Fluorescent Silver Clusters on Protein Templates: Understanding

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C: Physical Processes in Nanomaterials and Nanostructures

Fluorescent Silver Clusters on Protein Templates: Understanding Their Structure Tomash S. Sych, Zakhar V. Reveguk, Vladimir A. Pomogaev, Andrey A. Buglak, Anastasiia Andreevna Reveguk, Ruslan R. Ramazanov, Nikolay M. Romanov, Elena V. Chikhirzhina, Alexander M. Polyanichko, and Alexei I Kononov J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b08306 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

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The Journal of Physical Chemistry

Fluorescent Silver Clusters on Protein Templates: Understanding Their Structure Tomash S. Sych,a Zakhar V. Reveguk,a Vladimir A. Pomogaev,b,c Andrey A. Buglak,a Anastasiya A. Reveguk,a Ruslan R. Ramazanov,a Nikolay M. Romanov,a Elena V. Chikhirzhina,d Alexander M. Polyanichko,a and Alexei I. Kononov*a a - Saint Petersburg State University, Saint-Petersburg 199034, Russia. b - Department of Physics, Tomsk State University, Tomsk 634050, Russia. c - Department of Chemistry and Green-Nano Materials Research Center, College of Natural Sciences, Kyungpook National University 1370 Sankyuk-dong, Buk-gu, Daegu 702-701, Republic of Korea d - Laboratory of Molecular Biology of Stem Cells, Institute of Cytology of the Russian Academy of Sciences, Saint-Petersburg 194064, Russia.

Abstract Luminescent metal nanoclusters (NCs) stabilized by natural proteins are of special interest in bioimaging applications. However, the detailed structure of the protein-templated NCs and the nature of their emissive states remain poorly understood. A fair amount of nonluminescent metal ions and clusters complexed to the proteins hinders probing of the structure of the emitting clusters using mass spectroscopy, infrared, or other conventional spectroscopy methods. In this respect, only luminescent excitation spectra distinguish the emitting NCs. In this experimental

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and theoretical joint study, we modeled the fluorescent excitation and excitation anisotropy spectra of protein-based silver (Ag) NCs. We varied the synthesis conditions and studied the spectral properties of Ag clusters on bovine serum albumin (BSA) and lysozyme, which had already been used as templates, as well as on HMG box (HMGB)1 and histone H1 (H1) proteins. We also calculated the electronic spectra of quantum mechanics–optimized Ag–thiolate, Ag– semiquinone, and Ag–formaldehyde complexes with two confined electrons using second-order algebraic diagrammatic construction [ADC(2)] and resolution-of-identity approximate coupledcluster singles-and-doubles (RI-CC2) methods and compared them with the experimental spectra. We propose a model for the fluorescent Ag–protein complexes in which two reduced Ag atoms are sufficient to form the fluorescent core of the complex. The proposed structural model of the luminescent centers in the Ag–protein complexes differs from the common view that the fluorescent metal NCs in proteins contain about 10 or more metal atoms. The fluorescent Ag clusters formed on the four investigated natural protein matrices exhibited two different spectral and structural patterns. Deprotonated free cysteine residues stabilized the fluorescent Ag3+1 core formed in the BSA matrix. The second type of fluorescent center was realized in the H1, HMGB1, and lysozyme protein matrixes. In this case, tyrosine residues probably stabilize the fluorescent Ag2 centers.

Introduction In the last decade, metal nanoclusters (NCs) have aroused growing interest in chemistry, biology, and material science due to their unique properties. Exhibiting quantum behavior1–4, they can be applied in catalysis, chemical sensing, bioimaging, single-molecule optoelectronics, and other


