Mode of Protein Complexes on Gold Nanoparticles Surface: Synthesis

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Mode of Protein Complexes on Gold Nanoparticles Surface: Synthesis and Characterization of Biomaterials for Hemocompatibility and Preferential DNA Complexation Poonam Khullar, Manoj Kumar Goshisht, Lovika Moudgil, Gurinder Singh, Divya Mandial, Harsh Kumar, Gurinder K. Ahluwalia, and Mandeep Singh Bakshi ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b02373 • Publication Date (Web): 24 Oct 2016 Downloaded from http://pubs.acs.org on October 25, 2016

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Mode of Protein Complexes on Gold Nanoparticles Surface: Synthesis and Characterization of Biomaterials for Hemocompatibility and Preferential DNA Complexation Poonam Khullar3*, Manoj Kumar Goshisht3,4,# Lovika Moudgil5,# Gurinder Singh5, Divya Mandial3, Harsh Kumar4, Gurinder Kaur Ahluwalia2, Mandeep Singh Bakshi1* 1

Department of Natural and Applied Sciences, University of Wisconsin - Green Bay, 2420

Nicolet Drive, Green Bay, WI 54311-7001, USA. 2Nanotechnology Research Laboratory, College of North Atlantic, Labrador City, NL A2V 2K7 Canada. 3Department of Chemistry, B.B.K. D.A.V. College for Women, Amritsar 143005, Punjab, India. 4Department of Chemistry, Dr. B. R. Ambedkar National Institute of Technology, Jalandhar-144011, Punjab, India. 5Faculty of Applied Sciences, UIET Panjab University Regional Centre, Hoshiarpur 146001, Punjab, India. #Authors with equal contribution.

Corresponding Authors *E mail: [email protected] (P.K.) *E mail: [email protected] (M.S.B)

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Abstract By using in situ synthesis of gold nanoparticles (Au NPs) in the presence of binary mixtures of cytochrome c (Cyc,c) and bovine serum albumen (BSA) model proteins, we demonstrated a new method of studying protein – protein interactions on the surfaces of nanomaterials. Such interactions were simultaneously evaluated and supported by the molecular dynamics studies in terms of protein docking. Both experimental and theoretical studies collectively indicated a strong complexation among Cyc,c and BSA on the surface of Au NPs with multipoint anchoring mechanism to Au surface. They also highlighted that Cyc,c – BSA complex exhibited much stronger surface adsorption rather than Cyc,c or BSA alone. Biofunctional Au NPs thus obtained were tested for hemocompatibility for their possible applications as drug delivery vehicles in systemic circulation by employing the hemolysis. The hemolysis was done for the Au NPs which were coated with entire mixing range of Cyc,c – BSA mixtures to explore the most appropriate mixing compositions of Cyc,c – BSA mixtures for hemocompatibility. In addition, protein coated Au NPs demonstrated strong complexation with DNA which were significantly pronounced for the Cyc,c – BSA complex coated NPs rather than Cyc,c or BSA alone coated NPs. Cyc,c – BSA docked complex on Au NP surface behaved like a typical Helix-Turn-Helix motif because of the size disparity between a much larger BSA and smaller Cyc,c protein that resulted in stronger complexation with DNA in comparison to surface adsorbed Cyc,c or BSA alone. These finding bear important relevance in biotechnology in terms of gene expression and transcription factors. Key words: Protein coated nanoparticles, protein docking, molecular dynamics, hemolysis, DNA complexation.

Introduction Protein – protein interactions play a vital role in various biochemical processes occurring at both cellular and molecular levels.1-4 They are also responsible for some of the most potential critical illnesses such as Alzheimer5,6 and cancer7. Various experimental techniques and theoretical methods have been employed to understand their complex interactions in the bulk phase as well as on the solid surfaces.8-10 In contrast to the bulk, protein – protein interactions are


