Article pubs.acs.org/Langmuir
Direct Evidence of Intrinsic Blue Fluorescence from Oligomeric Interfaces of Human Serum Albumin Arpan Bhattacharya,† Soumitra Bhowmik,† Amit K. Singh,‡ Prashant Kodgire,‡ Apurba K. Das,*,† and Tushar Kanti Mukherjee*,† †
Discipline of Chemistry, Indian Institute of Technology Indore, Khandwa Road, Indore 453552, Madhya Pradesh, India Centre of Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Khandwa Road, Indore 453552, Madhya Pradesh, India
‡
S Supporting Information *
ABSTRACT: The molecular origin behind the concentration-dependent intrinsic blue fluorescence of human serum albumin (HSA) is not known yet. This unusual blue fluorescence is believed to be a characteristic feature of amyloid-like fibrils of protein/peptide and originates due to the delocalization of peptide bond electrons through the extended hydrogen bond networks of cross-β-sheet structure. Herein, by combining the results of spectroscopy, size exclusion chromatography, native gel electrophoresis, and confocal microscopy, we have shown that the intrinsic blue fluorescence of HSA exclusively originates from oligomeric interfaces devoid of any amyloid-like fibrillar structure. Our study suggests that this low energy fluorescence band is not due to any particular residue/sequence, but rather it is a common feature of selfassembled peptide bonds. The present findings of intrinsic blue fluorescence from oligomeric interfaces pave the way for future applications of this unique visual phenomenon for early stage detection of various protein aggregation related human diseases.
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responsible for their intrinsic blue fluorescence.15 In another work, Sharpe et al. have studied the blue autofluorescence from the amyloid-like cross-β-sheet structure of an octapeptide, GVGVAGVG by using FTIR and solid-state NMR spectroscopy and suggested that extensive delocalization of peptide bond electrons through the hydrogen bond networks of cross-β-sheet structure is the reason behind the origin of intrinsic blue autofluorescence.16 Similarly, Amdursky et al. have reported blue luminescence from dipeptides, H-Phe-Phe-OH, and highlighted the importance of quantum confinement effect of the self-assembled peptide nanotubes.17 The phenomenon has also been observed from protein amyloids that have been associated with various diseases.19 More recently, Pinotsi et al. have demonstrated a detailed study on the structure-specific visible fluorescence in hydrogen bond-rich Aβ1−42 and Aβ33−42 peptides by combining ab initio molecular dynamics simulations and fluorescence spectroscopy.25 It has been shown that the low energy excitation and visible fluorescence from small protein fibrils is directly related with the charge delocalization along with proton transfer across the hydrogen bond networks. In particular, their experimental results show that the proton transfer is directly coupled to the intrinsic visible fluorescence of these peptide sequences.25 Moreover,
INTRODUCTION The intrinsic fluorescence of proteins lacking any fluorescent prosthetic groups appears in the wavelength range of 305−355 nm due to the presence of aromatic amino acids (Trp, Phe, and Tyr). This intrinsic fluorescence by virtue of its high sensitivity finds enormous application in biophysical research. It has been extensively used to understand various protein−ligand interactions1−5 as well as folding and unfolding kinetics of various proteins.6−12 Recently, another kind of intrinsic blue fluorescence has been observed from protein crystals and fibril-forming peptides.13−26 The phenomenon is unusual as individual amino acids do not show fluorescence in the visible region of the electromagnetic spectrum. Earlier, Shukla et al. have first observed this blue fluorescence from protein crystals and large polypeptides in solid and solution phase.13 It has been proposed that this low energy electronic transition is not owing to aromatic side chains but, rather, due to delocalization of peptide electrons through intramolecular or intermolecular hydrogen bond networks. Notably, early attempts to explain protein activity highlighted the role of water mediated proton transfer mechanism from the standard peptide structure to a peptenol form.14 Later, several groups have reported similar intrinsic blue fluorescence from fibril forming short peptide sequences. For example, Mercato et al. have observed intrinsic blue-green fluorescence from elastinrelated polypeptide and proposed that charge transport through the hydrogen bond networks of self-assembled nanofibrils is © 2017 American Chemical Society
Received: July 15, 2017 Revised: September 7, 2017 Published: September 20, 2017 10606
DOI: 10.1021/acs.langmuir.7b02463 Langmuir 2017, 33, 10606−10615
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Scheme 1. (A) Representation of Native Human Serum Albumin Structurea and (B) Schematic Representation of Fibril Formation via Intermediate Oligomers
a
The crystal structure of HSA is obtained from the Protein Data Bank (http://www.rcsb.org./pdb).
