Hidden Dityrosine Residues in Protein-Protected Gold Nanoclusters

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The Hidden Dityrosine Residue in the Protein-Protected Gold Nanoclusters Lei Su, Tong Shu, Jianxing Wang, Zhenyun Zhang, and Xueji Zhang J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.5b03224 • Publication Date (Web): 04 May 2015 Downloaded from http://pubs.acs.org on May 11, 2015

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The Journal of Physical Chemistry

The Hidden Dityrosine Residue in the ProteinProtected Gold Nanoclusters Lei Su,* Tong Shu, Jianxing Wang, Zhenyun Zhang, and Xueji Zhang* Research Center for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, China.

ACS Paragon Plus Environment

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Abstract. The protein ligand shells of fluorescent protein-protected gold nanoclusters play an important role in the physiochemical properties and sensing applications of the nanoclusters. Recently, more and more attention has been paid on the investigation of the changes in the protein structure elements induced by the introduction of the nanoclusters in the proteins. In this work, the strategy of removal of the encapsulated gold nanoclusters from the protein ligand cages was proposed for the first time, producing the “hollow” (or possibly “imprinted”) proteins for investigations. Nontoxic cysteamine was used as the etchant. With bovine serum albumin, lysozyme and ovalbumin as model proteins, it was found that the dityrosine cross-links exist in the protein-protected gold nanoclusters.


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1. INTRODUCTION Gold nanoclusters (GNCs) comprising few to several hundreds of atoms bridge small organogold complexes and plasmonic gold nanoparticles.1-5 GNCs typically have a core-shell structure that consists of an Au core and a ligand shell. Among various ligand shells, proteins are of particular attraction as the ligands for synthesizing GNCs, because proteins are environmentally-benign reducing and stabilizing molecules and offer natural biocompatibility.6,7 The protein ligand shells have been known to exert a great influence on the stability, core-shell interface structure, and optical properties of the encapsulated GNCs.6-12 Moreover, the protein ligand shells are the outer layer of the encapsulated GNCs and directly interacts with the environmental substances. Therefore, the protein ligand shells also play a strong role in several other important aspects of the GNCs, including the biocompability, surface immobilization, and sensing applications.6,7,13,14 Interestingly, a few recent studies have revealed that the growth of GNCs within the protein cages may reversely impose great influence to the structure of proteins as well as their functions.15 For instance, structural changes such as significant decrease in the helical content of bovine serum albumin (BSA)16 and lysozyme15,17 occurred on the formation of GNCs because of the breakage of disulfide bonds, and the internal motions of lysozyme that are important for its function were found to be altered because of the embedded GNCs.15 These recent findings have thus attracted a strong interest in investigating the changes in the protein structure elements induced by the introduction of the GNCs in the proteins. In the present study we report the presence of dityrosine (diTyr) cross-links in the proteinprotected GNCs systems. Figure S1 shows the molecular structure of diTyr. Biologically, exposure of proteins to oxidative stress, such as UV light, γ-irradiation, oxygen radicals and other oxidants, may lead to the changes in every level of protein structure from primary to