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fields5–17. Among the great variety of NCs, gold (Au) NCs are the most extensively investigated due to their chemical stability15. However, fluorescent silver (Ag) clusters are the brightest, exhibiting high quantum yield and large absorption cross-section13. Silver NCs can be prepared by reducing Ag ions using various polymer templates, which determines their stability in aqueous solution8–11,18–24. Although the structures of some non-fluorescent clusters have been revealed25,26, the detailed structures of the fluorescent complexes of Ag clusters with ligands remain unknown. Biomolecule-stabilized clusters are of special interest in bioimaging applications. DNAstabilized fluorescent Ag clusters have been synthesized in a wide spectral range in visible and near infrared regions9–11,17,27,28. Theoretical calculations suggest the extended structure of DNAbased clusters29,30. Different structural models of DNA-stabilized Ag clusters have recently been proposed31–34. DNA-protected Ag clusters have excellent photostability, high absorption crosssection, and fluorescence quantum yield. DNA sequence and environment modifications can tune their spectral properties. NC synthesis on protein templates also has many advantages for biological applications35–37. Protein-based Au clusters have been synthesized using as templates various proteins such as bovine serum albumin (BSA)38,39, lysozyme39–41, trypsin39, lactotransferrin42, insulin43, pepsin39,44, and horseradish peroxidase45. In contrast to Au clusters, there have been only a few studies on protein-templated fluorescent Ag clusters. Light-emitting Ag clusters have been synthesized using chymotrypsin46, BSA47–53, human serum albumin54, ovalbumin54, and lysozyme55. A few amino acids and peptides have also been explored in the synthesis of fluorescent Ag clusters56–59. In the meantime, neither the nucleation sites nor the size of Ag clusters within the protein matrices are clear. The same can be said of Au clusters. The size of


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the fluorescent NCs in proteins is typically proposed based on mass spectroscopy (MS) analysis. For BSA, the literature reports 8–15 Ag atoms47,48,52,53 for red-emitting clusters. Based on x-ray photoelectron spectroscopy (XPS) analysis, Ag atoms are supposed to bind with sulfur atoms in the protein48. However, not all Ag atoms in the complex form emitting clusters. A huge amount of “dark” (non-emissive) clusters and non-reduced Ag ions mask the fluorescent fraction in absorption, MS, and XPS analyses so that the spectra produce only the average picture, with nonfluorescent species contributing the most28,32. Only fluorescence excitation spectra can distinguish fluorescent clusters. Based on quantum mechanical–molecular mechanical (QM/MM) simulations of the fluorescence excitation spectra of some Ag–DNA complexes, we have proposed detailed structures of complexes with chromophoric cores consisting of 3–6 Ag atoms32,33. The modelling of large protein templates is however much more complicated, requiring additional information on the clusters’ location. In this respect, single amino acids (AAs) can be explored as model systems for studying protein–cluster interactions. The complexes of small silver clusters with individual amino acids60–66 and also with thiols67 have been studied previously experimentally and theoretically in the gas phase. In particular, the absorption and photo-fragmentation spectra of the complexes of Ag3+ cluster with tryptophan60,61,63 and histidine65 were extensively studied. It was proposed that complexes between silver clusters of appropriate size and tryptophan could exhibit fluorescence61. Thiolateprotected silver clusters with two and four confined electrons were also suggested as potential fluorescent complexes67. In our recent study we demonstrate that only cysteine and tyrosine are able to form the fluorescent complexes with silver clusters in solution68. It should be noted that in proteins the interaction mechanism may be different. In proteins, α-carboxyl and α-amino


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groups are involved in peptide bonding. Also, sulfur atoms may form disulfide bonds, and sidechain groups may be charged or not depending on pH values. Uncertainty regarding the protein-based fluorescent NC structures renders further tuning of their emission properties difficult. It has been reported that the fluorescence quantum yield values of Ag clusters in proteins are about 1% or even less48,50,52. This seems surprising, considering the span of their fluorescence lifetime of several nanoseconds. However, a reasonable explanation might be that the chemical yield of the fluorescent complexes is extremely low and they are masked by the huge amount of dark species absorbing in the same spectral range. High chemical yield and stability under ambient conditions also remain major challenges in the synthesis of Ag clusters on protein templates. Here, we address the questions underlying the abovementioned issues to achieve a clearer understanding of fluorescent Ag–protein complexes. We varied the synthesis conditions and studied the spectral properties of Ag clusters on the proteins BSA and lysozyme, which have already been used as templates, and other proteins such as linker histone H1 (H1) and HMGBdomain protein (HMGB1). Linker histone H1 is a lysine-rich nuclear protein69. It contains 194 amino acid residues. Its central globular domain is flanked with short N-terminal and long Cterminal unordered tails. HMGB1 is one of the best known members of the HMGB superfamily70. It contains 215 amino acid residues, including three cysteines. Its amino acid sequence consists of three regions that form two DNA-binding domains (HMGB domains A and B) and an unordered regulatory C-terminal domain71,72. Originally, DNA structural organization in chromatin was thought to be the major function of HMGB proteins73. However, it was proved recently that they perform numerous regulatory functions in the nucleus74, cytoplasm75, and even outside the cell76. Lysozyme is a low–molecular weight (14 kDa) enzyme that contains 129