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dramatically affected when a protein adsorbed on the metallic nanoparticle (NP) surface interacts with an aqueous solubilized protein.11,12 A nanometallic surface adsorbed protein is always in the unfolded state. In order to shield its aqueous exposed hydrophobic domains, it undergoes seeding with the aqueous solubilized protein. This seeding process is usually quite prominent and much more facilitated than that happens simultaneously in the bulk. Thus, protein – protein interactions become even more prominent and interesting when they are triggered by the nanometallic surface adsorbed protein. In fact, multiprotein complexes thus produced from the non-covalent interactions between different proteins are important to explore and mimic to those present in the biological systems and are known for their several important biological functions. We are presenting a unique route to explore protein – protein interactions operating between different proteins on nanometallic surfaces in view of their special interest in nanomedicine. Bioactive engineered NPs are the important components of nanomedicine where they are used as drug delivery vehicles and in biological devices. This can be best evaluated by choosing an appropriate combination of water soluble model proteins13,14 which are well studied and their physiochemical aspects such as size, shape, hydrophobicity, secondary structure, conformational changes, and most importantly complementarity with the nanosurfaces are well documented. To this end, a combination of cyctochrome c (Cyc,c) and bovine serum albumin (BSA) mixture is an ideal choice because both are highly water soluble and demonstrated strong potential to adsorb at nanometallic surfaces. Cyt,c consists of a single polypeptide chain of 104 amino acid residues which is covalently attached to a heme group with average molecular weight of 12,40015. The active heme center is surrounded by a tightly packed hydrophobic side chains and an outer covering of hydrophilic side groups. BSA, on the other hand, is much bigger protein with a molecular mass of 66 500 Da, and is composed of 580 amino acid residues.16,17 It is a versatile carrier protein with wide hydrophobic, hydrophilic, anionic, and cationic properties. In the folded state, both BSA and Cyc,c are expected to interact with each other through predominantly electrostatic interactions which can be dramatically changed when protein unfolds upon its adsorption on the nanometallic surface.18-22 Gold (Au) NPs are ideal choice to study the surface adsorption of proteins because they are highly stable and protein coated NPs do not undergo agglomeration over an extended period of time, thus, providing a wide time window to explore their characteristic features. At molecular level, the protein adsorption can be quantified by the modelling and simulations to compliment the experimental finding. Molecular dynamics



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and protein docking are other interesting theoretical studies which comprehend the experimental findings and elucidate the active amino acids which drive the protein – protein interactions and their surface adsorption on the nanometallic surfaces.23 This study leads to the formation of protein coated biomaterials with interesting characteristic features as well as applications.24-26 In order to demonstrate their biological applicability in the systemic circulation, haemolytic assay has been performed and results are reported for the NPs samples coated with different compositions of Cyc,c and BSA mixtures. In relevance to bionanotechnology, these NPs also show remarkable complexation ability with DNA, which is specific and pronounced only when the NPs are coated with binary protein mixtures rather than their individual components. Experimental Materials Cytochrome c (Cyt,c) from bovine heart muscle, bovine serum albumin (BSA), chloroauric acid (HAuCl4), and deoxyribonucleic acid sodium salt were purchased from Aldrich. Double distilled water was used for all preparations. Preparation of Cyc,c – BSA binary mixtures and their Au NPs surface adsorption during in situ reactions Stock solutions of Cyc,c and BSA were made by dissolving 10 mg / 10 ml in pure water. This was followed by the mixing of Cyc,c and BSA to produce Cyc,c – BSA binary mixtures covering the entire weight fraction (wCyc,c) range by keeping the total amount constant. The surface adsorption of Cyc,c, BSA, and their complexes was carried out by simultaneous synthesis of Au NPs under in situ reaction conditions. For this purpose two methods were adopted. In the first method, Au NPs were synthesized by taking 1 mM of HAuCl4 in 10 ml of each weight fraction in screw-capped glass bottles and kept in water thermostat bath (Julabo F25) at precise 70 ± 0.1 oC for six hours under static conditions. The aqueous protein solution initiated the reduction of Au(III) into Au(0) due to the weak reducing ability of unfolded protein that resulted in the color change from colorless to bright pink or pink-purple. After six hours, the samples were cooled to room temperature and kept for overnight. They were purified from pure water at least three times to remove unreacted protein. Purification of protein coated Au NPs was done by collecting the Au NPs at 8,000 – 10,000 rpm for 5 min after washing each time with distilled water.


