Understanding the Effect of Single Cysteine Mutations on Gold

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Understanding the Effect of Single Cysteine Mutations on Gold Nanoclusters as Studied by Spectroscopy and Density Functional Theory modeling Sayantani Chall, Soumya Sundar Mati, Indranee Das, Amrita Kundu, Goutam De, and Krishnananda Chattopadhyay Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01789 • Publication Date (Web): 06 Oct 2017 Downloaded from http://pubs.acs.org on October 7, 2017

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Understanding the Effect of Single Cysteine Mutations on Gold Nanoclusters as Studied by Spectroscopy and Density Functional Theory modeling

Sayantani Chall†1, Soumya Sundar Mati‡2, Indranee Das§3, Amrita Kundu†4, Goutam De§ and Krishnananda Chattopadhyay†*

†

Structural Biology & Bio-Informatics Division, CSIR-Indian Institute of Chemical

Biology, 4, Raja S. C. Mallick Road, Kolkata 700032, India. ‡

Department

of

Chemistry,

Govt.

General

Degree

College,

Keshiary,

PaschimMedinipur, 721135 §

CSIR-Central Glass and Ceramic Research Institute, 196, Raja S. C. Mullick Road,

Kolkata, 700032, India.

*Corresponding Author Email: [email protected]

**

Authors 1 & 2 contributed equally; Authors 3 & 4 contributed equally.

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Abstract: Fluorescent metal nanoclusters have generated considerable excitements in nano-bio technology, particularly in the applications of bio-labeling, targeted delivery, and biological sensing. The present work is an experimental and computational study to understand the effect of protein environment on the synthesis and electronic properties of the gold nanoclusters. MPT63, a drug target of Mycobacterium tuberculosis, has been used as the template protein to synthesize, for the first time, gold nanoclusters at a low micromolar concentration of the protein. Two single cysteine mutants of MPT63, namely MPT63Gly20Cys (Mutant-I) and MPT63Gly40Cys (Mutant-II) have been employed for this study. Experimental results show that cysteine residues positioned into two different regions of the protein induce varying electronic states characteristics of the nanoclusters depending on the surrounding amino acids. A mixture of five atoms and eight atoms clusters has been generated for both mutants, and the former has been found to be predominant. Computational studies, including the DFT (density functional theory), FMO (frontier molecular orbital) and NBO (natural bond orbital) calculations validate the experimental observations. The asprepared protein stabilized nanoclusters are found to have applications in live cell imaging.


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Introduction: The development of fluorescent noble metal nanoclusters is considered as one of the most exciting achievements of nanotechnology. This is because of the ability of metal nanoclusters to incorporate biological macromolecules as intrinsic structural or functional components1-7 Protein molecules are now used as templates to synthesize and stabilize fluorescent gold nanoclusters (FGNC), which possess enhanced biological specificity and functionality.1, 8-10 More importantly, the presence of protein environment renders significantly low cytotoxicity and high stability over a wide range of pH thereby making these materials useful for biological applications, e.g., bio-sensing, bio-labeling, bio-imaging and targeted drug delivery.1 In the protein based synthesis method of FGNC, Au ions are first sequestered by the protein molecule, which can then be in situ reduced at alkaline pH. Various proteins such as BSA,8,

11-13

HSA,14 human transferrin,15 ferritin,16 trypsin,17 pepsin,18 horseradish

peroxidase,19 insulin,20 and lysozyme9 have been employed for the preparation of FGNC. The structure of the nanoclusters and the resulting fluorescence are found to be modulated by the nature of the template proteins. Although there exist multiple articles describing the synthesis of fluorescent gold nanoclusters using different protein molecules21-24, it is still unclear how protein conformation affects the properties of gold nanoclusters. Carriet. al.12 have used the computational method to show that the binding process of peptides on gold surfaces can be changed by varying amino acids. An experimental attempt has been made by W. R. Golmnet. al.13 in which the authors used eight different protein scaffolds, which yielded varying optical characteristics. Although their syntheses were carried out under identical experimental condition, they used different template proteins, which differed significantly in the size, conformation and structural integrity. For example, myoglobin, a protein template they used is completely alpha helical, while another protein beta lactalbumin is predominantly beta sheet. Also, the former protein contains a heme co-factor whose effect is difficult to


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determine. We believe that a more robust understanding of the effect of protein environment can be obtained only by using a single template protein (and hence keeping the overall conformation identical) but using different regions of the template for the nanocluster synthesis. In this manuscript, we have addressed this by incorporating an Au binding center at two separate parts of a single protein template, MPT63 (Fig 1).25 MPT63 is a globular protein of 130 amino acids with an immunoglobulin like fold. It is a species specific protein found only in Mycobacterium tuberculosis, and it does not show any cross reactivity towards other Mycobacterium proteins. MPT63 has been demonstrated to induce the humoral immune response in infected guinea pigs. MPT63 has been found its use not only against Mycobacterium tuberculosis,26-29 but also as an active antiviral agent in a formulation developed against HIV/AIDS.15 The recent research effort in our laboratory has been concentrated on to produce nanoparticles based delivery system of MPT63. In addition to the primary objective as discussed above, the secondary aim of the present manuscript is to understand if the position dependence of nano-surface may play any role towards the stability of nanoparticle-drug candidate conjugation. Wild type MPT63 does not have any cysteine residue in its sequence, and hence we have generated two single cysteine mutants using site directed mutagenesis. In the first mutant (MPT63Gly20Cys, Mutant-I), a glycine residue at the position 20 has been replaced with a cysteine, while in the second mutant (MPT63Gly40Cys, Mutant-II), the replaced glycine is at the position 40. These two glycine residues (at positions 20 and 40) are situated on the connecting loops of anti-parallel strands on each β-sheet (Fig 1). Charge distributions at the surface of these two residues are different. We have found that the yield of recombinantly expressed protein can be a bottleneck, and a majority of the previous studies have been carried out using commercially available protein systems which can be obtained at high


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concentrations. To circumvent this shortcoming, we have optimized for the first time a synthesis procedure, which requires µM protein concentration. Syntheses of FGNC using these two mutants (Au-I with the Mutant-I, and Au-II with the Mutant-II) yield a mixture of nanoclusters (Au5 and Au8), with Au5 being the primary product. The synthesis of the FGNC was characterized by MALDI (Matrix Assisted Laser Desorption / Ionization)-TOF (Time of Flight) mass spectroscopy, fluorescence spectroscopy, X-Ray photoelectron spectroscopy (XPS), Transmission Electron Microscopy (TEM) and small angle X-Ray scattering techniques. Also, the use of DFT (density functional theory) was found to be useful to understand the nature of interactions between Au nanoclusters and cysteine mutants. DFT calculations assisted in the understanding of the experimental findings. The use of FMO (frontier molecular orbital) approach helped us to demonstrate the energy values between the HOMO-LUMO gaps of two clusters. To obtain an insight into the charge distribution of the intermolecular interactions, the geometries obtained from DFT calculations were used to perform the natural population analysis (NPA) and natural bond orbital (NBO) analysis.

