [30]. The

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and Discussion Section.

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concentrations of proteins and NR for each experiment are given in the text in the Results

Table 1. Extinction coefficients ε at appropriate wavelength λ for the investigated compounds λ, nm

ε, M-1 cm-1

Reference

HSA

280

3.5*104

[1]

BSA

295

4.4*104

[1]

NR

555

2.0*104

[29]

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Compound

2.2. UV-vis absorption Absorption spectra were measured using a Lambda 25 spectrophotometer (Perkin-Elmer, USA) in the 250 – 750 nm wavelength region. The bandwidth of both slits was set to 1 nm, the increment was 1 nm, and the scanning rate was 960 nm min -1.

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ACCEPTED MANUSCRIPT 2.3. Turbidity (optical density) Turbidity measurement is a convenient method for rapidly detecting protein aggregation. Here we used the value of optical density at 400 nm (OD400) as a turbidity indicator for protein solutions (without NR) because at 400 nm the absorption of proteins and EtOH is

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negligible.

2.4. Steady-state fluorescence

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Fluorescence spectra were measured using spectrofluorometer Fluoromax-4 (Horiba

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Jobin Yvon, France). The excitation wavelengths as well as the corresponding emission

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wavelength region used for investigated samples are listed in Table 2, the bandwidths of both slits for all experiments were set to 2 nm. Each fluorescence spectrum was the

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average of at least tree measurements.

Table 2. Parameters of steady-state fluorescence measurements Excitation, nm

Emission, nm

Trp in proteins

295

300-500

Tyr and Trp in protens

280

300-500

NR

510

570-750

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Fluorophore

Decomposition procedure was applied to protein intrinsic fluorescence spectra exited at 280 nm (I280(λ)) to obtain Tyr and Trp contributions. The description of this procedure is given in [26] and in references therein. 2.5. Time-resolved fluorescence Fluorescence lifetime measurements were performed using a custom-built fluorimeter. The setup consisted of a photomultiplier system with a Hamamatsu R5900 16-channel multi-anode photomultiplier (PML-16, Becker&Hickl, Berlin, Germany), polychromator 6

ACCEPTED MANUSCRIPT (12.5 nm/PML-16 channel) and a pulsed laser diode [26]. For intrinsic fluorescence of protein solutions excitation was performed with a pulsed 280 nm laser diode (Edinburgh Instruments, UK) delivering 700 ps FWHM, average power 0.8 μW pulses at a repetition rate of 10 MHz. To excite NR fluorescence a pulsed 510 nm laser diode (InTop, Russia) was used delivering 30 ps FWHM, average power 100 mW pulses at a repetition rate of

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50 MHz. Data acquisition for all time-resolved measurements was performed during an appropriate time to obtain the optimum signal for the deconvolution procedure [31]. The

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details of the procedure used for the analysis of fluorescence decay curves aimed at the

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assessment of tyrosine signal can be found in the Results and Discussion section and in [26].

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2.6. Circular dichroism

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Far-UV circular dichroism (CD) measurements were performed using using a Chirascan (Applied Photophysics, UK) automatic recording spectropolarimeter with Peltier-type

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thermostated cell holder. Quartz cuvettes with 0.05 cm path-length were used (Helma Analytics,

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Germany). CD spectra of BSA–PBS buffer and BSA–PBS buffer–ethanol mixtures were recorded between 190 and 260 nm at 20 C. Each spectrum was the average of four different runs

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and smoothed using Pro-Data software Savitzky-Golay algorithm.

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3. Results and discussion

3.1. Turbidity measurements To assess the effect of ethanol (EtOH) on the interaction between protein molecules in water-EtOH mixtures, turbidity of a set of solutions was measured for both human (HSA) and bovine (BSA) serum albumins (Fig. SI1). Based on these results we conclude that the aggregation process of HSA and BSA in water-EtOH mixtures starts at [EtOH]HSAagg = 40% (v/v) and at [EtOH]BSAagg = 45% (v/v), respectively. At these concentrations 7

ACCEPTED MANUSCRIPT intermolecular beta-structures formation starts, leading to the increase of OD400 due to light scattering that is in agreement with the literature [14]. Since the investigation of the aggregation process was out of the scope of this paper, all the experiments were performed at [EtOH] concentrations lower than 40% (v/v).