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amino acid residues with four S–S bonds55. It has been used for growing various functional nanomaterials for chemical and biochemical applications77–79. In particular, lysozyme-stabilized Au clusters can be used for detecting various analytes80,81. HMGB1 and linker histone H1 are very promising objects for “multi-purpose” fluorescent labeling, since various biological processes can be monitored using these proteins. Both HMGB1 and H1 were previously used as DNA carriers in gene transfer experiments82–85. Practical application of the proteins as the transfection agents would also greatly benefit if the carrier is fluorescently labeled. H1, HMGB1 and lysozyme have very different amino acid sequences and structural properties, which would help to reveal the mechanisms of the cluster formation. We calculated the electronic spectra of QM-optimized Ag–thiolate, Ag–semiquinone (sQ), and Ag–formaldehyde complexes with two confined electrons using second-order algebraic diagrammatic construction [ADC(2)] and resolution-of-identity approximate coupled-cluster singles-and-doubles (RI-CC2) methods, and compared them with the experimental spectra. Our observations suggest that two reduced Ag atoms are sufficient to form the fluorescent metal core, stabilized by free cysteine or tyrosine residues, in the proteins.

Experimental and Theoretical Methods Chemicals BSA (MW 66,437), lysozyme (MW 14,307), silver nitrate (AgNO3), and sodium borohydride (NaBH4) were obtained from Sigma-Aldrich Chemicals. H1 (MW 20,951) and the non-histone chromatin protein HMGB1 (MW 24,908) were isolated from calf thymus nuclei. The former was


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extracted with 5% perchloric acid, followed by precipitation with 3 volumes of acidic acetone at -20°C as described elsewhere86,87; HMGB1 was isolated according to an earlier-described procedure88. The purity of the products was tested with sodium dodecyl–sulfate polyacrylamide gel electrophoresis (SDS-PAGE)89. The H1 and HMGB1 concentrations were determined by UV absorbance using extinction coefficients ε230 = 41,000 M-1cm-1 (H1) and ε280 = 33,000 M-1cm-1, respectively90. Synthesis of clusters The synthesis parameters were varied (as indicated in SI) to determine the optimal synthesis conditions for preparing the Ag–protein fluorescent complexes. Typically, 0.1 M AgNO3 solution was added to 1 mg/ml protein solution (H1, 0.25 mg/ml; HMGB1, 0.2 mg/ml). To achieve the desired pH value of 12.5, sodium hydroxide was added to the protein solution before AgNO3 was added. The mixture was incubated for 1 h under vigorous stirring at room temperature. Subsequently, a freshly prepared 1 mM NaBH4 solution was added dropwise, and the mixture was kept at 4°C in the dark until further analysis (usually after 24 h for BSA and after 1 h for H1, HMGB, and lysozyme). The final molar ratios were as follows: BSA, 30:1 Ag:BSA and 1:10 NaBH4:Ag; H1, 1:1 Ag:H1 and 1:1 NaBH4:Ag; HMGB1, 7.5:1 Ag:HMGB1 and 1:1 NaBH4:Ag; and lysozyme, 1:1 Ag:lysozyme and 0.5:1 NaBH4:Ag. Spectral methods Fluorescence emission and excitation, excitation anisotropy spectra, and fluorescence decay were obtained at room temperature using a Fluorolog-3 (HORIBA Jobin Yvon) spectrofluorometer. The measurements were carried out using a 0.4-cm quartz cuvette. Longwave pass filters were used to remove scattering light. Fluorescence emission spectra were