exclusion chromatography, gel electrophoresis, electron microscopy, and confocal microscopy, we have shown that the blue fluorescence exclusively appears from the β-sheet rich oligomeric interfaces of HSA. To the best of our knowledge, this is the first report to show the intrinsic blue fluorescence from oligomeric interfaces of protein.
recent computational studies have highlighted the importance of nuclear quantum effects on the photophysical properties of several hydrogen-bonded systems.27−32 Nuclear quantum effects result in significant delocalization of protons along hydrogen bonds which can result in shifts and significant broadening of the optical spectra. Although, it is believed that this intrinsic blue fluorescence is a generic phenomenon of amyloid-like fibrillar structure of proteins and peptides, the origin behind the concentration-dependent intrinsic blue fluorescence of native human serum albumin (HSA) at physiological conditions is unknown. HSA is the most abundant protein in the blood plasma and performs various important physiological tasks.33,34 It contains a single polypeptide chain and consists of 585 amino acids with a molecular weight of ∼66.5 kDa. The secondary structure consists of ∼67% of α-helix and 17 disulfide bonds (Scheme 1A). Previously, by using spectroscopic techniques, we have demonstrated that HSA undergoes concentration-dependent reversible self-oligomerization as a result of soft protein− protein interactions. 35 It has been observed that the physicochemical properties and secondary structure of these self-associated oligomeric forms of HSA differ significantly from that of monomeric protein. It is known that irreversible aggregation of proteins proceeds with the formation of soluble oligomers, which subsequently form insoluble toxic amyloidlike fibrils (Scheme 1B) and may lead to various kinds of human diseases.36−38 Moreover, early reports suggest that the toxicity of Aβ and other amyloidogenic proteins arises mainly from the early oligomeric intermediates rather than insoluble fibrils.39,40 For example, by using oligomer-specific antibody, Kayed et al. have demonstrated the inhibition of in vitro toxicity of soluble oligomers of a wide variety of amyloidogenic proteins and peptides and proposed that the oligomer-specific antibody recognizes a unique common structural feature of the peptide backbone in the oligomers that is independent of the amino acid side chains.39 Similarly, Bucciantini et al. have previously reported that the soluble oligomers formed from nondisease related proteins are more cytotoxic than their mature fibrils due to the easy accessibility of the hydrophobic side-chains and other regions of the polypeptide chain.40 These reports clearly establish the fact that the early oligomers are the primary toxic species and they may have a common mechanism of pathogenesis. Therefore, facile detection and isolation of initial oligomers in the aggregation pathway of protein is of significant biological importance. In the present study, we have demonstrated facile and noninvasive visual detection of the oligomeric forms of HSA in a label-free manner by monitoring their intrinsic blue fluorescence. The main focus of the present work is to uncover the true origin behind the blue fluorescence of HSA. By combining the results of spectroscopy, size
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EXPERIMENTAL SECTION
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METHODS
Materials. Human serum albumin (HSA, lyophilized powder, essentially fatty acid-free), N,N,N′,N′-tetramethylethylenediamine (TEMED, ∼99%), acrylamide (≥99%), and ammonium persulfate (APS, ≥99%) were purchased from Sigma-Aldrich. Glycerol (≥99%), bromophenol blue, bis-acrylamide, and glacial acetic acid were purchased from MP Biomedical. Succinic anhydride was purchased from SDFCL, L-leucine, L-phenylalanine, L-tryptophan, L-tyrosine, HOBt (1-hydroxybenzotriazole), DCC (N,N′-dicyclohexylcarbodiimide) were purchased from SRL. Coomassie brilliant blue, methanol (≥99%), N,N-dimethylformamide (DMF, ≥99%), ethyl acetate, and hexane were purchased from Merck, India. Milli-Q water was obtained from a Millipore water purifier system (Milli-Q Integral). Synthesis of Peptide Bolaamphiphiles. Peptide bolaamphiphiles (i−iv) were synthesized by solution phase methodology (Scheme S1).41−43 For chemical reactions and purification of peptides, methanol, N,N-dimethylformamide, ethyl acetate, and hexane were dried according to the literature reports.41−43 Reactions were monitored by thin-layer chromatography (TLC) on Merck silica gel 60-F254 plates. Visualization was attained on TLC under UV light. All of the peptides were synthesized by conventional solution phase fragment condensation methodology using HOBt and DCC as antiracemization and coupling reagents, respectively. Hydrolysis of methyl ester was performed by saponification. All intermediates and final compounds were purified and well characterized by FT-IR, 1H NMR (400 MHz), 13C NMR (100 MHz), and mass spectral studies.41−43 Hydrogel Preparation. For gelation of peptide bolaamphiphiles i, ii, iii, and iv, 20 mmol L−1 of peptide bolaamphiphiles were suspended and dispersed in 1 mL of pH 8 solution using a vortex for 15 min. Then 0.1 N HCl was added drop by drop until the pH reached 5.5−6. Peptide bolaamphiphiles i and iii were capable of forming stable, selfsupporting hydrogels at room temperature, whereas bolaamphiphiles ii and iv were unable to form a gel under similar conditions. These hydrogels are stable over a wide range of pH (4−8) for more than three months at room temperature.