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3.2. Steady-state fluorescence of tryptophan (Trp) and tyrosine (Tyr) residues in BSA and HSA

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The dependences of tryptophan (Trp) fluorescence intensity on EtOH concentration obtained

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upon 295 nm excitation for HSA and BSA are presented in Fig. 1 a. For both proteins no

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alterations in fluorescence intensity take place up to [EtOH] = 15% (v/v), suggesting that the conformational changes of protein structures (if any) take place far from Trp residues in BSA

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and HSA, e.g. in domain III. As EtOH concentration increases the Trp fluorescence of homologous proteins exhibits different behaviors: the intensity for HSA increases while that for

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BSA drops down. The increase of Trp214 fluorescence in HSA, which is located in domain II of

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the protein, could be supposed to be due to a reduction of static fluorescence quenching by neighboring groups that takes place in the native protein structure [32]. The same removal of

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static quenching should occur for Trp213 in BSA as its local environment is similar to that of Trp214 in HSA [22-23]. Hence, the decrease of Trp fluorescence in BSA could be a consequence

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of conformational changes in the vicinity of Trp134, which is located in domain I and characterized by the lager absorption cross-section and lager fluorescence lifetime [33] and thus has the lager contribution to fluorescence intensity. At low EtOH concentrations ([EtOH]<15% (v/v)) the position of maximum of Trp fluorescence of the investigated proteins hardly changes while at higher concentrations ([EtOH]>15% (v/v)) the blue shift occurs and at [EtOH]=30% (v/v) the position of maximum for HSA and BSA are almost equal (Fig. SI2). The position of maximum of Trp fluorescence depends on the projection of the local electric field on the long axis of the amino acid side-chain: the lager the projection, 8

ACCEPTED MANUSCRIPT the lesser the blue shift [34-36]. As addition of EtOH to aqueous solution leads to a decrease of the bulk dielectric constant and polarity and the electric field in the vicinity of Trp residues couldn’t decrease due to a change in dielectric constant of bulk media, we suppose that the blue shift of fluorescence spectra is caused by rearrangements of the first hydration sphere and/or by

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protein conformational changes.

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Fig. 1. (a) Maximum of fluorescence intensity of Trp in HSA (red circles) and in BSA (black squares) as a function of EtOH concentration, λexc = 295 nm. (b) Maximum of fluorescence intensity of Tyr in HSA (red circles) and in BSA (black squares) as a function of EtOH concentration, λexc = 280 nm. HSA and BSA concentrations were 4.1 µM and 4.7 µM, respectively. Maximum fluorescence intensity was normalized to the value obtained at [EtOH] = 0% (v/v)

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ACCEPTED MANUSCRIPT The normalized fluorescence intensity of tyrosine (Tyr) obtained upon 280 nm excitation for HSA and BSA as a function of EtOH concentration is presented in Fig. 1 b. For both proteins the dependence of Tyr fluorescence was similar, suggesting similarity of conformational changes of the investigated proteins. At low EtOH concentrations ([EtOH] < 15% (v/v)) the intensity of Trp fluorescence hardly changes for both BSA and

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HSA, thus Tyr fluorescence increase reveals the conformational changes of protein

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molecules far from Trp residues, e.g. in domain III.

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3.3. Fluorescence lifetimes of tryptophan (Trp) and tyrosine (Tyr) residues in BSA and

Fluorescence

decay

curves

for

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HSA BSA

were

measured

using

a

custom-built

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spectrofluorimeter upon excitation at λexc = 280 nm [26]. We recently showed that the analysis of decay traces at short wavelengths (λem<320 nm) allows to obtain Tyr

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fluorescence lifetime [26]. The channel of the photomultiplier system which

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corresponded to the wavelength λemTyr ~ 318 nm was a good compromise between the proximity to the position of the maximum of Tyr fluorescence (ca. 308 nm) and a decrease of the signal/noise ratio of the detector. The channel of the photomultiplier

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system which corresponded to the wavelength λemTrp ~ 355 nm was chosen as the closest

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to the fluorescence emission maximum of the native protein. At longer wavelengths (λem > 340 nm) only Trp residues fluoresce significantly (Fig. SI3), thus the decay traces at λemTrp are solely determined by fluorescence relaxation of Trp residues. The minimal residual values for decay traces recorded at λemTrp were achieved by fitting using a sum of two exponents. It is well-known that fluorescence decay of free Trp in solution as well as that of Trp in proteins is biexponential [28, 32, 37-44]. As the analysis of biexponential nature of Trp fluorescence is out of the scope of this paper, we averaged the fluorescence lifetime for Trp residues using the following equation: 10

ACCEPTED MANUSCRIPT     i ai ,

(1)

i 1,2

where τi and ai are the lifetime and the corresponded normalized amplitude of the ith Trp fluorescence decay component, respectively. The minimal residual values for decay traces recorded at λemTyr were achieved by fitting