Experimental Section: Materials: AR grade salt Tetrachloroauric acid (HAuCl4) was purchased from Spectrochem. Sodium borohydride (NaBH4), sodium hydroxide granules (NaOH), were obtained from Merck, India. Throughout the experiment, high purity water (≈18.2 MU) was used. Dialysis tube (molecular weight cut off 90%), were pooled and dialyzed against 20 mM Sodium phosphate buffer of


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pH 7.5 and aliquots were stored at -20 ᵒC until required. For purification of the cysteine mutants, 10 mM DTT has been added to the protein solution. Before any experimental study, excess DTT has been removed by extensive dialysis of the protein sample. Characterization: Room-temperature optical absorption spectra were recorded by UV-VIS spectrophotometer (Shimadzu, UV-2450) using a cuvette of 1 cm path length. The steadystate fluorescence experiments were carried out using a PTI fluorescence spectrophotometer (Photon Technology International, USA) and Shimadzu spectrofluorimeter (model RF 5301). The fluorescence emission spectra were recorded at excitation wavelength 325 nm and 385 nm. Fluorescence lifetimes were determined from time-resolved intensity decay by the method of time correlated single-photon counting using a nanosecond diode LED at 370 nm (IBH, nanoLED-07) as the light source. The data stored in a multichannel analyzer were routinely transferred to the IBH DAS-6 decay analysis software. For all the lifetime measurements, the fluorescence decay curves were analyzed using the bi-exponential iterative fitting program provided by IBH as in Eqn (1)30 = exp − … … … 1

Where ai is the pre-exponential factor representing the fractional contribution to the time resolved decay of the component with lifetime τi. Average lifetimes 〈τ〉 for bi-exponential decays of fluorescence were calculated from the decay times and pre-exponential factors using the following equation31, < > =

+ … … … … … 2 +

All fluorescence spectra were corrected with respect to instrumental response. All measurements were repeatedly done, and reproducible results were obtained. The transmission electron microscopy (TEM) image was obtained using JEOL-JEM-2100F transmission electron microscope. For this purpose, the sample was placed on carbon coated


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Cu grids (normal) and analyzed. X-ray photoelectron spectroscopy (XPS) measurement of the sample was done on a PHI 5000 Versaprobe II XPS system with an Al Kα source and a charge neutralizer at room temperature, maintaining a base pressure at about 6×10-10 mbar and an energy resolution of 0.6 eV. MALDI-TOF mass spectroscopy was carried out using ultrafleXtreme MALDI-TOF/TOF (Bruker) mass spectrometer. Computational details: The ground state geometry of nanoclusters Au5 and Au8 were optimized employing density functional theory32-33 using the B3LYP34-35 functional with the standard basis set, LanL2DZ, for all atoms. All the structures corresponding to actual minima of the potential energy surface were confirmed by the vibrational frequency calculations. Electronic transition wavelengths and oscillator strengths were calculated using time dependent density functional theory (TD-DFT)36-37 using same functional and basis set. The above computations were carried out with Gaussian 0338 program package. Cell imaging Study: We have used cervical cancer cell line HeLa for the live cell experiments. HeLa cells were maintained in DMEM supplemented with 10% heatinactivated fatal bovine serum (FBS),110mg/L sodium pyruvate, 4mM l-glutamine, 100units/ml penicillin and 100 µg/ml streptomycin in humidified air containing 5% CO2 at 37°C. Cells were seeded in a 35mm poly-D-lysine coated plate (MatTek Corporation, Ashland, MA) and grown to ~75% confluency. After that these cells were treated with goldMPT63 nanocluster solution at a particular concentration before imaging. These experiments were carried out using a Zeiss LSM510 Meta confocal microscope equipped with CApochromat 40X (NA=1.20, water immersion) objective. The nanocluster was excited using a diode laser at 405 nm.

Results and Discussions: Synthesis and characterization of Au-MPT63 Nanoclusters:


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It may be noted that most of the previous syntheses (if not all) of protein stabilized nanoclusters were carried out using relatively high concentration of commercially available proteins. In contrast, the aim of this study required the use of a recombinantly expressed protein, which could be purified only at a low concentration. The use of low protein concentration necessitated optimization of the published protocol of protein-nanocluster synthesis used by Xie's group8. The synthesis optimization was achieved by varying different reaction parameters, including protein concentrations, gold concentrations, pH and temperature during the syntheses steps. After the synthesis, the solution was dialyzed to purify the synthesized product. The concentrations of cysteine mutants were varied between 0.12 mg mL-1 and 0.90 mg mL-1. Gold concentration was varied from 0.6 mM to 2.5 mM. 50 µl of 1M NaOH was added during each synthesis. The reaction condition was optimized by measuring the fluorescence intensity of the prepared nanoclusters. The maximum fluorescence intensity was observed using 0.9 mg/ml protein concentration and 2.5 mM gold concentration. A pH dependence within a pH range between 3 and 12 determined pH 8 to be the best for nanocluster yield and stability (Fig S1). The optimum temperature for the synthesis was found to be 37°C. Reaction time for the synthesis of the nanocluster was also optimized to be 16hours to complete the growth kinetics of the nanoclusters (Fig S2a). The stability of the as-prepared protein capped nanocluster was also checked for 7 days (Fig S2b). Optical spectroscopy has been extensively used to monitor the growth and structural feature of gold nanoclusters. In contrast to the nanoparticles, which are characterized by their surface plasmon resonance band, nanoclusters offer fluorescence properties. Absorption spectra of Au-I (Au-Mutant-I) did not show any characteristic surface plasmon resonance band (Fig 2a). However, the ground state behavior of the as-prepared materials was investigated using excitation spectral study.