NMR Spectroscopy. NMR spectra of peptide bolaamphiphiles were recorded on a Bruker AV 400 MHz spectrometer at 298 K.41−43 The concentrations of all of the peptides were in the range of 1−10 mmol L−1 in CDCl3 and DMSO-d6 for 1H and 13C NMR. Chemical shifts were expressed in parts per million (ppm, δ) relative to residual solvent protons as internal standards (CHCl3: δ 7.26, DMSO: 2.50 for 1H NMR, CHCl3: δ 77.00, DMSO: 39.50 for 13C).41−43 10607
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Figure 1. (A) Changes in the fluorescence spectra (λex = 375 nm) of HSA as a function of concentrations (2−500 μM) at 450 nm emission wavelength. Inset show the photographs of HSA solutions upon UV illumination (λex = 365 nm) with different concentrations. (B) Native PAGE of HSA in the concentration range of 2 to 150 μM; lane 1: 2 μM; lane 2: 5 μM; lane 3: 8 μM; lane 4: 50 μM; lane 5: 100 μM; lane 6: 150 μM. (C) Correlation plot of fluorescence intensity (λex = 375 nm) of HSA at 450 nm with ThT fluorescence (λem = 482 nm) as a function of concentrations of HSA. (D) Comparison of ThT fluorescence enhancement upon binding with different HSA samples monitored at 482 nm emission wavelength. Concentration of ThT used is 10 μM.
Mass Spectrometry. Mass spectra were recorded on a Bruker micrOTOF-Q II by positive mode electrospray ionization using acetonitrile as a solvent.41−43 Rheology. Oscillating rheology of peptide bolaamphiphile hydrogels was performed on a Paar Physica Modular Compact Rheometer (MCR 301, Austria). A 20 mmol L−1 solution of each peptide bolaamphiphile hydrogel was prepared for the measurements. A 25 mm cone plate with 1° angle configuration was used, and the temperature was set to be constant at 25 °C. Storage (G′) and loss (G″) moduli were measured at 0.1% strain with a true gap of 0.05 mm. To determine the exact strain for the frequency sweep experiment, the linear viscoelastic (LVE) regime was performed at a constant frequency of 10 rad s−1. FTIR Spectroscopy. FTIR spectra were recorded using a Bruker (Tensor-27) FTIR spectrophotometer. Measurements were done with KBr pellet techniques; the dried samples were placed in between two KBr pellets in a sandwich manner and scanned between 500 and 4000 cm−1. CD Spectroscopy. Circular dichroism (CD) spectra were recorded on a JASCO J-815 CD spectropolarimeter using a quartz cell of 1 mm path length. Scans were made with a slit width of 1 mm and speed of 20 nm/min. The far-UV CD spectra were analyzed using CDNN software. UV−vis Spectroscopy. Absorption spectra were recorded in a quartz cuvette (10 mm × 10 mm) using a Varian UV−vis spectrophotometer (Carry 100 Bio). Fluorescence Spectroscopy. The concentrations of HSA samples were determined spectrophotometrically by using molar extinction coefficient of 35 495 M−1 cm−1 at 280 nm44 and dissolving the required amount of HSA (MW = 66 437 g mol−1) in pH 7.4 PBS. The fluorescence spectra were recorded in a quartz cuvette (10 × 10 mm2) using Fluoromax-4
Spectrofluorimeter (HORIBA Jobin Yvon, model FM-100) with excitation and emission slit widths at 2 nm. The pH of the HSA solutions for fluorescence measurements was adjusted by adding aqueous HCl or NaOH using a pH meter (Eutech Instruments). Time-Correlated Single Photon Counting (TCSPC). Fluorescence decays were recorded on an HORIBA Jobin Yvon picosecond time-correlated single photon counting (TCSPC) spectrometer (model Fluorocube-01-NL). The samples were excited at 376 and 445 nm by picosecond diode laser (model Pico Brite-375L and DD-450L). The decays were collected with the emission polarizer at a magic angle of 54.7° by a photomultiplier tube (TBX-07C). The instrument response functions (IRF, fwhm ∼140 ps for 375 nm, and ∼160 ps for 445 nm) were recorded using a Ludox solution. The fluorescence decays were analyzed using IBH DAS 6.0 software by the iterative reconvolution method, and the goodness of the fit was judged by reduced χ-square (χ2) value. The decays were fitted, and decay components were analyzed by following earlier literature.45,46 Scanning Electron Microscopy (SEM). The SEM images were recorded by using a field-emission scanning electron microscope (FE-SEM), Supra 55 Zeiss. Peptide solutions were prepared by mixing required amount of peptides in 0.5 mL of methanol in individual vials. Then, the required volume of Milli-Q water was added to 0.5 mL of peptide solutions to make the final peptide concentration to 10 mmol L−1 and allowed to stand at room temperature. For SEM measurements, hydrogels (20 mmol L−1) and peptide solutions (10 mmol L−1) were drop cast on a cleaned glass slide and dried overnight in a desiccator. These dried samples were coated with gold. HSA solutions were prepared by adding required amount of HSA (MW= 66 437 g mol−1) at PBS buffer at pH 7.4. HSA fibrils 10608