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corrected for instrument sensitivity. Fluorescence excitation spectra were corrected for inner filter effect due to high sample absorbance in the UV range as described elsewhere33. Spectral data was obtained at constant bandpass (taken in the range of 3-10 nm depending on intensity) and presented in electron-volts scale. The fluorescence emission spectra were λ2-scaled. Fluorescence lifetime measurements were performed with LEDs with FWHM of about 2 ns. Emission bandpass was set at 14 nm. The quantum yield was measured by a direct method using integrating sphere Quanta-φ. Luminescence anisotropy was calculated as (I0 − G∙I90)/(I0 + 2G∙I90), where I0 and I90 are the intensities of vertically and horizontally polarized emission beam, respectively, at vertically polarized excitation light, and G is the instrument factor calculated as I0/I90 at horizontally polarized excitation light. Absorption spectra were obtained with a Specord 210 Plus double-beam spectrophotometer. Raman spectra were obtained with a Senterra micro-Raman spectrometer (Bruker) with an excitation wavelength of 785 nm. X-ray photoelectron spectra were acquired on a Thermo Fisher Scientific ESCALAB 250Xi spectrometer using a monochromated Al Kα X-ray source. The pass energy was 20 eV. Mixed ion/electron charge compensation was used. The samples were prepared as thin films on a HOPG (highly ordered pyrolytic graphite) substrate by depositing a few drops of the aqueous solution onto the surface, followed by drying with argon. The samples’ stoichiometry was controlled according to the well-described approach based on comparing the relative intensities of the corelevel signals91. Spectra were fitted using Gaussian–Lorentzian convolution functions with Shirley background subtraction92. The accuracy of the binding energies was ±0.1 eV. Theoretical calculations The geometries of all complexes with closed-shell structures were optimized using secondorder Møller-Plesset perturbation theory (MP2) with RI-MP2 performed in the ORCA 3.0


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program package93 and with the RI-CC2 method94. For RI-MP2 optimization, a 6-31G(d) basis set was used in all geometry optimizations; Ag atoms were treated with Los Alamos National Laboratory (LANL) effective core potential LANLTZ95. The hybrid basis sets, noted here as def2-TZVP/P, were used for the RI-CC2 optimization, where the def2-SVP, def2-TZVPP, and def2-TZVP basis sets96 were used for hydrogen, silver, and the remaining elements, respectively. The electronic absorption spectra of the abovementioned complexes were calculated using both the ADC(2) method97 and the RI-CC2 method accompanied by the def2-QZVP/P large basis sets, where the def2-TZVPP and def2-TZVP basis sets were replaced by the Karlsruhe def2QZVPP and def2-QZVP basis sets, respectively96. Also, we used time-dependent density functional theory (TDDFT) method with the M06-2X functional98 and the def2-TZVP/P basis set.


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Results and Discussion Ag clusters in BSA template

Figure 1. Fluorescence excitation, excitation anisotropy, and emission spectra of the Ag–BSA complex (λex 500 nm, λem 700 nm); absorption spectrum of Ag NCs was obtained as the difference between the absorption spectra of Ag–BSA and Ag+–BSA complexes. Fig. 1 shows the spectral properties of the Ag clusters obtained in 1 mg/ml BSA solution by reducing Ag+ ions with NaBH4 at 30 Ag/BSA and 0.1 NaBH4/Ag+ ratios at pH 12.5 in a wide spectral range. The Ag–BSA complex emitted at ca. 700 nm. The fluorescence emission spectrum was independent of the excitation wavelength (Fig. S1), suggesting only one type of fluorescent cluster. The multi-exponential fluorescence decay (Fig. S2) could be attributed to the excited-state relaxation on a nanosecond time scale rather than to heterogeneity. The same effects have been observed for some fluorescent DNA- and polymer-based Ag clusters99–101. The fluorescence excitation spectrum (Fig. 1) was measured using a dilute Ag–BSA solution to cover a wide spectral range of up to 5 eV, where the absorbance of the sample increased significantly. The fluorescence excitation spectrum and anisotropy curve exhibited four