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In the second method, calculated amounts of pre-synthesized citrate stabilized Au NPs (11 ± 3 nm with zeta potential of -18.5 ± 1.7 mV) were taken in small UV-visible cuvettes and then titrated with freshly prepared Cyc,c – BSA mixture by keeping constant the amount of Au NPs at 70 oC. Cyc,c – BSA mixtures demonstrated dramatic surface adsorption on NPs and was simultaneously monitored by the spectroscopic analysis. Similar experiments were carried out with pre-synthesized cetyltrimethylammonium bromide (CTAB) stabilized Au NPs of almost similar size and zeta potential of 23.7 ± 3.7 mV to demonstrate the nature of electrostatic interactions driving the protein surface adsorption. Methods Spectroscopic analysis: UV-visible (Shimadzu-Model No. 2450, double beam) measurements in the wavelength range of 200 – 900 nm were carried out to simultaneously monitor the reactions under the effect of temperature as well as reaction time to observe the influence of BSA – Cyc,c complex on the synthesis of Au NPs in terms of protein unfolding and surface adsorption. This instrument was equipped with a TCC 240A thermoelectrically temperature controlled Cell Holder that allowed to measure the spectrum at a constant temperature within ± 1oC. Microscopy and dynamic light scattering (DLS): Protein coated Au NPs were characterized by Transmission Electron Microscopic (TEM) analysis on a JEOL 2010F at an operating voltage of 200 kV. The samples were prepared by mounting a drop of a solution on a carbon coated Cu grid and allowed to dry in the air. DLS measurements were performed using a light scattering apparatus (Zetasizer, Nano series, Nano-ZS, Malvern Instruments) equipped with a built-in temperature controller with an accuracy of ± 0.1 oC. The measurements were made using a quartz cuvette with a path length of 1 cm. Average of 10 measurements were analyzed using the standard algorithms with an uncertainty of less than 7%. Molecular dynamics (MD) simulations: Docking simulations were carried out using Brownian dynamics (BD) technique. The calculations were performed using the SDA version 7 software27. The structures of BSA and Cyt,c were taken from the protein data bank with entry code 4F5S and 3NWV. Five thousand BD trajectories were computed starting with the two proteins positioned randomly with their centres at a distance of 80 Å, where the interactions between the two proteins were negligible, and the simulation time step of 0.50 ps. The proteinprotein encounter complexes obtained during a BD simulation trajectory were clustered with a



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clustering algorithm. Most populated structure obtained from the docking study was further investigated by MD simulations, in explicit water. The obtained structure was then solvated and subjected to 10 ns of MD simulations alone and in the presence of gold surface. The GolP (GoldProtein Force Field) force-field28,29 was used to model a gold slab (5.75 nm x 5.93 nm, thickness of five atomic layers). The GolP parameterized to be compatible with both the bioorganic forcefield OPLS/AA (Optimized Potentials for Liquid Simulations)30 and SPC/E (simple point charge) were used to model the protein and water, respectively. We used the simulation package GROMACS (Groningen Machine for Chemical Simulations) program (version 4.6.5)31 and the visualization package PYMOL. Simulations were done in parallelepiped boxes with periodic boundary conditions. We selected the box size in such a way that protein and its periodic image was separated by at least 12 Å and the proteins were not able to interact with its periodic images via van der Waals potentials. Simulations were performed in physiological ion concentration (150 mM NaCl). The system was pre-equilibrated using energy minimization subjected to steepest descent, until tolerance of 1000 kJ/mol was achieved. Firstly, all water molecules and ions along with proteins were energy minimized, followed by the minimization of proteins by fixing the main-chain and Cα atoms. Finally, the whole system was minimized. The system was then equilibrated with NVT (constant Number of atoms, Volume, and Temperature) ensemble at a target temperature of 300 K. Temperature coupling was performed by using Nose-Hoover thermostat.32,33 Classical MD simulations for time 10 ns were performed at T = 300 K. Electrostatic was treated with the Particle Mesh Ewald approach (PME)34 with a 1.2 Å grid. Lennard-Jones interactions was cut-off at d = 12 Å, with a smooth switching-off starting at d = 10 Å. A 1fs integration time step is used. The LINCS (Linear Constraint Solver) algorithm35 was used to constraint the bond lengths in the simulations. For the system cut-off for van der Waals was 1.2 nm. Hemolysis: Hemolytic assay was performed to evaluate the response of Cyc,c – BSA complex coated Au NPs on blood group B of red blood cells (RBCs) from a healthy human donor. Briefly, 5% suspension of RBCs was used for this purpose after giving three washings along with three concentrations (i.e. 25, 50, and 100 µg/ml) of each NPs sample. 1 ml packed cell volume (i.e. hematocrit) was suspended in 20 ml of 0.01 M phosphate buffered saline (PBS). The positive control was RBCs in water and it was prepared by spinning 4 ml of 5% RBCs suspension in PBS. PBS as supernatant was discarded and pellet was re-suspended in 4 ml of


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water. The negative control was PBS. All the readings were taken at 540 nm i.e. absorption maxima of hemoglobin. DNA complexation: Purified protein coated Au NPs were subjected for the DNA complexation from salmon testes. It was done by titrating the protein coated Au NPs samples with known amount of DNA in the concentration range of 10-6 M and simultaneously monitoring the color change and measuring the UV-visible absorbance at 25 oC.