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Excitation spectrum of Au-I (λem = 405 nm, Fig 2b,c) showed a predominant component peak at 325 nm with a small but prominent contribution at a higher wavelength (385 nm). A third component was observed at around 280 nm as a result of the contribution of aromatic residues of the protein (e.g., tyrosine and tryptophan). The emission maxima corresponding to these two excitations were found to be at 405 nm (excitation at 325 nm) and 445 nm (excitation at 385 nm). According to literature,39-41 5 atom gold nanocluster (Au5) corresponds to λex = 325 nm, λem = 405 nm, while 8 atom gold cluster (Au8) corresponds to λex = 385 nm, λem = 445 nm. Our inference is supported by the observation reported by C. Helmbrecht and his co-workers. The above assignment of the nanocluster atoms based on the emission wavelength can be backed up by the electron Jellium model. The Fermi energy (EFermi) of gold is approximately 5.5 eV (Fig 3).41 Fig 3 shows a simulated trend of the number of gold atoms against the emission wavelength, which has been developed using the spherical Jellium model (Eem = EFermi/N1/3). The marked points in Fig 3 show the expected emission wavelengths of 5 atoms (385 nm) and 8 atoms (450 nm) gold nanoclusters. The slight deviation of the λems from the Jellium model derived numbers (385 nm and 450 nm) from the experimental values (405 nm and 445 nm) comes from the influence of the protein environment on the gold atoms. The presence of protein environment has been reported to shift the emission wavelengths of metallic nanoclusters.42 Mass spectrum analysis: To ascertain clusters size, we used MALDI mass spectrometry of Au-MPT63 nanoclusters. Fig S3a shows the mass spectrum observed with Mutant-I (MPT63Gly20Cys), in which a peak at 16.3 kDa was obtained. In contrast, the mass analysis of Au-I was found to give peaks at 33.6 kDa and 34.3 kDa (Fig S3b). The experimental results affirmed the formation of ̴ Au5 and ̴ Au8 nanoclusters respectively for a 1:2 Au to protein complex (Fig S3b). The mass spectrometry result validates DFT calculations (see 10 ACS Paragon Plus Environment

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below). The observation of Au5 and Au8 clusters in mass spectrometry is in good agreement with the spectrofluorometric data. XPS analysis: Oxidation state and binding properties of synthesized nanoclusters were determined by XPS (Fig 4). After deconvolution of the Au 4f, distinct peaks of Au (0) for 4f7/2 and 4f5/2 components centered at 83.8 eV and 87.2 eV respectively, were observed (Fig 4a). Such result indicated that a major amount of Au3+ was reduced to Au (0) in the gold-MPT63 system during the synthesis procedure at basic environment (Fig 4a). A relatively less intense peak of Au1+ for 4f5/2 (at 89.9 eV) was also observed (~19.2 mol% calculated from XPS spectra). These Au+1 species were expected to be located at the surface of gold clusters facilitating strong Au-S bonding in the protein nanoclusters.43 In Fig4b, the peaks for binding energies of S 2p3/2at 162.3 and 167.6 eV could be attributed to the sulfur bound to gold and oxidized sulfur, respectively.44 The peak positioned at 162.3 eV corresponded to the covalent interaction of gold nanoclusters with the sulfur groups present in the cysteine residue of MPT63. Whereas, the formation of oxidized sulfur could be resulted due to degradation of protein in the presence of highly alkaline environment (pH ~11) during the synthesis of gold nanoclusters. TEM study:

The protein templated gold nanoclusters were further investigated by TEM (Fig 5). The bright field TEM micrograph showed the presence of many spherical gold nanoclusters (Fig 5a). Their size distribution (typically in the range of 1.08–3.2 nm) resulted an average diameter of ~2.08 nm (considering about 200 nanoclusters; inset of Fig 5a). It is noteworthy here that the size of the Au5 or Au8 clusters (existence of the nanoclusters was confirmed by mass spectroscopy) should normally be close to 1 nm.18 However, we observed the population of relatively larger nanoclusters in the bright field image of TEM (~1−3 nm). This 11 ACS Paragon Plus Environment

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is presumably because of the fact that these small clusters possessing high surface energy can coalesce to attain stability. During TEM grid preparation and also when exposed to electron beam, clusters have been shown to coalesce due to the solvent evaporation45. HRTEM image (Fig 5b) revealed nanoclusters with lattice fringes corresponding to (111) plane of gold (0.23 nm). A close look at HRTEM image showed additionally the presence of several twinning in these nanoclusters (shown by arrows in Fig5b).These data supported above mentioned hypothesis of the growth of nanoclusters as a result of coalescence of the ultra-small gold clusters.46,47,48 The effect of the position of the cysteine residues on the spectroscopic properties of the MPT63 clusters: Absorption spectroscopy indicated the absence of surface plasmon resonance bands in both Au-I (Au-Mutant-I, Mutant-I is MPT63Gly20Cys) and Au-II (Au-Mutant-II, Mutant-II is MPT63Gly40Cys). The analyses of the excitation and emission spectroscopy suggested that for Au-I and Au-II, there was a mixture of two clusters, namely Au5 and Au8. For both Au-I and Au-II, the major excitation was found to be at 325 nm, with small contributions from 385 nm (Fig 2 and S4). The corresponding emission maxima were found to be at 405 nm and 445 nm, which were expected for the Au5 and Au8 clusters respectively. Although both Au5 and Au8 were found to be present in two mutants, Au5 was always found to be the dominant species. This result further implied that the odd atom gold nanocluster (Au5) is more stable than the even number Au8. It has been shown theoretically that the odd atom gold nanocluster is more stable when interacting with prolines.49 The present study provides experimental support to that prediction. It was also observed that Au5-I had significantly high fluorescence intensity compared to Au5-II. It was demonstrated both by the fluorescence spectra and also by determining the relative quantum yields (0.042% and 0.013% for Au5 in Mutant-I and Mutant-II 12 ACS Paragon Plus Environment