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using a sum of tree exponents. In [26] for the BSA-sodium dodecyl sulphate (SDS) complexes the shortest lifetime component (τ3) was attributed to Tyr residues because of

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three reasons. Firstly, the two longer lifetime components (τ1 and τ2) depended on SDS

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concentration similar to that for Trp obtained at λemTrp. Secondly, the values of τ1 and τ2 were nearly equal to that for Trp obtained at λemTrp. Thirdly, the dependence on SDS

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concentration for τ3 was very different from that for τ1 and τ2 [26]. The same situation was

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observed for BSA in water-EtOH mixtures. In Fig. 2 the comparison between fluorescence intensity and fluorescence lifetime for Trp and Tyr residues is presented. From these data one can conclude that the decrease of fluorescence intensity is the

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consequence of the decrease of fluorescence lifetime while the absorption cross-section hardly varies. The analysis of absorption spectra of BSA in water-EtOH mixtures using derivative spectroscopy [26] proved this hypothesis (data not shown). In other words, the

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coincidence between the dependencies of fluorescence intensity of Tyr obtained from

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spectra decomposition and fluorescence lifetime of Tyr obtained from the analysis of decay traces at λemTyr represents evidence that the τ3 could be attributed to Tyr relaxation.

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Fig. 2. Maximum of fluorescence intensity (black squares) and the fluorescence lifetime

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(red squares) for Trp (a) and Tyr (b) residues in BSA. λexc = 280 nm. BSA concentrations was 15 µM. Maximum fluorescence intensity was normalized to the value obtained at

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[EtOH] = 0% (v/v)

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The alterations in fluorescence parameters of Trp and Tyr residues could be caused by conformational changes of the protein molecule. To test this hypothesis fluorescence measurements with a hydrophobic dye Nile Red (NR) were performed.

3.4. Fluorescence Nile Red (NR) in water-EtOH mixtures in the presence and in the absence of BSA Nile Red is a dye that has a very low quantum yield in polar solvents as the nonfluorescent excited state with charge transfer is energy preferable [29]. NR non12

ACCEPTED MANUSCRIPT covalently binds to hydrophobic regions in protein structure, that results in increase of its quantum yield as well as a blue shift of position of its emission maximum [30]. The positions of fluorescence maximum of free NR in water and in EtOH are 665 nm and 655 nm, respectively, while that of NR bound to HSA is 630 nm (Fig. SI4) [45-46]. Fluorescence properties of NR bound to a protein could provide information about the

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dynamics of hydrophobic parts of macromolecule due to ligand binding, intermediate formation or denaturation [30]. In the present work steady-state and time-resolved

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fluorescence measurements were performed to investigate the changes in the local

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environment of NR upon addition of EtOH in the case of the dye bound to BSA as well as in the case of free dye in solution. For free NR in water-EtOH mixtures a gradual blue

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shift of the position of fluorescence intensity maximum from 665 nm to 655 nm was

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observed accompanied by monotonous increase of intensity by ~12 times (Fig. SI4). On the contrary, for NR bound to BSA red shift (25 nm) of the position of fluorescence

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intensity maximum accompanied by a monotonous decrease of intensity by ~2.4 times

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occurs up to [EtOH] = 25% (v/v), and after that the fluorescence intensity increases by ~2 times with a slight blue shift (5 nm) (Fig. 3). It should be noted that steady-state fluorescence spectra for NR bound to BSA have at least two components while for free

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dye there is no any structure of the spectra. This fact could be a consequence of the

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presence of at least two different environments of NR bound in protein structure [47-48].

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Fig. 3. Averaged fluorescence lifetime for NR bound to BSA (black squares) and

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fluorescence lifetime of free NR (red circles) at registration wavelengths λ1 = 617 nm (closed symbols) and λ2 = 655 nm (open symbols) as a function of EtOH concentration,

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λexc = 510 nm. BSA and NR concentrations were 15 µM and 5.6 µM, respectively

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The presence of the two different environments of NR can be deduced from the analysis of fluorescence decay curves obtained at λ1 = 617 nm and λ2 = 655 nm. The latter

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wavelength corresponds to the maximum of fluorescence of NR bound to BSA at [EtOH]

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= 40% (v/v) and that of free NR in water-EtOH mixture at the same EtOH concentration, thus lifetime(s) obtained from fluorescence decay traces at 655 nm could provide

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information about the exposure of NR to solvent and/or about its release from BSA due to conformational changes of the protein. Another wavelength is at the blue edge of

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fluorescence spectrum of NR bound to BSA, where the contribution of the exposed dye is minimal.