Results and discussion Protein docking in the absence of Au NPs Protein docking among Cyc,c and BSA is studied by following the structures of proteinprotein encounter complexes generated through Brownian Dynamics (BD) simulations. BSA, a much larger protein in comparison to Cyc,c, is kept fixed while Cyc,c is allowed to find the most preferred orientation on the surface of BSA. The adsorption free energies of Cyc,c on the BSA have been computed for the structures resulting from the docking. Protein complexes thus obtained during a BD simulation trajectory are clustered together to identify different orientations of Cyc,c on BSA. During docking, the interaction energy of the proteins is driven by three major terms viz. van der Waals energy described by site-site Lenard-Jones (ELJ) interactions, electrostatic interaction energy (UEP), and the desolvation energy (Uds). Binding in the most populated complex generated from the docking studies is mostly due to the electrostatic term because LYS5, LYS8, LYS86, LYS87, and GLN12 residues of Cyc,c interact with GLU45, ASP56, SER58, HIS59, and PHE49 residues of BSA, and hence, the electrostatic interactions between the charged amino acids of different proteins drive the docking. The initial three dimensional structure of docking conformation of BSA and Cyc,c is shown in Fig 1a where it has been compared with the individual structures of BSA and Cyc,c to understand the Cyc,c – BSA complex. Interacting amino acid residues of both proteins are highlighted in Fig 1b and their appropriate orientations have been depicted in Fig 1c (labelled diagram is shown in the supporting information, Fig S1). Experimental studies, on the other hand, do not show clear Cyc,c – BSA interactions (Fig 1d), where two prominent peaks at 280 and 410 nm belong to the tryptophan and Soret band of Cyc,c, respectively, while another weak band between 500 – 600 nm is the Q-band of Cyc,c. The absorption maximum of these bands do not show any



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temperature (Fig 1d) as well as concentration (Fig S2a) dependence, however, the increase in the intensity of 280 nm band with temperature is due to a greater aqueous exposure of tryptophan residues upon temperature induced unfolding. Protein docking in the presence of Au NPs From the docking results, the most populated complex depicted in Fig 1a has been used for the molecular dynamic (MD) simulations for interaction with Au(111) in solution. Fig 2a shows the most optimized configuration of Cyc,c – BSA complex on the gold surface. The total energy versus simulation time provides a stable MD run (Fig S3). The root mean square deviation (RMSD) of all protein atoms of solvated Cyc,c and BSA in the absence and presence of gold stabilizes after 1 ns (Fig S4). It is larger in the presence of gold than alone which indicates that the flexibility of the two proteins is enhanced when they encounter the gold surface. In addition, the fluctuations are larger in BSA in comparison to Cyc,c (Fig S5) because BSA adsorbs through two sides in comparison to only one active site of adsorption of Cyc,c (see adsorbed positions of respective protein in Fig 2a, inset). The electrostatic potential calculated with an adaptive Poisson-Boltzman approach36 on the solvent accessible surface indicates that the patches with negative potential on the surface of BSA are responsible for anchoring the Cyc,c – BSA complex on the gold surface (Fig 2b). These patches comprise of amino acid residues ASP1, THR2, LYS 261, ASP 265, CYS 264, and LYS 275. Cyc,c, on the other hand, interacts through GLU21, LYS22, GLY23, GLY24, and HIS26. This is consistent with the fact that adsorption energies for charged amino acid residues is significantly larger, namely -57, -27, and 19 kcal/mol for ASP, LYS, and ARG, respectively, than those of neutral amino acid residues like SER, PRO, and VAL (-6, -4 and -3 kcal/mol, respectively).37 The same trend in adsorption energies is also reported using the consistent valence force field (CVFF) empirical potential energy.38 Thus, the charge transfer of charged residues clearly dominates the binding. Protein docking in situ The above results are tested from the in situ NP surface adsorption of Cyc,c – BSA complex during the synthesis of Au NPs (Fig 3a). As NPs growth proceeds, the Soret band of Cyc,c runs through a prominent isosbestic point at 393 nm with a large blue shift of ~ 20 nm due to heme degradation in the event of protein unfolding and loss of tertiary structure.39,40 It is