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recorded the excitation spectrum as a function of emission wavelengths. The excitation spectrum of the 340 nm emission band shows a single peak at 295 nm which is close to its absorption maximum (Figure S2B of the SI). However, excitation spectrum of the visible fluorescence band reveals the appearance of a new excitation band at 350 nm, which red shifts and broadens with an increase in the emission wavelength of HSA (Figure S2B of the SI). These observations clearly indicate that the progressive shift of the fluorescence maxima to a longer wavelength is due to the presence of various groundstate species that absorb light at and above 350 nm and emit at different wavelengths in the visible region of the electromagnetic spectrum.35 Interestingly, a similar kind of excitation peak at ∼340 nm has been observed earlier for other protein aggregates and polypeptides that exhibit intrinsic blue fluorescence.13−26 These results are in accordance with our previous observations and indicate the presence of various groundstate emissive species at high protein concentration.35 To know the nature of these emissive species at high protein concentration, we have performed native polyacrylamide gel electrophoresis (PAGE). Figure 1B shows the native PAGE of HSA in the concentration range of 2−150 μM. It is evident that, in the concentration range of 2−8 μM, a band appears at 66.5 kDa which corresponds to the monomeric protein and a very weak band at 133 kDa appears due to the presence of a negligible amount of dimers. However, multiple intense bands have been observed in the concentration range of 50−150 μM and can be assigned to dimers (133 kDa), trimers (200 kDa), tetramers (266 kDa), and higher order oligomers of HSA. These results indicate that the observed concentrationdependent blue fluorescence of HSA may be related with the oligomerization process and conformationally modified oligomers may be responsible for the blue emission. Notably, a good linear correlation (Pearson’s r = 0.9988) has been observed between the appearance of the blue fluorescence of HSA and fluorescence enhancement of thioflavin T (ThT) as a function of HSA concentrations (Figure 1C). ThT shows enhanced fluorescence upon binding with β-sheet rich structures.26,35,49−51 In the absence of HSA, ThT exhibits a very weak fluorescence band centered at 482 nm upon excitation at 412 nm in phosphate buffer (pH 7.4).35 While negligible enhancement in the fluorescence of ThT has been observed in the presence of 2 μM HSA, ∼26-fold enhancement has been observed in the presence of 150 μM HSA, suggesting the formation of β-sheet rich oligomeric interfaces at high HSA concentration (Figure 1D). Here, it should be noted that the fluorescence contribution of HSA at 482 nm emission wavelength was subtracted from the total fluorescence. Notably, ThT shows ∼90-fold enhancement in its fluorescence intensity upon binding with heat denatured HSA (150 μM HSA heated to 348 K), indicating more extensive β-sheet rich structural arrangements (Figure 1D). These observations correlate well with the far-UV CD and FTIR results. Far-UV CD measurements have been performed to estimate the β-sheet contents of monomers, oligomers, and fibrils of HSA. Here, it is important to mention that at high protein concentration (150 μM) the CD signal saturates and hence for the present study we have compared the secondary structure contents of 2 and 15 μM HSA samples. The far-UV CD spectra were analyzed using CDNN software. The CD spectrum of native HSA (2 μM) shows two characteristic minima at 208 and 222 nm (Figure S3 of the SI). Secondary structural analysis revealed a majority of
were prepared by heating at 348 K for 24 h. Then, the heat denatured protein sample was spin-cast with a spin-coater (Apex Instruments Spin NXG-P1) on a clean glass cover slide at 2000 rpm for 3 min and then dried in a desiccator overnight. The dried HSA sample was finally coated with gold. The coverslips and glass slides were first cleaned with 2% Hellmanex III (Sigma-Aldrich) and then with chromic acid. Each of these cleaning steps was followed by repeated washing with Milli-Q water. Finally, these washed slides were rinsed with methanol (Sigma-Aldrich) and dried in vacuum oven. Confocal Microscopy. The images were recorded with an inverted confocal microscope (Olympus, model no. FV1200MPE, IX-83) using an oil immersion objective (100× 1.4 NA). A 405 nm diode laser source was used to excite the samples using appropriate dichroic and filters in the optical path. The samples were immobilized on a cleaned cover slide by spin-casting at 2000 rpm for 3 min and fixed on a cleaned glass slide in a sandwich manner by using transparent nail polish before imaging. All of the experiments were performed in PBS buffer at pH 7.4. Size Exclusion Chromatography (SEC). The chromatographic system used was an AKTA protein purification system (GE Healthcare) with Sephacryl S-300 gel