If the environment of NR molecules was homogeneous, fluorescence decay traces should be monoexponential with the lifetime independent on the registration wavelength [49]. In our experiments this situation was held for free NR in water-EtOH mixtures (red symbols in Fig. 3). The increase of the fluorescence lifetime for free NR in water-EtOH mixtures coincides that of fluorescence intensity. This fact is a consequence of the decrease of the polarity of solvent with the increase of EtOH concentration [50-51]. 14

ACCEPTED MANUSCRIPT In the case of NR bound to BSA (black symbols in Fig. 3) the fluorescence decay traces were biexponential. The biexponential character of fluorescence decay traces of NR incorporated in proteins is usually explained by the presence of two different environments of the dye [47-49, 52]. As EtOH concentration increases up to [EtOH] = 25% (v/v) the higher lifetime for both registration wavelengths is nearly constant while its

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relative amplitudes decreases almost fourfold revealing conformational changes of BSA (Table SI1, SI2). A similar change of amplitudes of lifetime components of NR bound to

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proteins was obtained under protein denaturation by urea, guanidine hydrochloride

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(GndHCl) and heat [48-49]. In this paper we used NR as an effective probe to detect the presence of BSA conformational changes, thus we averaged the fluorescence lifetimes

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using the equation (1) similarly to the case of Trp fluorescence decay.

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Fig. 3 shows that the averaged lifetimes of NR bound to BSA don’t depend on the registration wavelength for the native protein. As the EtOH concentration increases up to

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[EtOH] = 12% (v/v) the lifetime obtained at the blue edge of the fluorescence spectra (λ1)

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falls down while the other lifetime obtained at the red edge of the fluorescence spectra (λ2) is almost constant. At high EtOH concentrations the averaged lifetimes become close to the lifetime of free NR in water-EtOH mixtures. Thus, conformational changes of BSA

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structure upon addition of EtOH lead to homogenization of the microenvironment of NR

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and to its exposure to the solvent. For characterization of conformational changes of BSA in water-EtOH mixtures the phase diagram approach was applied to fluorescence parameters of NR as well as to that of Tyr and Trp. The results of this analysis are presented in the next section (3.5).

3.5. Intermediate formation upon addition of EtOH monitored by phase diagram technique Construction of phase diagrams is one of the effective tools to monitor conformational changes in proteins. This technique is based on 2D-plot of two parameters of investigated 15

ACCEPTED MANUSCRIPT system which have a common origin. This approach was introduced by Burstein [53] and is commonly used to determine the presence of intermediates between native and unfolded states [54-59]. For instance, in case of Trp fluorescence the phase diagram could be the plot of fluorescence intensity at 365 nm versus the fluorescence intensity at 320 nm, which are indicative of changes in protein conformation [56]. Linear parts of phase

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diagram reflect all-or-none transitions of a protein structure. Strictly speaking, some intermediate states couldn’t be detected by this approach if the point corresponding to the

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intermediate lies on the line between the previous and the next state of the protein.

presence of distinct intermediate species.

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However, nonlinearity of the phase diagram is considered to be the evidence of the

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Fig. 4 shows the phase diagrams for the BSA-EtOH system built using extrinsic NR

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(Fig. 4 a) and intrinsic (Fig. 4 b) fluorescence. The presence of at least of three states can

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be deduced from these data. Fig. 5 shows CD spectra for these three states.

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Fig. 4. (a) Phase diagrams representing changes in microenvironment of NR bound to BSA

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(black circles) and free NR in water-EtOH mixtures (red squares) induced by EtOH. BSA and NR concentrations were 15 µM and 5.6 µM, respectively. (b) Phase diagrams representing

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conformational changes (N → I1) of BSA (black squares) and HSA (red circles) changes based on intrinsic fluorescence data. Maximum fluorescence intensity was normalized to the value

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obtained at [EtOH] = 0% (v/v). BSA and HSA concentrations were 4.7 µM and 4.1 µM, respectively.