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attributed to the surface adsorption of Cyc,c – BSA complex on Au NPs. A blue shift in Soret band indicates the increasing covalent interactions of heme with the gold surface (nucleating centres) which simultaneously break thioether bridges between the vinyl groups of heme and the sulfur atoms of two cysteine side-chains and result in unfolding. Almost similar results are obtained at pH 2.5 (Fig S2b) but with less clarity while practically no shift is observed when the same experiment is carried out at pH 10.5 (Fig S2c). Thus, the pH effect is not prominent in these experiments. It is more clear for wCyc,c (weight fraction of Cyc,c,) = 0.1 rather than wCyc,c = 0.8, i.e. in the BSA rich region than in the Cyc,c rich region as depicted in the intensity versus temperature profile (Fig 3b) where two break points correspond to the blue shift (Fig 3a) through isosbestic point. Thus, the docking of Cyc,c on BSA mainly through electrostatic interactions as depicted in Fig 1a-c causes the loss of tertiary structure of Cyc,c. However, the loss of native structure of both protein in the form of Cyc,c – BSA complex is further determined from the MD studies by using do_dssp as an interface to DSSP program. It is a standard method for assigning secondary structure within the atomic resolution coordinates of the protein.41 The secondary structure undergoes a large change in the presence of Au NPs rather than in the absence of NPs (Fig 3c,d) where the amount of alpha-helix is decreased whereas the amount of turns is increased (Fig S6). The fluctuations in both proteins in the presence of NPs are larger compared to the simulations of free proteins in the solution. Also, the secondary structure of BSA changes to a greater extent in comparison to the Cyc,c with a larger conformational change and greater number of open structures.42 Further, the covariance matrix corresponding to the Cα-atom coordinates is calculated and principal component analysis (i.e. essential dynamics analysis) is performed. The 3N eigenvalues (6183 eigenvalues) of the covariance matrix are ranked in a decreasing order of magnitude (Fig S7, S8) where 85 % of the total positional fluctuations are described by the first 50 eigenvectors of both models, while the first 2 eigenvectors alone represent about 40 %. Their displacement is shown in Fig 3e with significant and more intense fluctuations depicted by the residue 1 – 4 of BSA (corresponding 261 – 275), in comparison to the residue 21 – 26 of Cyc,c (corresponding 605 – 610). Similarly, dihedral angle of BSA (residue 1 – 4) undergoes large change in conformation between 0 and 180 degree (Fig S9 – S12). Protein docking on pre-synthesized Au NPs



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Since the surface adsorption of protein is brought about by the electrostatic interactions, thus, the charge transfer of charged residues can be evaluated by taking surface modified presynthesized Au NPs. For this purpose, citrate stabilized Au NPs of 11 ± 3.3 nm with zeta potential -18.3 mV (see experimental) have been titrated with Cyc,c – BSA (wCyc,c = 0.5), and a typical UV-visible profile is shown in Fig 4a. Pre-synthesized Au NPs show a clear absorbance close to 520 nm due to SPR which increases and red shifts with the increase in the amount of Cyc,c – BSA. After certain protein concentration, it starts decreasing indicating the onset of selfaggregation among the NPs. Fig 4b depicts the intensity and wavelength variations of Au NPs versus protein concentration. Initial increase in the intensity is due to the greater colloidal stability achieved by the NPs and the maximum thus obtained in the curve represents the minimum concentration of the protein (Cm) required to achieve maximum stability. It coincides with the marked red shift in the wavelength profile due to the onset of aggregation among the protein coated NPs. DLS profile (Fig 4c) shows a regular increase in the size of Au NPs with the increase in the amount of Cyc,c – BSA mixture which is significantly higher in comparison to that of Cyc,c or BSA alone. Thus, a large amount of both proteins in the form of their complex at wCyc,c = 0.5 is adsorbed on the NP surface which leads to a dramatic increase in the size. In other words, Cyc,c – BSA complex possesses greater surface adsorption ability in comparison to either of the proteins. The surface adsorption of protein shifts the zeta potential from negative (due to citrate stabilized pre-synthesized Au NPs) to high positive value (Fig 4d) that remains constant especially for Cyc,c because of its high isoelectric point (Ip ~ 10) which provides a global positive charge at low pH. Zeta potential decreases for both BSA alone as well as Cyc,c – BSA complex indicating the fact that the surface adsorption is primarily driven by BSA due to its double contact on gold surface as indicated by the MD studies in Fig 2. A decrease in the positive zeta potential value suggests a multilayer protein adsorption triggered by the seeding20,43,44 process because BSA is much bigger protein in comparison to Cyc,c, and hence, it happens for BSA as well as Cyc,c – BSA complex, and not for Cyc,c alone. Interestingly, no coagulation of NPs is observed if we take CTAB stabilized pre-synthesized NPs instead of citrate stabilized NPs, and titrate with Cyc,c – BSA (wCyc,c = 0.5) (Fig S13). This demonstrates that the protein adsorption is mainly driven by the oppositely charged electrostatic interactions as depicted in Fig 2.