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electronic transitions at 280, 320, 410, and 500 nm. The shape of the excitation spectrum was quite different from that of Ag clusters stabilized by DNA102 and synthetic polymers101, for which a single intense transition is observed in the visible range. This suggests that in the case of a protein matrix, the fluorescent clusters have a planar or globular shape rather than the threadlike shape proposed for DNA-based clusters32,33. We also obtained the Ag cluster absorption spectrum as the difference between the absorption spectra of the Ag–BSA complex before and after the addition of NaBH4 (Fig. 1). The spectrum obtained is thus very close to the fluorescence excitation spectrum of the Ag cluster, suggesting that most of the reduced Ag atoms form fluorescent rather than dark Ag clusters. Typically, synthesizing fluorescent Ag clusters in proteins requires alkali pH conditions46–55. We studied the pH dependence of the chemical yield of the fluorescent Ag–BSA complexes. Fig. 2 presents the pH dependences of the fluorescence intensity of the Ag–BSA complex at 700 nm and of the intrinsic BSA fluorescence at 350 nm. The increased pH led to an increased fluorescent clusters yield, which peaked at about pH 12.5. The decreased yield at pH > 13 was probably due to AgOH formation. At the same time, the intrinsic protein fluorescence of tryptophan residues decreased with the increase of pH (Fig. 2), indicating that alkali pH conditions destroy the native protein structure103.


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Figure 2. pH dependence of the fluorescence intensity of BSA and Ag–BSA solutions. Blue: bare BSA (λex 280 nm, λem 350 nm), red: Ag–BSA (λex 500 nm, λem 700 nm), orange: Ag–BSA with TCEP. The chemical yield of the fluorescent clusters depends not only on pH, but also on the NaBH4/Ag+ (Fig. 3) and Ag+/BSA ratios (Table S1). The fluorescent quantum yield of the redemitting clusters synthesized at the optimal conditions appeared to be 20%. This value differs significantly from the values reported previously (Table S2). The difference in the chemical yields of the fluorescent clusters obtained in different conditions could explain the discrepancy in the obtained values.


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Figure 3. Dependence of the fluorescence intensity of the Ag–BSA cluster (λex 500 nm, λem 700 nm) on the NaBH4/Ag+ ratio.

Figure 4. Hydrolysis of disulphide bonds. The next question we addressed in this study relates to the cluster location site in the protein. Why do we need an alkali pH? In such conditions, some amino acid side chains groups lose protons, and hydrogen bonds break, leading to transition to a molten globule-like state103. Moreover, disulfide (S–S) bonds can also be subjected to alkaline hydrolysis104 (Fig. 4). Native BSA contains 17 S–S bridges and one free cysteine. S–S bridges in BSA are destroyed at alkali pH, which was proven by the corresponding Raman and XPS spectra. The Raman spectrum of native BSA exhibits a characteristic S–S stretching band around 500 cm-1

105,106.

In alkaline

solution, the intensity of this bond was strongly reduced, indicating the S–S bond breakage (Fig. S3). In Fig. 5, the S2p XPS spectra of the BSA complexes with Ag clusters (after adding NaBH4 at a 0.1 NaBH4/Ag ratio) are shown in comparison with the bare BSA. The bands II (S 2p3/2 at


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163.6) are attributed to the S–S bonds. At the low-energy edge of the bare BSA spectrum, there was also a component IV that might be assigned to free thiol groups. The bands III (S 2p3/2 at ca. 162 eV) were assigned to the Ag–sulfur bonds. For the Ag–BSA complex, the stoichiometric sulfur/Ag ratio was 0.76, which within the experimental error was consistent with the 30:1 Ag/BSA ratio used in the experiment. The fraction of Ag-bound sulfur atoms was estimated to be 0.57 (ca. 20 of the total number of cysteine residues). The bands I at the high-energy side (S 2p3/2 at ca. 166.0 eV) were due to the oxidized sulfur, which indicates hydrolysis of the S–S bonds in BSA. The sulfur in S–S bonds can also be reduced in the presence of TCEP [tris (2chloroethyl) phosphate]. In this case, the fluorescent clusters in BSA formed at pH 8, and the emission intensity peaked at pH 11 (Fig. 2).