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quaternary (if multimeric proteins).18,19 One of these changes is a protein-protein cross-linkage via tyrosine-tyrosine bonding, viz. diTyr cross-link, and the formed diTyr residue has been widely used as a marker for oxidative stress.18-20 On the other hand, recently, tyrosine has been used to stabilize GNCs.21,22 Tyrosine residue in BSA, which is the most intensely used protein in the GNCs@protein family, has also been suggested to participate in reducing Au ions to form GNCs in BSA molecular cages.7,23 Thus, we speculate the possible formation of diTyr crosslinks in the GNCs@proteins system. However, although, in a recent study, the tyrosine-tyrosine crosslinker was proposed as the result of Au ion and tyrosine reduction/oxidation,7,23 such a tyrosine-tyrosine crosslinker was referred to as the polytyrosine,7,24 rather than the diTyr. Other than that, so far there have been no reports on the diTyr residue existing in the fluorescent GNCs systems. diTyr can usually be distinguished by the intense fluorescence at about 420 nm, measurable upon excitation within either at 315 nm (alkaline solutions) or 280 nm (acidic solutions) absorption bands.18,19,20,25 To date, no reports have been published about the intense fluorescence of diTyr in the fluorescent GNCs systems, leading to the question of whether the reducing tyrosine are transformed to diTyr cross-links on the formation of GNCs. In this study, a strategy of removing the encapsulated GNCs from the BSA scaffolds was proposed for the first time, leaving behind the “hollow” (or “imprinted”) BSA molecules for investigations. To remove the encapsulated GNCs from the “imprinted” BSA cages, the route of decomposing GNCs through chemical etching of the BSA-protected GNCs (GNCs@BSA) was chosen. Although GNCs@BSA have been shown to possess ultrastability,26,27 the powerful etchants (e.g., CN-)28 could etch them and quench their fluorescence. In a recent study, we reported the potent etching activity of cysteamine (CSH) towards the [email protected] Moreover,


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CSH is nontoxic. So, in this study CSH was used to etch the GNCs, and then the optical spectra of the remaining BSA molecules were studied.

2. EXPERIMENTAL SECTION 2.1 Chemicals: Cysteamine, L-tyrosine, and HAuCl4·3H2O were purchased from SigmaAldrich. Bovine serum albumin (BSA) was obtained from Shanghai Biotech. Inc., China. Lysozyme was from MP Biomedicals Inc., U.S. and ovalbumin was from Sigma-Aldrich. Other chemicals of at least analytical reagent were obtained from Beijing Chemical Corporation (Beijing, China). Deionized water produced by Millipore-Q system was used for preparation of all aqueous solutions. 2.2 Synthesis of the GNCs@proteins: GNCs@proteins were prepared according to the previous report.26 Briefly, 20 mL HAuCl4 solution (37 oC, 10 mM used for BSA, and 4 mM used for lysozyme, and 4 mM used for ovalbumin, respectively) was added to equal volume of protein solutions (37 oC, 50 mg mL-1 BSA, 20 mg mL-1 lysozyme, and 20 mg mL-1 ovalbumin, respectively). Upon vigorous stirring at 37 oC for 2 min, 2 mL NaOH (1 M) was introduced, and the mixture was incubated at 37 oC for 12 h. 2.3 CSH-induced etching of the GNCs@proteins: According to our previous report,27 1 mL of the as-prepared GNCs@BSA solution was incubated with 3 mL of freshly prepared Tris buffer solution (pH 8.0) containing CSH of a final concentration of 50 mM at 55 oC for 1 h. After cooling to room temperature, the resultant mixture solution was ultra-filtrated through a Millipore filter (100 kD). The filtrates were collected and found to be nonluminescence under


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UV light (365 nm). The protein retentates were collected and re-dispersed in 4 mL of Tris buffer solution (pH 8.0). Treatments of ultrafiltration and redispersion were cycled for three times. The resultant protein-containing solutions produced by 50 mM CSH etching of the GNCs were colorless and transparent under visible light, designated as the BSA-diTyr stock solution. CSHinduced etching of the GNCs@lysozyme and GNCs@ovalbumin were carried out with the similar protocols with minor modification by adjusting the solution pH to pH 10.0. 2.4 Fluorescence quenching study: Decrease of fluorescence intensity of diTyr by quenchers (Fe2+, Fe3+ and borate) was monitored by mixing quenchers-containing buffer solution (pH 8.0) with an equal volume of the obtained protein-containing solution in a cuvette and exciting it using 325 nm wavelength. 2.5 Instrumentation: UV-vis absorption spectra were recorded on a Shimadzu UV-1800 spectrometer. Fluorescence spectra were recorded on an F-4500 spectrometer (HITACHI) with slit widths of 5 nm. Luminescence decay curves were measured on an F-900 spectrofluorometer (Edinburgh Instruments, UK). The product solution from the CSH-induced etching of GNCs@BSA without further ultra-filtration was used for dynamic light scattering (DLS) analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in order to avoid the possible aggregation due to the ultra-filtration process.3 DLS analysis was performed with a ZS90 laser light scattering system (Malvern Instruments Corporation). The samples for DLS assays were pre-treated by 220 nm filter to exclude large aggregates and particles. The DLS assays for each sample were performed six times and the significant average results were adopted. SDS-PAGE was performed according to the manufacture protocol with an 8% SDSpolyacrylamide gel. The position of the protein bands on the gel were visualized by Coomassie blue staining. All measurements were carried out at room temperature.