filtration column. The mobile phase was 1X PBS at pH 7.4. Prior to use this was filtered with 0.45 μm (pore size) filters (Millipore) and degassed. HSA was dissolved to initial concentration of 1 mM in 1X PBS. Fractions of proteins (monomer and oligomer) were collected, and the protein concentration was checked by measuring the optical density at 280 nm using a Nanodrop 2000 UV−vis spectrophotometer (Thermo Scientific). The collected fractions were analyzed by native polyacrylamide gel electrophoresis (PAGE). Native PAGE. Equal amounts of protein samples were mixed with 2X sample buffer (1 M Tris, pH 7.5), glycerol (50%), and Bromophenol blue (0.5%) and incubated for 15 min at 37 °C in water bath and resolved in 5% native polyacrylamide stacking gel (30% acrylamide, Tris pH 7.5, 10% APS and TEMED) running at 55 V for 2 h and 12% native polyacrylamide separating gel (30% Acrylamide, Tris, pH 7.5, 10% APS and TEMED) by running at 90 V for 3 h using Mini Protean tetra cell system (Bio-Rad). Native PAGE gels were stained with Coomassie Brilliant Blue staining solution (Coomassie Brilliant Blue, methanol 40% v/v, glacial acetic acid 10% v/v and sterile H2O 50%) for overnight on a shaker and then destained with destaining solution (methanol 40% v/ v, glacial acetic acid 10% v/v, and sterile H2O 50%) for 3 times for 30 min each. Destained gels were then visualized in Image Quant LAS 4000 gel doc system (GE Healthcare).
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RESULTS AND DISCUSSIONS Concentration-Dependent Blue Fluorescence of Human Serum Albumin. The intrinsic blue fluorescence of HSA is concentration-dependent (Figure 1A). The fluorescence intensity at 450 nm grows slowly in the concentration range of 2−10 μM of HSA, and beyond that range, it increases sharply (Figure 1A and Figure S1of the Supporting Information (SI)), suggesting the involvement of some concentration-dependent process behind this blue fluorescence. Moreover, this intrinsic red-shifted fluorescence strongly depends on the excitation wavelength (λex; Figure S2A of the SI), which is similar to that commonly observed in quantum dot (QD) system due to heterogeneous size distribution.47,48 To know the electronic excitation associated with this visible fluorescence, we have 10609
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Figure 2. SEM images of (A) 500 μM native HSA and (B) heat denatured HSA samples. (C) Size exclusion chromatogram of 1 mM HSA sample. (D) Native PAGE analysis of fraction 1 and 2 collected from SEC experiment. (E) Photographs of fraction 1 and 2 in solution upon UV illumination.
α-helix of 65.1% with nominal β-sheet content of 6.8% (Table S1 of the SI). However, the α-helix content decreases to 58.3% with concomitant increase in the β-sheet content to 8.9% for 15 μM HSA sample (Figure S3 and Table S1 of the SI). Notably, the β-sheet content and random coil increases to 24.1 and 32.1%, respectively, for heat denatured HSA fibrils (15 μM HSA heated to 348 K; Figure S3 and Table S1 of the SI). FTIR results further confirm the alteration of the secondary structure of native HSA upon oligomer and fibril formation (Figure S4 of the SI). The amide I band of native HSA (2 μM) appears at 1651 cm−1 which shifts to 1648 and 1633 cm−1 upon formation of oligomers and fibrils, respectively. Similarly, amide II band position shifts to a lower frequency region upon formation of oligomers and fibrils (Figure S4 of the SI). These spectral changes indicate the presence of a β-sheet rich structural motif in oligomers and fibrils of HSA.52,53 Together, these results strongly suggest that the progressive shift of the fluorescence maxima to a longer wavelength with increase in the excitation wavelength is due to the presence of various emissive oligomers of HSA, and the phenomenon of blue fluorescence is closely related with the formation of β-sheet rich oligomeric interfaces. Here it is important to mention that recent simulation and experimental studies have also demonstrated the presence of various early oligomeric conformers of Aβ peptides with βsheet rich structural motif.39,40,54−56 Scanning Electron Microscopy (SEM). Scanning electron microscopy (SEM) has been performed to visualize the morphologies of these oligomers and heat denatured aggregates. SEM image of 500 μM HSA reveals the presence of amorphous aggregates with size in the range of ∼250−750 nm (Figure 2A) without any detectable protofibrils or fibrils in the whole substrate. The absence of any fibril-like morphology supports the obtained low binding affinity of ThT toward these oligomers. Notably, these aggregates are morphologically and