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UV-visible profiles of surface adsorption of Cyc,c – BSA complexes over the whole mixing range on pre-synthesized Au NPs have been shown in Fig 5a. Cm values thus calculated from all the maxima of different mixtures are plotted in Fig 5b. They show large positive deviations from the linear (ideal) behavior suggesting that the surface adsorption is highly facilitated for Cyc,c – BSA complexes (especially close to the 1:1 ratios) formed at different mixing ratios in comparison to Cyc,c and BSA alone. Multipoint anchoring of Cyc,c – BSA complex on the gold surface as demonstrated in Fig 2 is considered to be the driving force for favourable surface adsorption of this complex. The surface adsorption in the form of thick protein coating (indicated by the bock arrows) is clearly evident from the TEM images (Fig 5c,d) along with some of the high resolution images shown in Fig S14. Fusogenic behavior26 of surface adsorbed protein layers brings the adjoining NPs in small groups. Fusogenic behavior is governed by predominantly hydrophobic domains and it becomes prominent in the dried state. A sharp contrast between the dark metallic NPs with maximum growth at {111} crystal planes as depicted by the corresponding XRD patterns (Fig 5e,f) and the light shaded protein is further evident from the high resolution TEM images (Fig 5g,h). Bio-applicability Hemolysis Cyc,c – BSA complex coated Au NPs have been further employed to understand their bio-applicability in terms of hemolysis and DNA complexation. Hemolysis is the first step towards understanding the biological applicability of pharmaceutical formulations for their administration in systemic circulation.19 Naked NP surface has high surface energy to interact with the cell wall of blood cell leading to the deformation and eventually hemolysis.45,46 Thus, naked NPs cannot be used as drug carriers in the systemic circulation. However, protein coated NPs can be used if they do not show hemolysis. Fig 6a shows representative UV-visible plots of heme absorption within 500 to 600 nm of wavelength range with positive and negative controls (see experimental section). Photos of all the samples with different amounts of doses over the entire weight fraction (wCyc,c) range are shown in Fig 6b. The percentage hemolysis = [(sample absorbance - negative control absorbance)/(positive control absorbance-negative control absorbance) x 100] for all samples is computed and depicted graphically in Fig 6c. It is low in the BSA rich region and significantly high in the Cyc,c rich region of the mixtures. Thus, Cyc,c



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– BSA coated NPs in the latter region possess greater ability to disrupt the blood cell membrane and cause hemolysis. In the Cyc,c rich region of the mixture, the Cyc,c – BSA complex is expected to be rich in Cyc,c rather than BSA and hence, bears greater affinity to interact electrostatically with the blood cell membrane which consists of mainly lipids and membrane proteins with small amount of carbohydrates. Such a combination of lipids and membrane proteins provides membrane potential of ~ -10 mV to the blood cell membrane that allows a favorable electrostatic interactions with Cyc,c – BSA complex coated NPs of positive zeta potential (Fig 4d). The NPs with net positive charge prefer to attach to the microvilli of the Caco2 cells.47 It causes the membrane deformation, cell internalization, and eventually hemolysis. Thus, Cyc,c – BSA coated NPs especially of BSA rich region are only considered to be suitable vehicles for the drug release in the systemic circulation because of their low degree of hemolysis. DNA complexation These NPs show remarkable DNA complexation when treated with aqueous sample of DNA. A typical titration of the protein coated NPs with DNA is shown in Fig 7a. Cyc,c – BSA complex coated Au NPs show a prominent absorbance at 550 nm and a weak absorbance at 280 nm due to the tryptophan residues. Addition of DNA dramatically suppresses the 550 nm absorbance with a significant red shift of ~100 nm and an appearance of a characteristic peak of DNA at 260 nm. A variation of intensity of Au NPs absorbance is plotted against the DNA concentration in Fig 7b where we compared the DNA complexation of Cyc,c – BSA complex coated Au NPs (empty circles) with that of Cyc,c (filled diamonds) and BSA (empty squares) alone coated NPs. BSA coated NPs get additional colloidal stability upon complexation with DNA as their intensity increases initially and passes through a maximum contrary to that of Cyc,c coated NPs where the intensity decreases as soon as DNA is added. However, the effect is much pronounced in terms of rapid decrease in the intensity as well as the onset of self – aggregation among the NPs when Cyc,c – BSA (wCyc,c = 0.5) coated NPs are treated with DNA. This is clearly visible from the sample photos (Fig 7c) where the NPs suspension turns dark (see tube with wCyc,c = 0.5 mixture). One can calculate the initial concentration of DNA required for the complexation with protein coated NPs from the break point in the curve (Fig 7b). It is 0.16 µM and 0.31 µM of DNA for the Cyc,c and BSA coated NPs, respectively, while 0.24 µM DNA