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3. RESULTS AND DISCUSSION According to our previous study,27 the etching of the GNCs@BSA was conducted with 50 mM CSH at 55 oC for 1 h and the removal of the GNCs from the BSA molecules could be indicated by the changes in the properties of the GNCs@BSA. For instance, the GNCs@BSA lost their characteristic fluorescence emission peak (λpeak = 625 nm) after etching (inset of Figure 1a). As a result, the possible interferences from the GNCs could be avoided.


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Figure 1. (a) Photoluminescent (PL) emission spectra of the GNCs@BSA before (black line) and after etching by 50 mM CSH (red line) when excited at 325 nm, and excitation spectrum of the resultant solution (blue line, λem =410 nm). Inset: Magnification of emission spectra ranged from 570 to 700 nm. The peak marked * arises from second-order Rayleigh scattering. (b) PL decay profiles of the resultant diTyr-modified BSA. (c) Effect of pH on emission spectra of the resultant diTyr-modified BSA. Inset: bar plot of the corresponding peak emission intensity versus pH. (d) Effect of 0.2 mM Fe2+ (red line) and 0.2 mM Fe3+ (blue line), respectively, on the emission intensity of the resultant diTyr-modified BSA (black line). (e) Emission spectra of the resultant diTyr-modified BSA in the absence (black line) and presence (red line) of 0.10 M borate/boric acid buffer (pH 8.0). As revealed from Figure 1a, after etching, the resultant solution exhibited an intense fluorescence emission at lower wavelength (410 nm) excited at 325 nm and an excitation peak at 330 nm monitored at 410 nm emission wavelength. This phenomenon is characteristic of diTyr!18,19,20,25 Note that the aromatic amino acid residues, e.g., phenylalanine, tyrosine, and tryptophan, are not excited at this wavelength.25 Thus, the 325 nm wavelength excitation exhibits specificity towards the diTyr residue of the BSA and the observed intense fluorescence emission at 410 nm indicates the modification of BSA molecules with diTyr residue. More evidences for the presence of diTyr residue were obtained, as follows. Figure 1b shows the fluorescence decay profiles of the resultant diTyr-modified BSA in Tris-buffer solution (pH 8.0). Two lifetime components for 400 nm emission of diTyr were previously reported in the studies of diTyr.29


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Indeed, the fluorescence decays were best fitted with two principal decay components τ1 (1.6 ns, 28.01%) and τ2 (4.7 ns, 71.99%) (χ2=1.097). The longer decay time, 4.7 ns, prevailed. This 4.7 ns component was similar to the 4.4 ns lifetime observed by Harms et al. in the dynamic study of diTyr for 400 nm emission at pH 8.0.29 Figure 1c shows the pH-dependent fluorescence emission spectra of the resultant diTyr-modified BSA. It can be seen that with the increase of pH value the emission intensity increases. It has been reported that the singly ionized diTyr chromophore, in which one of the two phenolic hydroxyl groups is dissociated, is responsible for the 410 nm range emission of diTyr.20 The phenolic hydroxide of the diTyr has a neutral pKa. Thus, the elevated pH values favor the enhanced emission, and accordingly, as revealed in inset of Figure 1c, there is a quasi-linear pH-dependence of the measured emission intensity near neutral pH. In addition, the 410 nm emission of the resultant diTyr-modified BSA could be quenched by 0.20 mM Fe2+ or Fe3+ ions, as shown in Figure 1d, in line with previous reports.25 According to the previous study, the Fe2+/3+ ions-induced quenching mechanism has been attributed to the formation of the nonluminescent Fe[diTyr] complexes.25 Furthermore, it is known that borate/boric acid is a specific quencher for diTyr due to its ability to form a complex with diTyr.30,31 As shown in Figure 1e, the observed 410-nm emission can be quenched in the presence of borate/boric acid. These results fit perfectly the features of the diTyr residue.