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respectively). To obtain further insights into the excited state dynamics of the nanoclusters, we performed time resolved measurements of Au5 and Au8 clusters present in Au-I and AuII. The life time values of small atom nanocluster Au5 exhibited a bi-exponential decay curve, whereas lifetime of Au8 exhibited tri-exponential decay (Fig 6, Table 1). The average lifetime of the four variants (i.e., Au5-I, Au5-II, Au8-I, and Au8-II) were found to be 0.17 ns, 0.034 ns, 0.25 ns and 0.16 ns respectively. The lifetime results suggested the following: first, Au5 and Au8 differed mostly by the presence of a long lifetime component. Second, the small sub-nanosecond lifetime component of Au5was significantly quenched in Mutant-II (0.02 ns) when compared to that in Mutant-I (0.16 ns). Although accurate measurement of 0.02 ns component was challenging in our setup, repetitive measurements established the validity of the large difference in the lifetime values. Structure and energetic of protein-nanocluster complex: DFT analysis Density functional theory calculations were used to understand and validate the experimental observation of Au-I and Au-II using geometric structure and energetic considerations. Geometry Configuration: For these calculations, it is assumed that the –SH residue of cysteine residue is the major contributor to the protein-nanocluster complex. The experimental proof of this assumption came directly from the XPS measurements. Further experimental verification was obtained by repeating the synthesis in the presence of TCEP (tris(2-carboxyethyl)phosphine). TCEP is typically used for –SH protection. This experiment made sure that, in the presence of TCEP, cysteine residue would be unavailable for nanocluster complexation.50 We observed a substantial decrease in fluorescence intensity, implying diminished yield of the desired nanoclusters (Fig S5).


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Considering an imaginary sphere of radius 5Å and keeping a cysteine residue at the center of the sphere (Fig 7), it was observed that Val and Glu were the nearest neighbor of gold surface in Au-I. For Au-II, these residues were Pro and Tyr. Consequently, a three residue fragment of the protein containing these residues was chosen for each nanocluster. For example, a sequence of Val-Cys-Glu was selected for Au-I, while for Au-II the chosen sequence was Pro-Cys-Tyr. By extensive computational studies with Au-I and Au-II (n = 5 and 8), eight possible geometry conformations could be proposed (Fig 8 and Fig S6). The initial four geometries of Au5/8-I and Au5/8-II were generated by placing one gold cluster near the cysteine site of one protein molecule (1:1 complex, Fig S6). After geometry optimization of these four (Au5-I, Au5-II, and Au8-I, Au8-II) 1:1 gold compounds, time dependent density functional theory (TD-DFT) was used to calculate their absorption wavelength maxima (λabsmax). It was found out that the estimated λabsmax for these four 1:1 complex structures were very different from the experimental findings (Table 2). The deviation between the calculated and experimental values ruled out 1:1 complex formation. Subsequently, another active cysteine site was introduced to optimize four 1:2 complex structures of the nanoclusters (Au5-I2, Au5-II2, Au8-I2, and Au8-II2). The calculated values of λabsmax of these 1:2 complexes matched with the experimental findings (Table 2). From the optimized structures, it can be seen that the active sites of the amino acids of Mutant-I and Mutant-II are amines, carboxylic acids and sulfur groups. These groups have electron rich nitrogen, oxygen and sulfur atoms, forming the anchoring bonds with Au clusters in the complexes, donating electron density to the 5d and 6s orbitals of Au via their lone pairs. Gold atom can also play the role of a proton acceptor forming nonconventional hydrogen bonds (H-bond, Au···H) with amines and/or the carboxylic acid groups of the amino acids. Two particularly important non-conventional hydrogen bonding interactions involved 100Au-64H


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(2.43 Å) of Au5-II2 and 51Au-16H (2.55Å) of Au8-II2. In these cases, Au5 and Au8 were found to act as proton acceptors, a result observed in earlier studies51,49. Moreover, a significant change in Au-S bond length was observed for these mutants (Table 3).More precisely, different chemical environments, (Val19-Cys20-Glu21) in Au-I and (Pro39-Cys40Tyr41) in Au-II formed Au-S bonds of different lengths; those of Au-II(2.43 Å and 2.45 Å for 5 atoms and 2.42 Å and 2.48 Å for 8 atoms cluster) were found less than those of Au-I (2.51Å and 2.54 Å for 5 atoms and 2.45 Å and 2.50Å for 8 atoms cluster) (Table 3) indicating significant role of the protein sequence on the overall stabilization of the nanoclusters. The other types of bond lengths are presented in Table 3. We subsequently calculated λabs for 1:3 complexation ratio. This possibility was ruled out because of the deviation of calculated λabs (= 298 nm for Au5-I3) from the experimentally observed value (λabs = 325 nm). Spectral Properties and Frontier Molecular Orbital (FMO) Analyses: The ground state geometry optimized using the above calculation was used as the inputs for the time dependent-DFT (TD-DFT) calculations. These calculations were used to obtain information related to the absorption spectrum and oscillator strength (f) of the gold nanocluster-protein complex structures (Au5-I2, Au5-II2, and Au8-I2, Au8-II2). It can be seen from the simulated absorption spectra that Au5 cluster complexes have λabsmax of 324 nm and 327 nm for Au-I and Au-II respectively. Alternatively, Au8 cluster complexes have λabsmax of 384 nm and 383 nm for Au-I and Au-II respectively. Hence, absorption spectra calculated using the TD-DFT method was found in accordance with the values obtained by experimental studies (Table 2). The TD-DFT method provided proper interpretation regarding electron transition from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO). This process can yield a qualitative overview of electronic property of the clusters regarding their ability of electron/hole transport. Fig 9 shows the positions of HOMO and


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LUMO regarding their energy and λabsmax values for the protein-nanocluster complexes. Au5 has H-L energy gap of 3.79 eV for Au-I, and 3.63 eV for Au-II whereas Au8 has a lower H-L gap of 3.19 eV for Au-Iand3.18 eV for Au-II. FMOs analysis (Fig 9) ascertained clearly that Au5 nanoclusters would be energetically more stable than Au8clusters.

Natural Bond Orbital (NBO) analysis: NBOs can generate the most accurate possible expression of molecular properties regarding ‘natural Lewis structure' of the wave function. NBO allows any aspect of the wave function to be expressed in terms of Lewis(one-center lone-pair or two-centers bond-pair) and nonLewis type (all remaining orbitals) contributions, enabling us to provide information about the interactions in both filled and virtual orbital spaces that facilitate the analysis of intra- and intermolecular interactions.49, 52The analysis of the detailed natural bond interaction of the four complexes Au5-I2, Au5-II2, and Au8-I2,Au8-II2, are presented in Table 4 in which each complex has two S–Au bonds. There is a possibility of the existence of both covalent and donor-acceptor bonds between SAu atoms. The covalent bond is the recombination of the unpaired electrons of the sulfur and Au atoms. In contrast, the donor–acceptor bond can be formed by the donation of the lone pair electrons of the sulfur atom to the empty 6p orbital of the Au atom. If we consider that the covalent Au-S bond formation occurs through the interaction of hybridized 6s/6p orbital of Au with the hybridized 3s/3p orbital of sulfur, then the observation of weak contribution of s and p orbital in the hybridization process (Table 4) diminishes the possibility of S-Au covalent bond formation. On the other hand, the donor-acceptor (bond-antibonding) interactions were taken into consideration by examining all possible interactions between “filled” (donor) Lewis-type NBOs and “empty” (acceptor) non-Lewis NBOs and then estimating their energies by second order perturbation theory. 16 ACS Paragon Plus Environment