In the case of extrinsic fluorescence, we used the average fluorescence lifetimes of NR an λ1 and λ2 to obtain the phase diagram of albumin transitions. As a control, the phase diagram for free NR in water-EtOH mixtures was obtained. As seen in Fig. 4 a there are no inflection points in this case, hence, polarity of NR microenvironment decreases gradually that is in accordance with the literature [50-51]. In the presence of BSA the first 17

ACCEPTED MANUSCRIPT inflection of phase diagram for NR occur at [EtOH] = 15 % (v/v). Up to this concentration the Trp fluorescence hardly changes while that of Tyr increases. CD spectra of I1 shows partial denaturation of BSA in accordance with the literature [14]. Hence, the formation of the first intermediate couldn’t be detected solely by means of Trp fluorescence, probably because the corresponding conformational changes are located far

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from Trp residues, e.g. in domain III. The next inflection of phase diagrams for both extrinsic and intrinsic fluorescence coincides again and occurs at [EtOH] = 35% (v/v).

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CD spectrum at this EtOH concentration shows the restoration of the secondary structure

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of native protein. However, the turbidity data show the start of the aggregation process,

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regarded with a certain precaution.

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hence, the point on the phase diagram corresponding to the second intermediate should be

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Fig. 5. CD spectra of BSA in PBS in the absence (black) and in the presence of EtOH ([EtOH] = 15% (v/v) – I1, blue line, [EtOH] = 35% (v/v) – I2, red line). BSA concentration was 0.76 µM.

3.6. The influence of conformational changes in domain III on tyrosine fluorescence The results presented above indicate that conformational changes of BSA and HSA upon addition of EtOH are locate far from Trp residues because Tyr fluorescence is sensitive to alterations in protein structure while that of Trp remains unchanged. One of the possible locations of these conformational changes could be the domain III which has four Tyr 18

ACCEPTED MANUSCRIPT residues in its structure and no Trp residues [22-23]. To investigate the sensitivity of Tyr fluorescence to conformational changes of domain III a direct denaturation of this domain was induced by guanidine hydrochloride (GndHCl) [60]. As it was shown by Ahmad and co-workers [60] the domain III of HSA is the most labile to GndHCl denaturation while the overall structure of other domains remained unchanged up to [GndHCl] = 1.4 M.

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Fig. 6 shows phase diagrams based on the intrinsic fluorescence of BSA and HSA. The presented data indicates that denaturation of serum albumins occurs in a sequential

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manner. During the first stage (up to [GndHCl] = 0.3 M) Trp fluorescence intensity of

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BSA slightly increases while that of HSA remains unchanged. During this stage the compactness of the protein structure increases due to the formation of alpha-structures in

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the domain I in the vicinity of Trp134 [46, 60]. During the next stage ([GndHCl] = 0.3 -

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0.9 M) corresponding to conformational changes in domain III [60] the Trp fluorescence only slightly decreases for both proteins while that of Tyr continues to increase. Further addition of GndHCl leads to significant conformational changes of the protein structure,

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namely, in this concentration range ([GndHCl] = 0.9 – 2.3 M) the disruption of the structures of domains I and II starts and the exposure of Trp residues increases. The analysis of the phase diagrams (Fig. 6) presented in this section shows that Tyr

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fluorescence increase is more pronounced than the alterations in Trp fluorescence during

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conformational changes in protein structure, particularly in domain III.

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Fig. 6. Phase diagrams representing conformational changes (N → I1 → I2 → I3) of BSA (a) and

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HSA (b) changes based on intrinsic fluorescence data. Maximum fluorescence intensity was normalized to the value obtained at [GndHCl] = 0. BSA and HSA concentrations were 4.0 µM

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and 4.3 µM, respectively.

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4. Conclusions

Collectively, our data suggest that the initial conformational changes of albumin induced by EtOH are located in domain III, which lacks Trp residues both in BSA and HSA. Interestingly, the presence of four Tyr residues in domain III makes Tyr fluorescence a sensitive indicator of rearrangements in this domain as seen in the case of EtOH and verified in the experiment with GdnHCl. As shown previously by the example of albumins interaction with SDS [26], Tyr fluorescence could be indicative of local rearrangements of protein structure that could possibly find applications in the assessment 20

ACCEPTED MANUSCRIPT of proteins conformational changes in biofluids [27]. Moreover, the introduced procedure for Tyr lifetime determination allows the use of phase diagrams in the Tyr-Trp lifetimes coordinates, that extends the capabilities of intrinsic fluorescence in monitoring structural transitions.

Acknowledgements

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The reported study was supported by Russian Foundation of Basic Research (grant №16-

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32-00804) and Russian Science Foundation (grant №14-15-00602).

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Graphical abstract

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Highlights  Tyr fluorescence was used to monitor conformation of albumin with EtOH and GdnHCl  Tyr-Trp fluorescence diagram reveals intermediates in protein folding  Tyrosine fluorescence is sensitive to changes in domain III of albumin

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