Figure 5. S2p XPS spectra of bare BSA (bottom) and the Ag–BSA complex (at 0.1 NaBH4/Ag+ ratio). The roman numerals indicate the state of sulfur: I – oxidized; II – S-S bridge; III – Agsulfur bonds; IV – free thiol groups. The above results indicate that breakage of the S–S bridges is necessary for effective fluorescent cluster growth in BSA. The presence of free sulfur atoms in protein appears to be the necessary condition for stabilizing the emitting Ag clusters. The next question we addressed was how many silver and sulfur atoms are needed to form a AgnSm fluorescent center. The dependence of the


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fluorescence intensity of the Ag–BSA complex (in fact, the chemical yield of the fluorescent clusters) on the NaBH4/Ag+ ratio (Fig. 3) had a maximum at about 0.1. The total amount of Ag ions taken per protein molecule is 30. On average, it yields three reduced Ag atoms per protein, which suggests a small size of the fluorescent metal core, in contrast to the previously proposed models involving up to 15 Ag atoms47,48,52,53. Ag clusters in HMGB1, H1, and lysozyme We also investigated other proteins with different sulfur atom content. The Ag clusters on H1, HMGB1, and lysozyme were obtained in conditions similar to that of BSA by reducing Ag ions with NaBH4. This is the first time H1 and HMGB were used as templates for NCs synthesis. Surprisingly, although H1 contains no cysteine residues, it appears that fluorescent clusters emitting in the red spectral range can be formed. Fig. 6A shows the spectra obtained for the fluorescent Ag–H1 complex. The complex had an emission peak at ca. 770 nm. The fluorescent excitation spectrum had a sharp peak at 373 nm and a long-wavelength tail. The fluorescence excitation anisotropy spectrum revealed a weak lowest-energy electronic transition at about 600 nm. The absorption spectrum differed from the excitation spectrum by the presence of a significant amount of non-fluorescent Ag species, suggesting a relatively low chemical yield of the fluorescent clusters. The excitation spectrum of the clusters in H1 differed significantly from that of the Ag–BSA complex, suggesting a somewhat different structure of the cluster. Indeed, as the protein has no cysteines, as was the case for BSA, other functional groups stabilized Ag clusters in H1.


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Figure 6. Absorption, fluorescence excitation, excitation anisotropy, and emission spectra of the Ag NCs stabilized by the proteins: A: H1 (λex 370 nm, λem 700 nm), B: HMGB1 (λex 380 nm, λem 750 nm), C: lysozyme (λex 370 nm, λem 800 nm). Absorption spectra of Ag NCs were


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obtained as the difference between the corresponding absorption spectra of Ag NCs –protein and Ag+–protein complexes. The fluorescence emission spectrum of the synthesized Ag–HMGB1 complex had a peak at about 780 nm (Fig. 6B). The fluorescence decay curve of the cluster exhibited two components: 1.5 ns and 5.7 ns (Fig. S4). As with H1, the fluorescence excitation and absorption spectra of the cluster on HMGB1 had a peak at 380 nm and a long-wavelength tail. The fluorescence excitation anisotropy curve also revealed a lowest-energy state at about 600 nm. The spectral properties of the Ag–HMGB1 complex are very similar to those of the Ag–H1 complex. The fluorescence emission spectrum of the Ag clusters on lysozyme peaked at about 800 nm (Fig. 6C). The fluorescence decay curve of the cluster had two main components: 1.1 ns and 4.4 ns (Fig. S5). As with H1 and HMGB1, the fluorescence excitation and absorption spectra exhibited a sharp peak at about 400 nm and a long-wavelength tail. The Ag clusters on the H1, HMGB1, and lysozyme templates exhibited similar spectral patterns (a narrow, ca. 0.5 eV, peak at 3.0-3.3 eV and a low-intensity low-energy transition), suggesting similar structures. The spectral properties of the clusters in these proteins differ from that of the BSA-based clusters, thus implying a different structure. In contrast to cysteine-rich BSA, in which cysteine residues stabilize the Ag clusters, H1 has no cysteines at all, and HMGB1 and lysozyme have only three and eight cysteine residues, respectively. Thus, it can be concluded that the fluorescent Ag clusters in H1, HMGB1, and lysozyme are stabilized not by cysteines, but by other amino acid residues. It is also worth noting that the maximum chemical yield of the fluorescent clusters in these proteins was also observed at low Ag/protein ratios