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For each donor NBO (i) and acceptor NBO (j), the stabilization energy E(2) associated with delocalization i to j is estimated by Eqn 3:53 E(2) = ∆Eij = qiF(i,j)2/(ɛi - ɛj) ........... (3) Where qi is the donor-orbital occupancy, εi and εj are diagonal elements, and F(i,j) is the offdiagonal NBO Fock matrix element. The donors in S-Au bonds are either 3p lone pair electrons of sulfur atoms (ns) or σs-c bond, and the acceptors are 6p empty orbital (n*) of gold atoms. The second-order perturbation stabilization energies or E(2) thus calculated are shown in Table 5, which was found to be more than 5 kcalmol-1 for all four complexes (Au5-I2, Au5II2, Au8-I2, andAu8-II2). Two types of donor–acceptor interactions show that the donation of electrons from ns→ n* of Au is energetically more favored than from the σs-c bond. Thus the donation of ns of sulfur atom to the n* of Au is the primary charge transfer channel for all the four complexes. In Au8-I2 the largest second-order perturbation stabilization energies (E2) of S-Au bonds are 6.69 and 6.12 kcal mol-1, which were smaller than 7.44 and 6.12 kcal mol-1 observed for Au5I2(Table 5). Thus, the donation of electrons from S atom to the Au5 and Au8 atoms for Au-I is the primary charge transfer channel and mostly favorable for Au5 for higher perturbation stabilization energies. On the other hand, if we consider the perturbation energies for Au-II, it can be observed that E(2) values of S-Au bond are 8.16 and 10.78 kcal mol-1 for Au5-II, which were slightly lower than those observed for Au8-II(10.66 and 13.40 kcal mol-1). However, there existed three nonconventional hydrogen bonding interaction in Au5II2whereas only one was found inAu8-II2. The presence of nonconventional hydrogen bonding further provided extra stability towards Au5-II. Thus, all these above investigations (geometry optimization, FMO analysis and second order perturbation energy calculations) nicely corroborate our proposed thought that amino acid sequences, as well as residue position, may have a significant influence on the electronic 17 ACS Paragon Plus Environment

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interaction between a protein and a nanocluster. Positional differences in the cysteine residue caused a significant change in S-Au bond length, bond hybridizations, and E(2) values. All these reasons ultimately resulted in the formation Au5 as the primary product. We observed that Au5-I had higher relative fluorescence than Au5-II. The overall interaction of gold surface with cysteine and associated amino acid residues may be the most influential factor for the said observation. Again, higher values of oscillator frequency (f) of electronic transition supported the observed greater fluorescence intensity of Au5-I compared to the other one. Application to imaging: Since the template protein MPT63 can be a potential therapeutic agent for tuberculosis, we also investigated the capabilities of mammalian cells to absorb/uptake of these protein-gold nanoclusters conjugates. To inquire into this, we cultured HeLa cells and treated them with 2.5 mM of gold nanocluster solution at 37 °C. After four hours of treatment; we washed the cells with PBS to proceed for confocal imaging. Fig 10 shows the presence of fluorescent nanoclusters inside HeLa cells. Overlay images show that those fluorescent nanoclusters were incorporated throughout cell cytosol. The healthy shape and size of HeLa cells suggested that this treatment was not harmful to the cells. The ease of cellular incorporation and fluorescent properties would make these gold nanoclusters reported here a possible candidate for biomedical applications.

Conclusions: Proteins are usually a well-studied template for the synthesis of fluorescent gold nanoclusters. The present work aims to determine how protein sequences and conformation influence nano cluster synthesis and properties, which is an unexplored problem till date. We chose MPT63 as our template for its role as anti-mycobacterial and anti-viral agent, and there is an urgent


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need towards the development of its nano-formulation. We noted that a clear bottleneck towards studying protein position dependence originated from a relatively small yield of recombinantly expressed protein in the laboratory scale. To overcome this, we developed, for the first time, a synthesis optimization which could be applied at a low micromolar concentration. While a battery of techniques was used for the detailed characterization of the synthesized nanoclusters, steady state and time resolved fluorescence spectroscopy inferred the formation of Au5 and Au8 clusters in a mixture, with the former being the major contributor. This inference was confirmed by mass spectroscopy. The experimental findings were complemented and validated by different theoretical calculations, including DFT, FMO, and NBO. Both experimental and theoretical observations detailed in this study show convincingly that protein environment can play an important role in modulating nanocluster properties. Additionally, we demonstrated here the cellular uptaking of the complex conjugate without inducing significant cellular stress.

Acknowledgement: Authors S.C. and S.S.M have equal contributions and authors A.K. and I.D. have equal contributions. Author S.C. acknowledges DST Nanomission (JNC/AO/A-0610/(27)/20151964) for providing postdoctoral fellowship. Author K.C. acknowledges the SERB funding (EMR/2016/000310). Authors A.K. and I.D. acknowledge UGC and CSIR respectively for providing their SRF. KC thanks the director, CSIR-IICB for his encouragements and supports.

Supporting Information: Fig S1represents pH effect on the gold nanoclusters preparation, and Fig S2 represents stability and growth of nanocluster. Fig S3 represents MALDI-TOF mass spectra analysis of protein and protein stabilized gold nanocluster. Fig S4 describes excitation and emission


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spectra of Au5/8-II. Fig S5 depicts fluorescence spectra of Au NC in the absence and presence of TCEP. Fig S6 shows 1:1 geometrically optimized structure of Protein-Nanocluster complex.


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References: 1.

Chen, L.-Y.; Wang, C.-W.; Yuan, Z.; Chang, H.-T. Fluorescent gold nanoclusters:

recent advances in sensing and imaging. Anal. Chem. 2015, 87, 216-229. 2.

Li, J.; Zhu, J.-J.; Xu, K. Fluorescent metal nanoclusters: From synthesis to

applications. TrAC, Trends Anal. Chem. 2014, 58, 90-98. 3.

