Article pubs.acs.org/acssensors
Discrimination between Single Protein Conformations Using Dynamic SERS Thibault Brulé,*,† Alexandre Bouhelier, Alain Dereux, and Eric Finot Laboratoire Interdisciplinaire Carnot de Bourgogne (ICB), UMR 6303 CNRS, Université de Bourgogne Franche-Comté, F-21078 Dijon, France S Supporting Information *
ABSTRACT: In biomedicine and biophysics, the discrimination of protein conformations is of critical importance for identifying the unfolding states in the diagnosis of neurodegenerative diseases. We develop a dynamic Raman spectroscopic approach based on a statistical analysis of the time series of spectral fingerprints of single protein. We show that the unfolded state of bovine serum albumin can be identified in the time series using the fluctuations of the Raman bands of some amino acids, tryptophan, tyrosine, leucine, and histidine, acting as biomarkers. The statistical analysis induces also the sorting between physisorption and chemisorption events. This is confirmed by the spectral analysis of the different characteristic spectra highlighted based on the amino acids fingerprints following, notably, the hydrophobicity KEYWORDS: Raman spectroscopy, dynamic SERS, bovine serum albumin, folding-unfolding conformations, single-protein detection, statistical analysis
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plasmonics8−10 have been suggested but remain dependent on the surface functionalization. An optical spectroscopic method such as the Surface Enhanced Raman Spectroscopy (SERS)11 is a good way because it is mainly based on gold or silver nanostructures without chemical or biological functionalization. The SERS has already proved its abilities in terms of sensitivity, reaching the single molecule regime,12 and selectivity, through the detection of different molecules in a mix.13 We explore here the potential of SERS to study protein conformations. The chosen protein is the most abundant in the circulatory system: Bovine Serum Albumin (BSA). Its high concentration in plasmaaround 50 mg/mLinduces the high importance of characterizing this protein well for further investigation in blood. BSA has been widely studied by SERS,14−19 but conformational modifications were never observed with this technique. We show in the case of protein conformations where the spectral variations are relatively small that an up to date dynamic spectroscopic method at the single protein level coupled with careful multivariate statistical analysis, the Principal Component Analysis,19−21 is necessary.
complex medium is constituted by a large number of smaller noncomplex entities. This is especially the case as regards the structure of proteins built from 20 amino acids, or the blood constituted of many proteins. The level of complexity is proportional to the dependence criterion imposed between entities. Variables of the biological medium of proteins have typically to be taken into account: the concentration, the purity, the salinity of the solution, the presence of biological contributors/inhibitors, the temperature, the pH, etc. More precisely concerning proteins, all of these factors may involve drastic modifications in terms of protein conformations of the global structure and therefore its functionality. The misfolding of protein in the brain was shown to be the source of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Creuzfelt−Jacob.1 It is therefore important to determine the protein conformation under genuine physiological conditions using the least invasive methods for assessing its functionalities. Optical techniques are very good solutions for this investigation. Protein conformations were meanly investigated by circular dichroism.2 But this technique was limited to the study of a specific protein in a well-controlled medium. Infrared spectroscopy has also been used for this application,3−5 but the absorption of water in the infrared domain reduced the application field. Alternatively, protein conformations have been studied by fluorescence microscopy, and more precisely the Forster Resonant Energy Transfer (FRET)6 by monitoring the positions of the fluorescent labeling group and much more rarely using the intrinsic fluorescence of the protein.7 However, the labeling might modify the molecular structure or at least limit its deformation. Label-free alternatives based on © XXXX American Chemical Society
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MATERIALS AND METHODS
Our SERS active substrate is based on a self-assembly of raspberry-like gold nanoparticles (AuNPs) immobilized on a glass substrate and capped with microfluidics channels as described previously in ref 21. A Received: February 10, 2016 Accepted: April 7, 2016
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DOI: 10.1021/acssensors.6b00097 ACS Sens. XXXX, XXX, XXX−XXX
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ACS Sensors AuNP capping agent (HEPES) is reduced after deposition using an oxygen plasma cleaner and rinsing with deionized water. This substrate has demonstrated its interests in ref 22. Excited by a 784 nm laser diode, its enhancement factor is estimated around 104−107 and the limit of detection around 10 pM for a 512 s acquisition. Dynamic SERS measurements are conducted using a homemade confocal Raman setup. The substrate is illuminated by the laser through a water immersion objective (60×, NA = 1.20). The Raman scattering is recorded simultaneously on an avalanche photodiode and a spectrometer associated with a cooled CCD camera cadenced at 1 s acquisition time. BSA was diluted in PBS (pH = 7.2) at a concentration of 50 μg/mL (0.77 μmol/L) and was injected in a microfluidic channel at a flow rate of 2 μL/min driven by a peristaltic pump. Time series have been recorded on different hot spots with the same laser power (around 100 μW). It results in a global acquisition of 1500 s.
spectrum to the different dynamical conformations of single protein on the gold surface. Multivariate Statistics. The next step is to sort the individual spectra into groups on the basis of the similarities and correlation between spectra. The multivariate analysis based on the Principal Component Analysis (PCA) comes under this objective. The PCA applied on 1500 spectra shows that all spectroscopic data result from a combination of two reference spectra. Figure 2a shows the eigenspace of the projection based on the two first eigenvectors, i.e., the two first principal components. The two branches in the graph, colored in blue and red, allow sorting into two groups. The set of black center points corresponds to noise spectra.