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(Tables S3–S5). For H1 and lysozyme, the maximum was reached at the ca. 1:1 ratio, suggesting very small cluster sizes. Probable structure of fluorescent Ag clusters in proteins It has been shown earlier67 that Ag-ligand complexes with two confined electrons are the probable candidates for experimental realization of the fluorescent complexes. For BSA, free cysteine residues are necessary for stabilizing the fluorescent Ag clusters. In BSA, up to four free sulfur atoms can be in close proximity to each other. As was shown above, the metal core contains about three silver atoms. Therefore, it is reasonable to suggest that 2–4 sulfur atoms stabilize the fluorescent metal core with two confined electrons. We considered the Ag–thiolate complexes [nAg + (n-1)L]-1, where L = SCH3 ligands, with two confined electrons investigated earlier at the TDDFT/CAMB3LYP level of the theory67. We added several other Ag–thiolate complexes in this study: [2Ag + L]-1, [2Ag + 2L]-2, and [2Ag + 2L + F]-1 with a formaldehyde (F) residue. We calculated the electronic spectra of the complexes using high-level ab initio methods that would eliminate uncertainties related to charge-transfer interactions that the TDDFT method may not account for properly. To reduce computation time, the SCH3 ligand was chosen as a model of deprotonated cysteine residues. We focused on the first 5–6 transitions and calculated the corresponding vertical excitation energies and oscillator strengths using the RICC2 and ADC(2) methods. Also, we calculated spectra in a wider range at the TDDFT/M06-2X level of theory, which was shown to give an accurate description of excited states in Ag3+-DNA complexes107. We also compared the spectra of two complexes calculated using the ADC(2) and the SCS-ADC(2) (spin-component scaling) approaches. The difference between them appears to be insignificant (Table S6).


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The optimized structure of the Ag5(SCH3)4 complex is presented in Figs. 7 and S6. In this complex, the silver core, Ag3+1, contains three Ag atoms. The electronic absorption spectra calculated at the ADC(2) and TDDFT/M06-2X levels of the theory appeared very close to the experimental fluorescence excitation spectrum of the Ag–BSA complex (Fig. 7). The calculations made by the ADC(2), RI-CC2 and TDDFT/M06-2X methods are consistent with each other, validating the calculation approach (Table S7). For comparison, the electronic transitions calculated for other complexes are also presented in Table S7. Of course, in the protein, the geometry of the cluster stabilized by the neighboring cysteine residues may be somewhat different from that presented, but this requires additional robust QM/MM calculations. In any case, our calculations show that just two reduced Ag atoms appear sufficient to form the fluorescent cluster in the protein. This closely resembles the models of the DNA-based Ag3+1 fluorescent clusters we have proposed previously32. Small three-atom Ag clusters have also been identified as fluorescent centers in rare gas matrices108 and zeolites109,110.


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Figure 7. Absorption spectra of the Ag5(SCH3)4 complex calculated at ADC(2)/def2QZVP/P (first six transitions) and TDDFT/M06-2X/def2QZVP/P level of the theory and the experimental fluorescence excitation spectrum of BSA-templated Ag clusters. The calculated spectrum is broadened by a Gaussian with a width of 0.6 eV. As concluded above, the fluorescent Ag clusters in H1, HMGB1, and lysozyme are liganded not by cysteines but by other amino acid residues. In our recent study we show that only cysteine and tyrosine are able to form the fluorescent complexes with silver clusters in solution68. Along with cysteine, tyrosine has the highest binding Gibbs free energy values with Ag2 clusters66. Thus, it is reasonable to suggest that the fluorescent Ag clusters in H1, HMGB1, and lysozyme are stabilized by tyrosine rather than other amino acid residues. The excitation spectra of the clusters (Fig. 6) reveal a strong electronic transition at about 400 nm and a weak lowest-energy band in the red spectral range. The radiative decay from this lowest-energy state is responsible