Lin, C.-A. J.; Lee, C.-H.; Hsieh, J.-T.; Wang, H.-H.; Li, J. K.; Shen, J.-L.; Chan, W.-

H.; Yeh, H.-I.; Chang, W. H. Review: Synthesis of fluorescent metallic nanoclusters toward biomedical application: recent progress and present challenges. J. Med. Biol. Eng. 2009, 29, 276-283. 4.

Lin, C.-A. J.; Yang, T.-Y.; Lee, C.-H.; Huang, S. H.; Sperling, R. A.; Zanella, M.; Li,

J. K.; Shen, J.-L.; Wang, H.-H.; Yeh, H.-I. Synthesis, characterization, and bioconjugation of fluorescent gold nanoclusters toward biological labeling applications. ACS nano 2009, 3, 395-401. 5.

Slocik, J. M.; Wright, D. W. Biomimetic mineralization of noble metal nanoclusters.

Biomacromolecules 2003, 4, 1135-1141. 6.

Zheng, J.; Nicovich, P. R.; Dickson, R. M. Highly fluorescent noble-metal quantum

dots. Annu. Rev. Phys. Chem. 2007, 58, 409-431. 7.

Shang, L.; Dong, S.; Nienhaus, G. U. Ultra-small fluorescent metal nanoclusters:

synthesis and biological applications. Nano Today 2011, 6, 401-418. 8.

Xie, J.; Zheng, Y.; Ying, J. Y. Protein-directed synthesis of highly fluorescent gold

nanoclusters. J. Am. Chem. Soc. 2009, 131, 888-889. 9.

Lin, Y.-H.; Tseng, W.-L. Ultrasensitive sensing of Hg2+ and CH3Hg+ based on the

fluorescence quenching of lysozyme type VI-stabilized gold nanoclusters. Anal. Chem. 2010, 82, 9194-9200.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

10.

Xavier, P. L.; Chaudhari, K.; Baksi, A.; Pradeep, T. Protein-protected luminescent

noble metal quantum clusters: an emerging trend in atomic cluster nanoscience. Nano Rev. Expt. 2012, 3. 11.

Wen, X.; Yu, P.; Toh, Y.-R.; Tang, J. Structure-correlated dual fluorescent bands in

BSA-protected Au25 nanoclusters. J. Phys. Chem. C 2012, 116, 11830-11836. 12.

Wen, X.; Yu, P.; Toh, Y.-R.; Hsu, A.-C.; Lee, Y.-C.; Tang, J. Fluorescence dynamics

in BSA-protected Au25 nanoclusters. J. Phys. Chem. C 2012, 116, 19032-19038. 13.

Liu, Y.; Ai, K.; Cheng, X.; Huo, L.; Lu, L. Gold‐Nanocluster‐Based Fluorescent

Sensors for Highly Sensitive and Selective Detection of Cyanide in Water. Adv. Funct. Mater. 2010, 20, 951-956. 14.

Yan, L.; Cai, Y.; Zheng, B.; Yuan, H.; Guo, Y.; Xiao, D.; Choi, M. M. Microwave-

assisted synthesis of BSA-stabilized and HSA-protected gold nanoclusters with red emission. J. Mater. Chem. 2012, 22, 1000-1005. 15.

Le Guével, X.; Daum, N.; Schneider, M. Synthesis and characterization of human

transferrin-stabilized gold nanoclusters. Nanotechnology 2011, 22, 275103. 16.

Zhang, L.; Swift, J.; Butts, C. A.; Yerubandi, V.; Dmochowski, I. J. Structure and

activity of apoferritin-stabilized gold nanoparticles. J. Inorg. Biochem. 2007, 101, 1719-1729. 17.

Liu, J.-M.; Chen, J.-T.; Yan, X.-P. Near infrared fluorescent trypsin stabilized gold

nanoclusters as surface plasmon enhanced energy transfer biosensor and in vivo cancer imaging bioprobe. Anal. Chem. 2013, 85, 3238-3245. 18.

Kawasaki, H.; Hamaguchi, K.; Osaka, I.; Arakawa, R. pH‐Dependent synthesis of

pepsin‐mediated gold nanoclusters with blue green and red fluorescent emission. Adv. Funct. Mater. 2011, 21, 3508-3515.


Page 22 of 39

Page 23 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

19.

Wen, F.; Dong, Y.; Feng, L.; Wang, S.; Zhang, S.; Zhang, X. Horseradish peroxidase

functionalized fluorescent gold nanoclusters for hydrogen peroxide sensing. Anal. Chem. 2011, 83, 1193-1196. 20.

Liu, C. L.; Wu, H. T.; Hsiao, Y. H.; Lai, C. W.; Shih, C. W.; Peng, Y. K.; Tang, K.

C.; Chang, H. W.; Chien, Y. C.; Hsiao, J. K. Insulin‐Directed Synthesis of Fluorescent Gold Nanoclusters: Preservation of Insulin Bioactivity and Versatility in Cell Imaging. Angew. Chem. Int. Ed. 2011, 50, 7056-7060. 21.

Popa, T.; Paci, I. Structure and Chirality in Sulfur-Containing Amino Acids Adsorbed

on Au (111) Surfaces. J. Phys. Chem. C 2015, 119, 9829-9838. 22.

Lin, W.; Insley, T.; Tuttle, M. D.; Zhu, L.; Berthold, D. A.; Král, P.; Rienstra, C. M.;

Murphy, C. J. Control of Protein Orientation on Gold Nanoparticles. J. Phys. Chem. C 2015, 119, 21035-21043. 23.

Roth, K. L.; Geng, X.; Grove, T. Z. Bioinorganic Interface: Mechanistic Studies of

Protein-Directed Nanomaterial Synthesis. J. Phys. Chem. C 2016, 120, 10951-10960. 24.

Iori, F.; Corni, S.; Di Felice, R. Unraveling the interaction between histidine side

chain and the Au (111) surface: a DFT study. J. Phys. Chem. C 2008, 112, 13540-13545. 25.

Goulding, C. W.; Parseghian, A.; Sawaya, M. R.; Cascio, D.; Apostol, M. I.; Gennaro,

M. L.; Eisenberg, D. Crystal structure of a major secreted protein of Mycobacterium tuberculosis—MPT63 at 1.5‐Å resolution. Protein Sci. 2002, 11, 2887-2893. 26.

Manca, C.; Lyashchenko, K.; Wiker, H. G.; Usai, D.; Colangeli, R.; Gennaro, M. L.