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for 1:1 (Cyc,c – BSA mixture) protein coated NPs. It suggests that the DNA complexation with protein coated NPs is quite quantitative with respect to the amount of surface adsorbed protein. The spiral structure of DNA possesses major and minor grooves which act as ideal harbours for the site specific interactions with protein. Usually, C-terminal α-helix is inserted into the major groove and is referred as ‘recognition helix’ while the flexible N-terminal mediates additional DNA contacts in the minor groove. Both the recognition helix and the Nterminal arm form base-specific interactions. One can easily differentiates between the DNA – Cyc,c interactions from that of DNA – BSA interactions in Fig 7b. DNA – Cyc,c interactions are clearly electrostatic in origin and mainly contributed by positively charged lysine residues with the negatively charged backbone of the DNA molecule.48 It results in the instantaneous decrease in the intensity of Cyc,c coated Au NPs (Fig 7b). While the complexation of BSA with DNA seems to be usual protein/DNA interactions, where the major groove of DNA is the most favored docking site for the protein because it is easily accessible through specific hydrogen bonds with bases.49 Such a complexation provides addition colloidal stability to the BSA coated NPs with the result intensity of Au NPs increases initially. However, further deposition of more DNA causes the self-aggregation (Fig 7b). Interestingly, when we take Cyc,c – BSA complex coated NPs, the DNA complexation is highly pronounced with significant decrease in the intensity of Au NPs. It shows that the DNA – (Cyc,c – BSA complex) interactions are even stronger and different from both DNA – BSA as well as DNA – Cyc,c interactions in a much similar manner as we observed stronger adsorption of Cyc,c – BSA complex on the Au surface (Fig 2). DNA complexation with Cyc,c – BSA complex coated Au NPs arises from the combination of usual DNA – protein interactions based on hydrogen bonding between DNA and BSA, site specific electrostatic interactions between positively charged Cyc,c and negatively charged backbone of DNA, and the contributions from the hydrophobic interactions. Cyc,c – BSA surface adsorbed complex shown in Fig 2a interacts with DNA as a whole and the proposed mechanism is demonstrated in Fig 8. In this situation, several other factors participate to enhance such interactions.50 Protein interface with DNA is predominantly clouded by the positive potential mainly contributed by the lysine and arginine of protein. It interacts with negative potential of charged phosphate residues of DNA.51 The electrostatic interactions are not sequence specific since the negative charges are evenly distributed along the DNA. Thus, the interfacial



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geometry comprehends the optimization of DNA – (Cyc,c – BSA complex) interactions. DNA binding adopts regular as well as various distorted conformations while a protein (more specifically Cyc,c – BSA complex) that can bend provides several interaction sites to the DNA backbone. It induces DNA kinking which further promotes the intercalation of aqueous exposed hydrophobic domains. The Helix Turn Helix motif is the most commonly used secondary structure for specific DNA recognition, and in the present case, NP surface adsorbed Cyc,c – BSA complex very well acts as a recognition helix. It seems that the BSA part of Cyc,c – BSA complex is docked in the major groove because of its larger size where it makes base specific contacts through hydrogen bonds, Van der Waals, and hydrophobic interactions. A second helix (i.e. Cyc,c) stabilizes the recognition helix by adjusting the width of the minor groove through helix twist which increases the negative electrostatic potential and thus, proves to be a favourable harbour. This mode of complexation reduces the electrical double layer required for the colloidal stability of protein coated NPs and hence, induces strong self-aggregation as we observed in Fig 7b. Thus, Cyc,c – BSA complex coated NPs are much better choice for the DNA complexation and recognition rather than either of Cyc,c or BSA coated NPs.

Concluding remarks Cyc,c and BSA proteins are used for understanding their docking in the absence and presence of Au NPs to design appropriate biofunctional nanomaterials for DNA complexation. Molecular dynamics and experimental studies are performed to compare the results. Fine correlation is observed between the two studies. Protein docking resulting in the Cyc,c – BSA complex formation is clear and more pronounced on the surface of Au NPs rather than in their absence. In addition, the Cyc,c – BSA complex demonstrates stronger protein adsorption in comparison to their individual components due to its multipoint anchoring mechanism to the gold surface. The adsorbed Cyc,c – BSA complex behaves like a typical Helix-Turn-Helix motif during the complexation with DNA and hence, results in a much stronger complexation rather than the individually adsorbed protein. Thus, the above results indicate that better bioactive nanomaterials can be synthesized by choosing an appropriate docked protein complex rather than single protein for DNA complexation and recognition.


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Acknowledgment: These studies were partially supported by the financial assistance from DST under nanomission research project [ref no: SR/NM/NS-1057/2015(G)], New Delhi. Dr Gurinder Kaur thankfully acknowledges the financial support provided by the Research and Development Council (RDC) of Newfoundland and Labrador, NSERC, and the Office of Applied Research at CNA.