size-wise quite different from the monomeric protein with a mean size of ∼140 nm (Figure S5 of the SI). Figure 2B shows the SEM image of 500 μM HSA heated to 348 K. It is evident that HSA forms long fibrillar assemblies at 348 K with twisted filaments of ∼30−70 nm wide and up to several micrometers in length. Similar fibrils of serum albumin have also been observed previously at high temperature.57,58 Therefore, SEM images clearly reveal that the reversible oligomers of HSA are structurally quite different from that of nanofibrillar aggregates having extensive cross-β-sheet networks. Size Exclusion Chromatography (SEC). Next, we have performed size exclusion chromatography (SEC) to separate different oligomers from monomers of HSA. SEC has been performed with an initial protein concentration of 1 mM. The chromatogram shows two clear peaks due to the presence of monomers and different oligomers (Figure 2C). As the oligomerization process of HSA is concentration-dependent, the fraction collected at the monomer peak with an effective protein concentration of 35.3 μM shows the presence of oligomers in native PAGE (Figure S6 of the SI). In order to avoid high protein concentration in the eluted fractions, we have mainly focused on two factions collected at the shoulder of the two peaks, namely fractions 1 and 2 (Figure 2C). The native PAGE analysis of fraction 1, which is collected at the shoulder of the oligomer peak, shows an intense dimer band at 133 kDa with a noticeable amount of monomers at 66.5 kDa. Fraction 1 also contains a minor amount of higher order oligomers. In contrast, fraction 2, which is collected at the shoulder of the monomer peak, shows a single band at 66.5 kDa in native PAGE (Figure 2D). The photographs of fractions 1 and 2 in pH 7.4 PBS upon UV irradiation is shown in Figure 2E. It is evident that fraction 1 with the majority of dimers and higher order oligomers exhibits intense blue fluorescence while the same has not been observed from fraction 2. These 10610
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Figure 3. Confocal microscopy images of (A) 500 μM, (B) 2 μM, and (C) heat denatured 500 μM HSA samples. The left panel shows the bright field images, the right panel shows the fluorescence images, and the middle panel shows the merged images.
Figure 4. (A) Chemical structures of four peptide bolaamphiphiles. (B) Photographs of peptides in solution and gel phase upon UV illumination. (C) SEM images of four self-assembled peptides in the methanol−water mixture. (D) Schematic representation of hydrogen-bonded oligomeric interfaces of HSA.
observations correlate well with the fluorescence and excitation spectra of fractions 1 and 2 (Figure S7 of the SI). Hence, these results clearly establish the fact that the blue fluorescence of HSA exclusively appears from oligomers and not from monomeric units.
Confocal Microscopy. Laser scanning confocal microscopy has been performed to directly visualize this intrinsic fluorescence of HSA oligomers. Images were recorded upon excitation with a 405 nm diode laser, and the fluorescence was collected with an appropriate emission filter. Distinct blue 10611
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Table 1. Fluorescence Lifetime Components of 150 μM HSA, HSA Fibrils, Peptide i, ii, iii, and iv (λex = 375 nm, λem = 450 nm) sample
τ1 (ns)
a1
τ2 (ns)
a2
τ3 (ns)
a3
⟨τ⟩ (ns)
χ2
HSA HSA fibrils peptide i peptide ii peptide iii peptide iv
0.25 0.20 0.35 0.31 0.25 0.25
0.51 0.72 0.52 0.66 0.72 0.59
1.44 1.24 1.86 1.84 1.30 1.90
0.32 0.21 0.37 0.24 0.21 0.27
5.99 5.40 7.54 6.42 5.80 6.55
0.17 0.07 0.11 0.10 0.07 0.15
1.60 0.77 1.69 1.30 0.86 1.64
1.06 1.16 1.20 1.09 1.07 1.15
fluorescence has been observed from the 500 μM HSA sample, and this blue fluorescence colocalizes with the amorphous aggregates (Figure 3A). In contrast, no such intrinsic fluorescence has been observed from the dilute 2 μM HSA sample (Figure 3B). On the other hand, the heat denatured HSA sample shows clear fibril networks with distinct blue-green fluorescence (Figure 3C). The merged image shows that the blue-green fluorescence originates exclusively from the fibrillar networks. Here it is important to compare the color of the visible fluorescence that originates from oligomers and fibrils of HSA. While oligomers show distinct blue color, fibrils of HSA exhibit blue-green color. Hence, we can easily differentiate between oligomers and fibrils of HSA by monitoring their intrinsic visible fluorescence. Although similar intrinsic visible fluorescence has been observed earlier from protein crystals and self-assembled peptide fibrils,13−26 the same has not been observed from small protein oligomers. To the best of our knowledge, this is the first observation of intrinsic blue fluorescence from protein oligomers without any amyloid-like fibrillar structure. Photophysical Study of Self-Assembled Peptide Bolaamphiphiles. A key question that remains to be addressed is whether this intrinsic fluorescence originates from any particular aromatic residue (Trp, Phe, and Tyr) or it is due to the inherent structural feature of the oligomeric interfaces of HSA. To