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RESULTS AND DISCUSSION Univariate Analysis. The short-time acquisition of SERS spectra reveals a fluctuating dynamics of spectra which is attributed first to the regime of single or few proteins. The average spectrum of this record presents less information than single spectra (Figure 1a). Simple descriptive statistics with a
Figure 1. (a) Average spectrum of BSA. (b) Mandel M-Factor spectrum of BSA.
univariate analysis consists of calculating the Mandel MFactor21,23 from the ratio between the variance and the average of the intensity at each wavenumber. M is a useful parameter to determine the main Raman bands associated with the presence of the BSA on several hot spots of different enhancement factors. The resulting Mandel spectrum (Figure 1b) is a global fingerprint of 32 Raman bands, for which a large number can be identified based on the compilation of the various data in the literature.14−19 This can be interpreted as a classical signature of BSA in SERS. It principally results from phenylalanine and tyrosine amino acids or from α-helix proof (S−S bond, C−S stretching, α-helix global vibration..., Table S-1). The Mandel spectrum at the single protein level provides also substantially many more bands than the survey of an ensemble of proteins, especially below 600 cm−1. We attribute the richness of the M
Figure 2. (a) Projection of each spectrum recorded for BSA in the eigenspace formed by the two first principal components of this record. Colors are affected by position in this space, i.e., by spectral similarities. (b) Reference spectrum of BSA for blue family identified in a. (c) Reference spectrum of BSA for red family identified in a. B
DOI: 10.1021/acssensors.6b00097 ACS Sens. XXXX, XXX, XXX−XXX
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partly explain the increase in the total intensity of the signal. The spectral analysis helps to conclude. Spectral Proof of the Unfolded Form of the Protein. The first stair is characterized by two Raman spectra (Figure 4)
The blue group is attributed to the natural folded state natural BSA usually observed at pH 7. Its reference spectrum (Figure 2b) is compared with the BSA spectra of the literature.14−19 The results are summarized in Table S-2. Thus, it appears clearly that the reference spectrum of the blue family corresponds to a natural form of BSA as previously observed. Inversely, the red reference spectrum is different (Figure 2c). We intend to investigate this point further. Once each spectrum is attributed to one group, the sequence of events was analyzed by coloring the time series of the total intensity of each spectrum (Figure 3a). The blue events, with a maximal duration close to 20 s, are clearly faster than the red event that is longer than 50 s.
Figure 4. Spectral signatures of different steps of the red event of BSA. Each spectrum is associated with a form happening in time at different moments localized on Figure 3b. Colored bands are associated with identified amino acids. (Gray) lysine, (red) tryptophan, (orange) leucine and histidine, (blue) tyrosine in plane, (cyan) tyrosine out of plane.
A and C acquired before (time = 892 s) and after (time = 930 s) the topmost stair identified with spectrum B. Spectra A and C are similar to the same level of background intensity at 1800 cts/s. Spectrum B (Figure 4) differs first with an increased background signal, which may explain by itself the peak of event B. Contrary to the principal band of the three spectra that is the same (1410 cm−1 − lysine),25 spectrum B differs from spectra A and C with some bands attributed to others amino acids having very low probabilities to be observed in folded conformation. The lysine band at 1410 cm−1 is present on each spectrum due to the large distribution of lysine at the protein surface (gray band in Figure 4). The additional bands observed in spectrum B come from tryptophan (red in Figure 4), leucine, histidine (both in orange), and tyrosine (variations of blue). Tryptophan is a specific biomarker for the unfolded conformation of the BSA because it has only two tryptophans in its structure. The main interest in these amino acids is not only from their very spatial localization but also from their high Raman activity coming from their benzoic structure. One of the tryptophans is at the protein surface, and the second is in the hydrophobic core of the protein.26 During the red event, the associated vibration is the bending of the benzene ring of this amino acid (1436 and 1556 cm−1).25,27,28 The bending of the benzene ring of tryptophan is only accessible when BSA is in its unfolded conformation because the opening of the protein unprotects the inside tryptophan while the ring of the one on the top of the protein is still blocked by its surrounding amino acids. These bands are therefore good markers of the inside tryptophan. Structurally, leucine and histidine are two inside
Figure 3. (a) Colored time series of the total intensity of each spectrum of the acquisition. Colors are affected based on Figure 2a. (b) Zoom in of the temporal signature of the red event of BSA.