Supporting Information: UV-visible and molecular dynamics profiles, and TEM images. This information is available free of charge via the Internet at http://pubs.acs.org.



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Figure caption Fig 1. (a) Molecular structure of Cyc,c – BSA complex obtained after docking studies. Insets show their individual structures. (b) and (c) show the interacting amino acids of both proteins during docking. They are identified in Fig S1. (d) UV-visible plots of a Cyc,c – BSA mixture (wCyc,c = 0.5) with temperature from 20 – 70 oC. See details in the text. Fig 2. (a) The most optimized configuration of Cyc,c – BSA complex on the gold surface. Insets show their individual configurations. (b) Complementary electrostatic surface potential on the solvent accessible surface of Cyc,c – BSA from explicit water simulations. Image did not show the entire simulation box to facilitate the display. Water molecules are omitted for clarity. Simulations are performed in physiological ion concentration (150 mM NaCl). See details in the text. Fig 3. (a) UV-visible plots of in situ reaction of the synthesis of Au NPs (HAuCl4 = 1mM) in the presence of Cyc,c – BSA (wCyc,c = 0.5) with temperature from 20 to 70 oC. Inset shows the isosbestic point along with the red shift in the Soret band of Cyc,c. (b) Plot of intensity of Soret band versus temperature. (c) and (d) Secondary structure change of Cyc,c – BSA in the presence and absence of gold at the end of the simulation, respectively. (e) Displacements of the components of the first eigenvector of Cyc,c – BSA in the presence of gold slab. See details in the text. Fig 4. (a) Plot of the UV-visible absorbance of the titration of Au NPs with different concentrations of Cyc,c – BSA mixture (wCyc,c = 0.5) at 70 oC. (b) It depicts the variation in the intensity of Au NPs absorbance and wavelength with the concentration of Cyc,c – BSA mixture (wCyc,c = 0.5). (c) and (d) Plots of the variation in the DLS size and zeta potential, respectively, of Au NPs upon titrating with Cyc,c, BSA, and wCyc,c = 0.5. See details in the text. Fig 5. (a) Plots of the variation of the intensity of the Au NPs upon titrating with Cyc,c – BSA mixtures of different compositions. (b) Plot of Cm for Cyc,c – BSA mixture with respect to wCyc,c. (c) and (d) TEM images show protein coated Au NPs while (e) and (f) images show their respective XRD patterns depicting the fcc crystal structure of gold. (g) and (h) High resolution TEM images of few NPs showing thick protein coating.



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Fig 6. (a) Typical UV-visible profiles of heme absorption of different doses of protein coated Au NPs along with positive and negative controls. (b) Photos of all samples of Cyc,c – BSA complex coated NPs at different compositions (wCyc,c). “1”, “2”, “3”, “4”, and “5”, represent the positive control, negative control, 25, 50, and 100 µg/ml of Au NPs, respectively. (c) Plots of hemolysis % versus wCyc,c in Cyc,c – BSA complex coated Au NPs. The dose range of protein coated NPs is 25 µg/ml, 50 µg/ml, and 100 µg/ml. See details in the text.

Fig 7. (a) Plots of the absorbance of Cyc,c - BSA(wCyc,c = 0.5) coated Au NPs titration with DNA at 25 oC. (b) Plots of the variation of intensity of Cyc,c - BSA(wCyc,c = 0, 1, and 0.5) coated Au NPs with the concentration of DNA. (c) Sample photos of purified different Cyc,c – BSA coated Au NPs before the titration (upper frames) and after the titration (lower frames) with DNA. See details in the text.

Fig 8. Schematic flow diagram of the proposed mechanism of the complexation of Au NPs with Cyc,c – BSA complex based on the molecular dynamics (Fig 1 and 2) and experimental studies (Fig 3, 4, and 5), and their subsequently complexation with DNA based on (Fig 7). See details in the text.


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Fig 4 4

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-6

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TOC

Mode of Protein Complexes on Gold Nanoparticles Surface: Synthesis and Characterization of Biomaterials for Hemocompatibility and Preferential DNA Complexation Poonam Khullar*, Manoj Kumar Goshisht, Lovika Moudgil, Gurinder Singh, Divya Mandial, Harsh Kumar, Gurinder Kaur Ahluwalia, Mandeep Singh Bakshi*

Binary protein coated gold nanoparticles demonstrate fine hemocompatibility and strong DNA complexation as possible biofunctional nanomaterials with important relevance to biotechnology.


Mode of Protein Complexes on Gold Nanoparticles Surface: Synthesis

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