address this point, we have synthesized and studied the photophysical properties of four peptide bolaamphiphiles having different amino acid sequence, namely, (i) HO-Phe-Leu-Suc-Phe-Phe-OH, (ii) HO-Trp-Leu-Suc-LeuTrp-OH, (iii) HO-Tyr-Phe-Suc-Phe-Tyr-OH, and (iv) HOLeu-Leu-Suc-Leu-Leu-OH (Figure 4A and Scheme S1 of the SI). Although, several earlier reports in the literature have shown that the blue-green fluorescence from short amino acid residues is mainly due to the presence of an extended β-sheet rich fibrillar structure,13−26 the focus of our study with peptide bolaamphiphiles is to just mimic the oligomeric interfaces of HSA. Here it is important to note that peptide iv has been designed to selectively identify the role of aromatic residues behind the blue fluorescence. Out of these four peptide bolaamphiphiles, peptides i and iii form hydrogels in phosphate buffer. The mechanical properties of these hydrogels were characterized by the rheological study (Figure S8 of the SI).22 Interestingly, all four peptides exhibit intrinsic blue fluorescence in solution (10 mmol L−1) upon UV illumination (Figure 4B and Figure S9 of the SI). Similar intense blue fluorescence has also been observed from hydrogels of peptides i and iii (Figure 4B). These results suggest that the phenomenon is not related to any particular residue or peptide sequence; rather it may be related to some inherent structure that is common for these peptides and HSA oligomers. The SEM images of these peptides reveal distinct self-assembled morphologies (Figure 4C). While, peptides i and iii show fiber-like networks, peptides
ii and iv display uniform vesicle-like morphologies (Figure 4C). Hydrogels of peptides i and iii show dense fiber-like networks (Figure S10 of the SI). The observed sequence-dependent morphological arrangements of these peptides mainly arise as a consequence of different extent of noncovalent interactions in the self-assembled nanostructures. Here it is important to note that peptides i and iii contain the majority of aromatic residues which can take part in π−π stacking along with H-bonding interactions and lead to a compact fibrillar network. On the other hand, peptides ii and iv contain the majority of aliphatic Leu residues which favor vesicle-like molecular arrangements mainly due to the dominant hydrophobic interactions instead of π−π stacking interactions. In addition, solvent polarity and dielectric constant may also play an important role in these selfassembly processes.23 The secondary structure of these selfassembled peptides has been probed using Fourier transform infrared spectroscopy (FTIR). All four peptides show amide I and II bands in the range of 1634−1643 and 1533−1545 cm−1, respectively (Figure S11A of the SI). Similarly, the hydrogels of peptides i and iii show amide I and II bands in the range of 1633−1641 and 1534−1543 cm−1, respectively (Figure S11B of the SI). These spectral features reveal predominantly β-sheet conformation of these peptides in the self-assembled structures.16,18,26 Although the self-assembly process of these peptides is highly sequence-dependent, they all show the intrinsic blue fluorescence, suggesting that this phenomenon may be a generic and characteristic feature of self-assembled peptide bonds. Fluorescence lifetime measurements further support this argument. It is well-known that fluorescence lifetime is a very sensitive and characteristic photophysical parameter for a particular fluorophore.59 The fluorescence decay profile of HSA oligomers exhibits non single exponential decay kinetics having an average lifetime of 1.60 ns with three lifetime components of 0.25 (51%), 1.44 (32%), and 5.99 ns (17%; Table 1). In addition, the lifetime of this visible fluorescence of 150 μM HSA strongly depends on the excitation and emission wavelength (Table S2 of the SI), indicating the presence of various emissive species in the solution. This observation is similar to the excitation wavelength-dependent emission observed in our steady-state experiments. Hence, the observed non single exponential decay kinetics mainly originates due to the presence of different emissive oligomers such as dimer, trimer, and higher order oligomers of HSA in solution. Interestingly, HSA fibrils and all four peptide bolaamphiphiles (10 mmol L−1) exhibit similar decay kinetics as well as lifetime components (Table 1), suggesting that the blue fluorescence is a generic phenomenon of self-assembled peptide bonds. Moreover, a similar excitation and emission wavelengthdependent fluorescence lifetime has also been observed for HSA fibrils, signifying the presence of different aggregates (protofibrils) in the heat denatured HSA sample (Table S3 of the SI). In general, peptide electrons show an electronic 10612