A Stepwise Process in the Long Event. Unlike blue short events, red events are characterized by their long duration, typically more than 40 s as shown in Figure 3b. The temporal shape of the event exhibits a stepwise process with two or three stairs. This observation agrees closely with those of Pang and Gordon24 discussing the optical transmission of a BSA protein trapped in a nanohole. Based on the steric hindering modification in the nanohole, they attributed these steps to two conformations of BSA. The highest transmission corresponds to the natural form of BSA, also called the folded form. The lower step was attributed to the unfolded form of BSA corresponding to the open form of the protein. Back to our case, the step distribution can also be interpreted as the change of conformation of the BSA when adsorbed on gold: leading to a transient unfolding observed during peak B. Nevertheless, the additional inlet of another protein may also C
DOI: 10.1021/acssensors.6b00097 ACS Sens. XXXX, XXX, XXX−XXX
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spectral signatures, one for the in-plane orientation and one for the out of plane orientation that never appear together during this protein−metal interaction. This hydrophobic interaction between BSA and the surface has been previously reported in infrared spectroscopy,5,31 SPR,10 and AFM.32
amino acids that surrounded the inside tryptophan. These two amino acids give spectral responses in spectrum B that are not present in spectra A and C, at 1145 and 1454 cm−1 for leucine and 1274, 1442, and 1576 cm−1 for histidine.25 The detection of the spectral fingerprints of leucine and histidine is tangible proof that hydrophobic amino acids located inside the protein structure are also enhanced by the gold surface. The most plausible explanation is that BSA changes its conformation by opening its structure and thereby giving access to inner amino acids which develop electrostatic affinity with gold, such as histidine. Spectral fluctuations are also observed between the different forms with tyrosine. Tyrosine is a hydrophobic molecule but is also distributed at the protein surface. The Fermi tyrosine doublet (830−850 cm−1) is used to distinguish the folded and unfolded forms.29 The 830 cm−1 band is observed for the folded form. This band is associated with the in-plane breathing of the phenol ring. The deformation of the protein when it opens induces a change of the orientation of the tyrosine with the electromagnetic field. The preferred excited vibration is the out of plane deformation causing the shift to 850 cm−1, observed in the unfolded form of BSA. Another doublet at 1170−1200 cm−1 for this amino acid is also observed between both forms, but in this case the out of plane ring vibration has its spectral fingerprint at 1170 cm−1 and the folded form is assigned to the 1200 cm−1 band. Spectrum B then provides information about the transient unfolding of the BSA on the gold surface over 10 s. The fact that peak B is composed of multiple short steps suggests a discontinuous motion of the unfolding with various discrete distances of the scattering spots from the gold surface. The reversible folding back to its natural form (spectrum C) is also observed before the protein desorption. This unfolded form of BSA is very rare at pH 7.2. This explains why this red event appears only once during the 1500 s of the acquisition. However, the two conformations of BSA are detectable by dynamic SERS in a single protein regime using the intrinsic biomarkers of BSA. It is important to notice that these two conformations are not sorted by the monovariate statistical analysis (Mandel factor). In the opposite, the multivariate statistical method (PCA) enables highlighting of the rarest conformation with the most important spectral fluctuations. The PCA also gives an important sorting between physisorption and chemisorption in this case. Indeed each family identified by the PCA is associated with a specific interaction of BSA with the gold nanoparticle. Concerning the blue family, BSA molecules are physisorbed: molecules undergo the Brownian diffusion close to the metallic surface, resulting in a repeatable spectrum of the different orientations probed during each second of the acquisition. This interpretation explains why the blue family distribution is quite large in the PCA projection (Figure 2a). It is also argued by the interpretation of the different spectral bands that were founded in different studies at large concentration of BSA. Concerning the red family, the single BSA molecule is chemisorbed: the molecule stays on the metallic surface by hydrophobic− hydrophobic reactions.30 Indeed, the gold surface of the nanoparticles is hydrophobic, and when the molecule comes to react with it, the hydrophobic amino acids are favored, like tryptophan, leucine, histidine, or tyrosine. The optimization of the interaction between the protein and the surface in terms of surface energy minimization induces the conformation modification of the protein. This is confirmed following the Tyrosine doublet decomposition in two different singlet
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CONCLUSIONS We have shown that the dynamic SERS analysis of a single protein can investigate the protein conformations in the case of BSA. The multivariate analysis by PCA of the dynamic SERS record enables discriminating the physisorption and the chemisorption events. This is confirmed by the spectral fingerprint analysis of the different characteristic spectra obtained, notably following the hydrophobic amino acids fingerprints, like tryptophan, tyrosine, leucine, and histidine. A better spectral resolution can monitor the fluctuations of the SERS fingerprint, thereby extracting rare protein conformations just based on its intrinsic biomarkers (amino acids) and their spectral fingerprints modifications.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.6b00097. Tables with Raman bands assignments of Mandel MFactor and blue family spectra (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Present Address †
Département de Chimie, Université de Montréal, CP 6128 Succ. Centre-Ville, Montréal, Canada.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the European project SPEDOC (FP7-ICT-2009-4) under Grant Agreement No. 248835 in cooperation with the Labex ACTION program (contract ANR11-LABX-01-01). This work was performed in the context of the European COST Action MP1302 Nanospectroscopy.
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