Molecular cloning, purification, and serological characterization of MPT63, a novel antigen secreted by Mycobacterium tuberculosis. Infect. Immun. 1997, 65, 16-23. 27.

Paramanik, B.; Kundu, A.; Chattopadhyay, K.; Patra, A. Study of binding interactions

between MPT63 protein and Au nanocluster. RSC Advances 2014, 4, 35059-35066.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

28.

Kundu, A.; Kundu, S.; Chattopadhyay, K. The Presence of Non‐Native Helical

Structure in the Unfolding of a Beta Sheet Protein MPT63. Protein Sci. 2016. 29.

Ghosh, R.; Mukherjee, M.; Chattopadhyay, K.; Ghosh, S. Unusual optical resolution

of all four tryptophan residues in MPT63 protein by phosphorescence spectroscopy: assignment and significance. J. Phys. Chem. B 2012, 116, 12489-12500. 30.

Lakowicz, J. R. Fluorescence anisotropy. In Principles of fluorescence spectroscopy;

Springer, 1999, pp 291-319. 31.

Chall, S.; Mati, S. S.; Konar, S.; Singharoy, D.; Bhattacharya, S. C. An efficient,

Schiff-base derivative for selective fluorescence sensing of Zn2+ ions: quantum chemical calculation appended by real sample application and cell imaging study. Org. Biomol. Chem. 2014, 12, 6447-6456. 32.

Hohenberg, P.; Kohn, W. Inhomogeneous electron gas. Phys. Rev. 1964, 136, B864.

33.

Kohn, W.; Sham, L. J. Self-consistent equations including exchange and correlation

effects. Phys. Rev. 1965, 140, A1133. 34.

Becke, A. D. Density‐functional thermochemistry. III. The role of exact exchange. J.

Chem. Phys. 1993, 98, 5648-5652. 35.

Lee, C.; Yang, W.; Parr, R. G. Development of the Colle-Salvetti correlation-energy

formula into a functional of the electron density. Phys. Rev. B 1988, 37, 785. 36.

Gross, E.; Kohn, W. Time-dependent density-functional theory. Adv. Quantum Chem.

1990, 21, 255-291. 37.

Marques, M. A.; Gross, E. K. Time-dependent density functional theory. Annu. Rev.

Phys. Chem. 2004, 55, 427-455. 38.

Frisch, M.; Trucks, G.; Schlegel, H.; Scuseria, G.; Robb, M.; Cheeseman, J.;

Montgomery, J.; Vreven, T.; Kudin, K.; Burant, J. Gaussian 03, revision C. 02. 2008.


Page 24 of 39

Page 25 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

39.

Goswami, N.; Yao, Q.; Luo, Z.; Li, J.; Chen, T.; Xie, J. Luminescent metal

nanoclusters with aggregation-induced emission. J. Phys. Chem. Lett. 2016, 7, 962-975. 40.

Helmbrecht, C.; Lützenkirchen-Hecht, D.; Frank, W. Microwave-assisted synthesis of

water-soluble, fluorescent gold nanoclusters capped with small organic molecules and a revealing fluorescence and X-ray absorption study. Nanoscale 2015, 7, 4978-4983. 41.

Zheng, J.; Zhou, C.; Yu, M.; Liu, J. Different sized luminescent gold nanoparticles.

Nanoscale 2012, 4, 4073-4083. 42.

Chen, T.-H.; Nieh, C.-C.; Shih, Y.-C.; Ke, C.-Y.; Tseng, W.-L. Hydroxyl radical-

induced etching of glutathione-capped gold nanoparticles to oligomeric Au I–thiolate complexes. RSC Advances 2015, 5, 45158-45164. 43.

Chaudhari, K.; Xavier, P. L.; Pradeep, T. Understanding the evolution of luminescent

gold quantum clusters in protein templates. ACS nano 2011, 5, 8816-8827. 44.

Le Guével, X.; Hötzer, B.; Jung, G.; Hollemeyer, K.; Trouillet, V.; Schneider, M.

Formation of fluorescent metal (Au, Ag) nanoclusters capped in bovine serum albumin followed by fluorescence and spectroscopy. J. Phys. Chem. C 2011, 115, 10955-10963. 45.

Qian, H.; Zhu, M.; Wu, Z.; Jin, R. Quantum sized gold nanoclusters with atomic

precision. Acc. Chem. Res. 2012, 45, 1470-1479. 46.

Yuk, J. M.; Jeong, M.; Kim, S. Y.; Seo, H. K.; Kim, J.; Lee, J. Y. In situ atomic

imaging of coalescence of Au nanoparticles on graphene: rotation and grain boundary migration. Chem. Comm. 2013, 49, 11479-11481. 47.

Wang, Y.; Liang, W.; Geng, C. Coalescence behavior of gold nanoparticles. Nano.

Res. Lett. 2009, 4, 684. 48.

Li, J.; Wang, Z.; Chen, C.; Huang, S. Atomic-Scale Observation of Migration and

Coalescence of Au Nanoclusters on YSZ Surface by Aberration-Corrected STEM. Sci. Rep. 2014, 4.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

49.

Rai, S.; Singh, H. Electronic structure theory based study of proline interacting with

gold nano clusters. J. Mol. Model. 2013, 19, 4099-4109. 50.

Reisz, J. A.; Bechtold, E.; King, S. B.; Poole, L. B.; Furdui, C. M. Thiol‐blocking

electrophiles interfere with labeling and detection of protein sulfenic acids. FEBS Journal 2013, 280, 6150-6161. 51.

Aliakbar Tehrani, Z.; Jamshidi, Z.; Jebeli Javan, M.; Fattahi, A. Interactions of

glutathione tripeptide with gold cluster: influence of intramolecular hydrogen bond on complexation behavior. J. Phys. Chem. A 2012, 116, 4338-4347. 52.

Pakiari, A.; Jamshidi, Z. Interaction of amino acids with gold and silver clusters. The

J. Phys. Chem. A 2007, 111, 4391-4396. 53.

Weinhold, F.; Landis, C. R. Natural bond orbitals and extensions of localized bonding

concepts. Chem. Edu. Res. Pract. 2001, 2, 91-104.