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transition in the wavelength range of 190−220 nm due to n−π* and π−π* transitions. Notably, recent computational studies have shown that proton transfer across the hydrogen-bonded networks indeed lowers the electronic excitation energy.27−32 Moreover, previously it has been shown that nuclear quantum effects can further result in red shift and significant broadening of electronic absorption spectra of hydrogen-bonded systems via introducing new electronic levels in both the ground and excited state.27−32 Together, we propose that the red-shifted broad excitation peak observed at ∼350 nm from oligomers of HSA (Figure S2 of the SI) is due to the delocalization of peptide electrons through H-bonded networks at the oligomeric interfaces (Figure 4D). Here, it is important to comment on the β-sheet rich oligomeric interfaces of HSA. HSA contains 1 Trp, 18 Tyr, and 30 Phe residues along with other nonaromatic amino acids. While the single Trp residue (Trp214) is situated in the hydrophobic pocket of domain IIA, Tyr and Phe residues are distributed throughout the polypeptide chain.33,34 The oligomeric interfaces may contain several amino acids and stabilized by weak hydrogen bond and/or π−π stacking interactions (Figure 4D). The presence of Trp (Trp214) from domain IIA at the interface can be ruled out by considering its unchanged fluorescence spectrum upon oligomer formation (Figure S12 of the SI). However, we cannot rule out the presence of other aromatic and nonaromatic amino acids at the interface, as they are distributed throughout the polypeptide chain of HSA. Notably, the observed phenomenon of visible fluorescence remains unaltered at the isoelectric point (pI 4.7) of HSA (Figure S13 of the SI), suggesting that the phenomenon is independent of the overall surface charge. Moreover, it is known that HSA contains two principal ligand binding sites in the hydrophobic cavities of subdomain IIA and IIIA. Therefore, by considering the unchanged fluorescence peak position of Trp 214 which is situated at subdomain IIA, we believe that residues from domain III may be involved in the oligomeric interface formation through hydrophobic, H-bonding, and/or π−π stacking interactions.
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Article
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b02463. Synthesis scheme of peptide bolaamphiphiles; fluorescence spectra and intensity plot of HSA as a function of concentration; wavelength-dependent fluorescence and excitation spectra of HSA; CD spectra of HSA monomer, oligomer, and fibrils; FTIR spectra of HSA monomer, oligomer, and fibrils; SEM image of 2 μM HSA; size exclusion chromatogram and the corresponding nativePAGE image; fluorescence and excitation spectra of different fractions collected from the size exclusion chromatography; dynamic rheology of peptide hydrogels; fluorescence and excitation spectra of peptide bolaamphiphiles; SEM images of peptide hydrogels; FTIR spectra of peptides and peptide hydrogels; normalized fluorescence spectra of 2 and 150 μM HSA; normalized fluorescence spectra of 150 μM HSA at pH 7.4 and 4.7; CD spectral analysis data for HSA monomer, oligomer, and fibrils; fluorescence lifetime components of 150 μM HSA at different excitation and emission wavelengths; and fluorescence lifetime components of HSA fibrils at different excitation and emission wavelengths. (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail: tusharm@iiti.ac.in. Phone: +917312438738. ORCID
Apurba K. Das: 0000-0002-8141-6854 Tushar Kanti Mukherjee: 0000-0001-6242-2434 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank IIT Indore for providing experimental facilities, and financial support. acknowledge SIC, IIT Indore for instrumental authors thank Department of Science and Government of India, for financial support.
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CONCLUSIONS
In summary, our present study clearly demonstrates that the intrinsic blue fluorescence of HSA exclusively originates from the β-sheet rich oligomeric interfaces. These oligomers are structurally different from the amyloid-like fibrillar assemblies of heat denatured HSA aggregates. The observed excitation wavelength-dependent fluorescence originates mainly due to the presence of various oligomers such as dimer, trimer, tetramer, and high order oligomers. The observation of this phenomenon in different self-assembled peptides with diverse morphologies establishes the fact that the phenomenon is a common feature of self-assembled peptide bonds. Moreover, it has been shown that neither the aromatic residues nor the amyloid-like fibrillar networks are essential for this phenomenon to occur. We believe that the present study will opens up a new dimension for better understanding and facile detection of various toxic oligomers in the aggregation pathways of various proteins.
infrastructure, The authors facilities. The Technology,
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