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Figure 2. (a) Spectral overlap between the donor diTyr emission (red line) spectrum and acceptor GNCs@BSA absorption (black line). (b) UV-vis absorption spectra of the GNCs@BSA before (black line) and after etching (blue line). (c) Schematic illustration of the formation of the diTyr residue due to protein oxidation via the formation of the GNCs inside the protein cage (step i) and the subsequent removal of the GNCs by CSH-induced etching process (step ii), leading to the observation of the PL emission of diTyr.


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These observations clearly indicate that after removing the GNCs, the resultant products could exhibit the fluorescent characteristics of the diTyr, indicating the formation of the diTyr residue in the BSA molecules and highlighting the necessity and importance of the removal of the GNCs from the interior of the BSA cages. However, the presence of the diTyr residue in the BSA molecules being used to stabilize GNCs poses an interesting question about the GNCs system: why don‘t the diTyr residue formed during the preparation of the GNCs emit their intense fluorescence in the presence of the GNCs? The possible reasons were explored. Figure 2a shows the spectral overlap of the fluorescence emission of the resultant diTyr-modified BSA (red line) and the absorption of the GNCs@BSA (black line). Because diTyr has been known as an energy transfer donor to the acceptors such as fluorescent Lucifer yellow tethered to the protein structure,32 and the GNCs have been reported to be used as an energy transfer acceptor,33,34 fluorescence resonance energy transfer (FRET) between the diTyr and the GNCs was firstly considered to account for the disappearance or quenching of diTyr emission in the GNCs@BSA system. FRET is an electrodynamic phenomenon. Energy transfer results from long range dipoledipole interactions between the donor and acceptor and the transfer efficiency is associated with the lifetimes of the donor. However, the measured average lifetime before and after CSHinduced etching was 3.48 (Figure S2) and 3.83 ns (Figure 1b), respectively. As a result, the transfer efficiency was calculated to be 0.091, obviously lower than calculated from the steady state data (0.84), indicating that FRET played a minor role in the PL quenching of the diTyr. Thus, other quenching mechanisms must also be considered, for instance, inner filter effect (IFE). IFE involves the absorption of the photon by the absorber and requires that the absorption band of the absorber possesses a complementary overlap with the excitation and/or emission bands of the fluorophore to some extent.35 The results shown in Figure 2a, 2b and Figure S3 meet this


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requirement of the IFE. In addition, IFE is known to have no effect on the excitation lifetime of the fluorophore. The lifetime results measured in this study, as aforementioned, were also in line with this feature of the IFE. On the other hand, after removing the GNCs via etching, as shown in Figure 2b, the absorption around 410-nm wavelength decreased remarkably; meanwhile, diTyr emission was observed (Figure 1a). These results indicated that in the presence of the GNCs IFE could play a crucial role in quenching the fluorescence emission of the diTyr residue. Figure 2c shows schematic illustration of the formation of the diTyr residue due to protein oxidation by HAuCl4 and the effect of the presence and absence of GNCs inside the protein cage on the observation of the PL emission of the diTyr residue. In principle, the procedure established in this study for exploring the diTyr residue in the BSA cages used for stabilizing GNCs may apply to other GNCs@proteins systems. GNCs@lysozyme and GNCs@ovalbumin as typical examples were analyzed using the same procedure established above, including the CSH-induced etching of the embedded GNCs followed by measuring the fluorescence emission at lower wavelengths. Figure S4 and S5 show the fluorescence emission spectra of the resultant solution (pH 8.0) containing the lysozyme and the ovalbumin, respectively. It can be seen that after the removal of the GNCs from the protein cages, both of these two protein-containing solutions exhibited the intense fluorescence emission centered at ca 410 nm, indicating the formation of the diTyr residue in the lysozyme and the ovalbumin structures, respectively, which resulted from the oxidative stress by the oxidant, chloroauric acid. In addition, as revealed by Figure S6 and S7, the spectral overlap of the fluorescence emission of the diTyr-modified proteins and the absorption of the GNCs occurred.