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for the red/near infrared emission of the clusters. We modeled the Ag clusters with two confined electrons stabilized by tyrosine residues in the phenolate ion (Ph) and sQ forms and also by the formaldehyde ligand. Under alkaline conditions, tyrosine enables electron transfer from Ph to the Ag cations, resulting in the phenolic form of tyrosine converting to the sQ form111. Therefore, the sQ form of the phenolate (O=C6H6) was chosen as a ligand in the complexes with Ag2 clusters. We also used the formaldehyde ligand as a model of the asparagine, aspartate, glutamine, and glutamate residues for comparison. Fig. S6 shows the structures of all studied complexes. The corresponding energies and oscillator strengths of the first six transitions are presented in Table S8. The complexes of Ag2 clusters with Ph, sQ, and formaldehyde exhibited strong transition at about 400 nm. The complexes with sQ, however, were the only complexes with a low-intensity, low-energy state, which is the characteristic feature of the excitation spectra of H1, HMGB1, and lysozyme. The calculated excitation and fluorescence anisotropy spectra of the complex of Ag2 clusters with the sQ appeared close to the experimental spectra for H1, HMGB, and lysozyme. Fig. 8 shows the calculated and experimental spectra of the complex of Ag NCs with HMGB1. It should be noted that there might be an additional band at ca. 280 nm in the experimental excitation spectra of HMGB1 and lysozyme due to possible energy transfer from tryptophan residues to the clusters. The lowest-energy charge-transfer transition in the calculated spectrum (marked by the red arrow) has low oscillator strength. However, the geometry of the complex of Ag2 clusters with tyrosine in the surrounding protein may differ from that found in vacuum. The oscillator strength of the lowest transition in the complex can thus be altered. A reasonable agreement between the calculated and experimental spectra allows one to propose that Ag2–tyrosine complexes can be


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luminescent centers in H1, HMGB1, and lysozyme. It is worth to note that two-atom Ag2 clusters may form in solution112,113. They exhibit strong absorption at 450 nm and emission at 540 nm.

Figure 8. Absorption spectrum of the Ag2sQ complex calculated at ADC(2)/def2QZVP/P level of the theory and the experimental absorption and fluorescence excitation spectra of HMGB1templated Ag clusters. The calculated spectrum is broadened by a Gaussian with a width of 0.4 eV. Conclusions We investigated the synthesis conditions and spectral properties of fluorescent Ag clusters stabilized by different protein templates. We also calculated the geometries and electronic spectra of some cysteine- and tyrosine-protected clusters with two confined electrons at ADC(2)/def2QZVP/P level of the theory. We propose a model for fluorescent Ag–protein complexes, in which two reduced Ag atoms are sufficient to form the fluorescent core of the complex. The structural model of the luminescent centers in the Ag–protein complexes we propose differs from the common view that the fluorescent metal NCs in proteins contain about 10 or more metal atoms. The fluorescent Ag clusters formed on the four investigated natural


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protein matrices have two different spectral and structural patterns. Deprotonated free cysteine residues stabilize the fluorescent Ag3+1 core formed in the BSA matrix. The second type of fluorescent centers is formed in H1, HMGB, and lysozyme protein matrixes. In this case, tyrosine residues probably stabilize the fluorescent Ag2 centers. ASSOCIATED CONTENT Supporting Information. Fluorescence emission spectra of the Ag-BSA complex; Raman spectra of BSA and the Ag-BSA complex; Fluorescence decay curves of the Ag-protein complexes; Dependence of the fluorescence intensity of the Ag-protein complexes on the ratios of reagents; Electronic spectra calculated for the silver-ligand complexes; Structures of the silver-ligand complexes (PDF). AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors acknowledge RSF for research grant 16-13-10090. This work was carried out using equipment from the Centre for Optical and Laser Materials Research and Centre for Physical Methods of Surface Investigation of Saint Petersburg State University. The reported calculations were carried out on the Stallo supercomputer at the University of Tromsø.


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Fluorescent Silver Clusters on Protein Templates: Understanding

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