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List of Tables: Table 1: Time resolved analysis of Au5-I, Au5-II. Au8-I and Au8-II. λex=325 nm, λem = 405nm (Au5)

Au-I Au-II

τ1 (ns)

τ2 (ns)

0.16 (93.0 %) 0.02 (87.0%)

1.63 (7.0%) 1.38 (13.0%)

λex=385 nm, λem = 445nm (Au8)

τav(p s) 170

χ2

τ1 (ns)

τ2 (ns)

τ3 (ns)

1.10

34.6

1.15

1.09 (23.4%) 1.11 (21.6%)

0.18 (66.5%) 0.11 (66.1%)

4.58 (10.1%) 6.38 (12.3%)


χ2

τav( ps) 252

1.00

161

1.04

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Table 2: Comparison of simulated and experimental λabs for Au5/Au8-II; here, II indicate (proline-cysteine-tyrosine) residue. Protein-Au NC complex Au5-(proline-cysteine-tyrosine) (1:1)

Theoretical (λabs) 453 nm

Au5-(proline-cysteine-tyrosine)2 (1:2)

323 nm

Au8-(proline-cysteine-tyrosine) (1:1)

423 nm

Au8-(proline-cysteine-tyrosine)2 (1:2)

383 nm

Experimental (λabs)

Au5 = 325 nm Au8 = 385 nm


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Table 3: Bond lengths of various types of bonds including Au-H nonconventional hydrogen bond.

Au5-I2 Covalent bond 24S-21C 75S-72C 49Au-51Au 48Au-49Au 48Au-24S 51Au-75S Au5-II2 Covalent bond 22S-19C 96S-67C 97Au-99Au 100Au-101Au 100Au-96S 97Au-22S Nonconventional H-bond 64H-100Au 97H-95Au 97H-95Au

Au8-I2 Covalent bond 24S-21C 70S-67C 93Au-100Au 94Au-98Au 94Au-70S 93Au-24S Au8-II2 Covalent bond 104S-75C 22S-19C 48Au-46Au 50Au-47Au 46Au-22S 49Au-104S Nonconventional H-bond 51Au-16H

Bond length (Å) 1.92 1.93 2.86 3.00 2.51 2.54 Bond length (Å) 1.91 1.90 2.68 2.70 2.43 2.45

2.43 3.38 3.70


Bond length (Å) 1.92 1.90 2.68 2.78 2.45 2.50 Bond length (Å) 1.92 1.90 2.85 2.70 2.42 2.48

2.55

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Table 4: Natural bond orbital analysis of Aun-I/II complexes (n = 5 and 8) Complex

Bond

S-Au σ bond Composition

Au5-I2

24S-48Au

0.890 (sp4.26)S + 0.507 (sp0.09d0.07)Au

75S-51Au

0.894 (sp4.47)S + 0.449 (sp0.12d0.06)Au

22S-97Au

0.862 (sp6.92)S + 0.507 (sp0.03d0.10)Au

96S-100Au

0.839 (sp8.34)S + 0.545 (sp0.04d0.11)Au

70S-94Au

0.863 (sp6.76)S + 0.506 (sp0.05d0.07)Au

24S-93Au

0.888 (sp8.12)S + 0.459 (sp0.04d0.08)Au

22S-46Au

0.846 (sp8.00)S + 0.507 (sp0.09d0.07)Au

104S-49Au

0.896 (sp3.61)S + 0.507 (sp0.06d0.09)Au

Au5-II2

Au8-I2

Au8-II2


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Table 5: Second-order perturbation stabilization energies for Aun-Au-I/II, n = 5 and 8.

Au5-I2

Au5-II2

Au8-I2

Au8-II2

Charge transfer

E2

n24S→n*48Au σ21C-24S→n*48Au n75S→ n*51Au σ72C-75S→n*51Au n22S→ n*97Au σ19C-22S→n*97Au n96S→ n*100Au n70S→ n*94Au n24S→ n*93Au σ21C-24S→ n*93Au n22S→n*46Au n104S→ n*49Au n104S→ n*48Au

7.44 0.94 6.12 0.71


8.16 0.55 10.78 6.12 6.69 1.16 13.40 4.30 10.66

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List of Figures:

Fig 1. Representative model structure of the β-sheet protein MPT63. (RCSB protein Data Bank, PDB ID:1LMI, MPT63 crystal structure.14

Fig 2. (a) Absorption spectrum of Au-I (Au-Mutant-I) nanocluster; deconvolution of excitation spectra of Au-I for (b) λem = 405 nm and (c) λem = 445 nm. Both 1b and 1c represent proportional contribution of wavelengths 280 nm, 325 nm and 385 nm.


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Fig 3. Correlation diagram between emission energy and number of gold atoms, N, per cluster. As the number of atoms increase, emission energy decreases. The correlation of emission energy with N is quantitatively fit with EFermi=N1/3, which is predicted by the spherical Jellium model.


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(b) 4f7/2

Au 4f Au (0)

4f5/2

+

Au

4f5/2

80

82

84

86

S 2p

2p3/2

Intensity (a. u)

(a)

Intensity (a. u)

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88

90

158

92

160

162

164

166

168

170

Binding energy (eV)

Binding energy (eV)

Fig 4. XPS spectra: (a) 4f7/2 and 4f5/2 peaks for gold nanoclusters showing the formation of Au (0) and relatively small amount of Au+ after NaOH treatment. (b) The peaks near 162.3 and 167.5 eV of S 2p3/2 confirm the presence of sulphur bound to gold and oxidized sulphur, respectively.

Fig 5. (a) Bright field TEM image showing the presence of goldnanoclusters. Inset in (a) represents the size distribution of nanoclusters. (b) HRTEM and FFT (inset) pattern reveal the existence of lattice fringes with twinning.


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Fig 6.Time resolved study of (a) Au-I and (b) Au-II

Fig 7. An imaginary sphere for (a) Mutant-I and (b) Mutant-II, representing selection of amino acid residues for DFT investigations within 5Å.


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Fig 8. Geometrically optimized structure of 1:2 nanocluster-amino acid complex (i.e., Aun-(3 chain amino acid residue)2) of (a)Au5-I2; (b)Au8-I2; (c)Au5-II2; and (d)Au8-II2.


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Fig 9. Frontier molecular representation of Au5/Au8 synthesized within Mutant-I and MutantII protein template.


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Fig 10. Confocal images of HeLa cells incubated with Au-I solution for 4 hours; A) fluorescence emission after excitation at 405nm, B) DIC images of the HeLa cells treated with Au-I for 4 hours and C) Overlay image showing incorporation of Au-I inside HeLa cells. Scale bar is 10 nm.


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Langmuir

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Understanding the Effect of Single Cysteine Mutations on Gold

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