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Wavelength /nm Figure 3. Dynamic light scattering (DLS) spectra of GNCs@BSA before (red line) and after (blue dotted line) etching by 50 mM CSH. The presence of the diTyr residue in these GNCs@proteins systems makes us aware that the GNCs are encapsulated actually by the protein shells with the altered structural elements, rather than the pristine proteins. Therefore, some observations of the GNCs@proteins that have not been well addressed in the literature might be related with this property of the GNCs@proteins, for instance, the observed sizes of the GNCs@proteins using DLS analysis and the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results. A relatively wide hydrodynamic size distribution of the GNCs@BSA ranged from 10.1 to 21.9 nm was reported by several groups.36-38 The GNCs@BSA used in this study also showed a relatively large mean hydrodynamic diameter of 13.5 nm, as shown in Figure 3 (red line). As for the SDS-PAGE, Yan et al reported recently that the GNCs@BSA exhibited the separated but continuous bands of various relative molecular masses on the gel.39 The GNCs@BSA used in this study also showed a similar SDS-PAGE, as shown in Figure S8. Both these previous results and present observations indicate the presence of the GNCs-containing protein aggregates, which might be


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attributed to the formation of protein-protein cross-linkage via diTyr cross-links. In addition, interestingly, as shown in Figure 3, the mean hydrodynamic diameter of the resultant diTyrmodified BSA was measured to be 18.9 nm (blue dotted line), slightly larger than that (13.5 nm) of the GNCs@BSA. This result indicates that in the presence of GNCs, BSA, which is bulky in nature, may completely wrap the Au cluster through gold-thiol interactions,7 forming a relatively compact shape, while due to removal of Au cluster from the BSA, the resultant denatured BSA might have a stretched conformation.

4. CONCLUSION In the present report we have unveiled the presence of the diTyr residue in the proteins used for synthesizing GNCs by using the strategy of removal of the GNCs from the protein ligand cages. The formation of GNCs within the protein ligand cages indeed can alter the structural elements of the proteins, e.g., the breakdown disulfide bonds in proteins, which has been well-known, and the formation of diTyr cross-links, which has been unveiled in this study. Although FRET process could exist between the diTyr residue and the GNCs, contributing to the fluorescence quenching of the diTyr residue, however, IFE should be the main mechanism for the fluorescence quenching of the diTyr residue in the presence of the GNCs. Further investigation of GNCs@lysozyme and GNCs@ovalbumin systems led to the relatively general conclusion of the formation of diTyr residue in these GNCs@proteins systems. Moreover, some observations of the GNCs@proteins that have not been well addressed in the literature, for instance, the observed hydrodynamic sizes of the GNCs@proteins using DLS analysis and the SDS-PAGE results, might be related with this attribute of the GNCs@proteins.


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ASSOCIATED CONTENT

Supporting Information Förster resonance energy transfer study, molecular structure of diTyr, PL emission spectra of the GNCs@ovalbumin and GNCs@lysozyme before and after etching, spectral overlap between donor emission and acceptor absorption spectrum, and the SDS-PAGE results are available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding author * E-mail: [email protected]; [email protected]. Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS

This work was financially supported by the National Natural Science Foundation of China (No. 21175010 and 21275017), the grant from Beijing Municipal Science and Technology Commission (z131102002813058), the Project-sponsored by SRF for ROCS, SEM, and the Fundamental Research Funds for the Central Universities (230200906108037).


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TOC GRAPHICS


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Hidden Dityrosine Residues in Protein-Protected Gold Nanoclusters

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