Effect of Gold Nanoparticle Conjugation on Peptide Dynamics and Structure

Molecular dynamics simulations were used to characterize the structure and dynamics for several peptides and the effect of conjugating them to a gold nanoparticle. Peptide structure and dynamics were compared for two cases: unbound peptides in water, and peptides bound to the gold nanoparticle surface in water. The results show that conjugating the peptides to the gold nanoparticle usually decreases conformational entropy, but sometimes increases entropy. Conjugating the peptides can also result in more extended structures or more compact structures depending on the amino acid sequence of the peptide. The results also suggest that if one wishes to use peptide-nanoparticle conjugates for drug delivery it is important that the peptides contain secondary structure in solution because in our simulations the peptides with little to no secondary structure adsorbed to the nanoparticle surface.


Introduction
Nanoparticles (NPs) are of interest due to their use in applications such as sensing [1], imaging [2][3][4], drug delivery, novel therapy [5], and control of protein structure and activity [6].In the emerging field of nanomaterial-biomolecule research, gold NPs are ideal drug-delivery agents because of their well-known chemical inertness and their minimal toxicity [7,8].Functionalized gold NPs often conjugate with certain proteins, antibodies, or peptides [9,10].Thus, these conjugates can be designed to target specific cells such as cancer cells and then deliver drugs to the targeted cells, which can reduce the dose and thus possible side effects.Many technologies based on biomolecule conjugation to NP have been developed in the past two decades.For example, aptamer bioconjugated NPs have shown a very high specificity for drug delivery in prostate cancer chemotherapy [11,12].Platinum-based anticancer drugs on gold NPs have demonstrated an unusual ability to penetrate the nucleus in lung cancer cells [13].
Several studies have shown that it is more effective to use gold NPs as drug delivery agents than using the drugs via traditional means.Cheng et al. found that use of a drug-gold NP conjugate reduced drug delivery times significantly compared to the free drug [14].Thomas and Klibanov showed that conjugating polyethylenimine chains to gold NPs enhanced the ability of polyethylenimine to transfer plasmid DNA into mammalian cells [15].Joshi et al. observed a significant reduction of blood glucose levels when insulin was delivered using gold NPs as carriers by the transmucosal route in diabetic rats [16].
In order to more effectively utilize NPs for drug delivery it is important to understand and visualize how biomolecules interact with NPs.It is known that conjugation of proteins to NPs can affect the protein structure and function.Aubin-Tam and Hamad-Schifferli have shown that the structure and function are influenced by the chemistry of the NP ligand, the NP size, the NP material, the stoichiometry of the conjugates, and the labeling site on the protein and the nature of the linkage [17,18].They also showed that surface-coating ligands on the NPs are labile and can adopt multiple conformations.Verma et al. have observed that while initial electrostatic complementarity mediates binding, further stabilization is achieved through additional favorable interactions on the surface of NP and the peptide [19].It is also known that the ligands can rearrange to optimize the interaction with a protein [20].
Computer simulation has emerged as a particularly valuable tool for characterizing and visualizing biomolecule-NP conjugates due to the difficulty in obtaining actual experimental data for such systems [21,22].Schulten and collaborators used molecular dynamics to predict the structure of an engineered polypeptide on a gold surface [23].Simulations by Hoefling et al. showed that the binding affinities are dependent on the chemical character of the amino acids when adsorbed to a gold surface [24].Verde et al. performed simulations to investigate the adsorption and mobility of peptides on a gold surface [25].Duchesne et al. devised a method to estimate the proximity of peptides on a gold NP surface.Other researchers have used simulation to investigate DNA conformations while bound to a gold surface [26][27][28].
For the current study we hypothesized that conjugating peptides to gold NPs induces changes in both the peptide structure and peptide dynamics.We studied six peptides: two sequences that were used in a cellular uptake study by Hill and colleagues [29], and the other four sequences which are For the unbound case the peptides were solvated in a 50.0 Å length cubic water box.Each system was then ionized with 0.5 mol/L ion concentration using a mixture of Na + and Cl í ions such that each system had zero net charge.
For the case of the peptide bound to the gold NP the peptide was first placed in water such that the sulfur atom in the cysteine was 3.0 Å away from the surface of a 5.0 nm diameter gold NP.The coordinates for the gold atoms were obtained by trimming a (111) symmetry crystal structure to form a 5.0 nm diameter sphere.The distance of 3.0 Å was chosen so that the system energy could be minimized effectively since Jiang et al. showed that the S-Au covalent bond length is around 2.8 Å (Figure 1b) [32].To reduce computational cost most of the gold atoms that could not interact with the peptide atoms were not included in the simulation.The system was then solvated in a rectangular water box and ionized with 0.5 mol/L ion concentration using a mixture of Na + and Cl í ions such that each system had zero net charge.The box size was chosen such that the distance between the edge of the box and the nearest gold or peptide atoms was at least 24.0 Å (twice the interaction cutoff distance).
All the molecular dynamics simulations were performed using NAMD 2.7b1 [33] with the TIP3P water model [34] and CHARMM force field [35,36].There are several forcefields for peptide-gold interactions that have been reported in the literature (e.g., [37,38]).We used parameters describing the interactions for Au-Au and S-Au from Vila Verde et al. and Miao and Seminario [25,36].Assigning these forcefield parameters neglects possible confinement effects on NP electronic structure that could change NP reactivity.These parameters also ignore possible interactions between the peptide and NP features such as edges and vertexes.For our simulations partial charges for the gold atoms were set to zero and the atoms were forced to remain in fixed positions during the simulation.For each of the two cases (unbound and conjugated) minimization was performed for 1,000 steps, followed by 20.0 ns of equilibrium, and 20.0 ns of production simulation.For equilibration and production Langevin dynamics [39] was used with a constant temperature of 300 K and the pressure was maintained at 1.0 atm [40,41].The SHAKE algorithm was implemented to allow a 2.0 fs timestep [42].Particle mesh Ewald was utilized for electrostatics with a real-space cutoff of 12 Å [43].Van der Waals interactions were cut off at 12 Å with a switching function between 10 and 12 Å.
In order to compare structures and dynamics between the unbound and conjugated cases for each peptide we used the trajectory produced during the 20 ns production simulation to analyze the solvent accessible surface area (SASA), root-mean-square fluctuation (RMSF) and conformational entropy, and performed clustering for the production simulations.The SASA and RMSF were measured by analyzing the molecular dynamics trajectories obtained from the production simulations using VMD [44].SASA is the solvent accessible surface area of the peptide and the RMSF of the CĮ quantifies the peptide dynamics.Before performing the RMSF analysis all the trajectory frames were aligned to the last frame of the trajectory.Clustering of the trajectory structures was performed by first generating the RMSD matrix, and then using the cutree function in the software package R [45].The number of clusters used for our analysis was chosen such that any increase in the number of clusters did not change the largest cluster.For each peptide, the structure in the largest cluster with a SASA value closest to the average SASA was then chosen to represent the most commonly seen type of structure in the simulations.To estimate the uncertainty of SASA and conformational entropy, the production simulation trajectory was divided into 20 equal size pieces, and the standard deviation of those pieces was calculated and used as an estimate of the uncertainty for each measure.The

Results a
To study compare the Figure 1: (i  For the N conjugated ( when conjug Figure 3) th esolved by NC and NC Figure 6).
The could reduce or eliminate specific desired interactions between the peptides and the cellular media.Thus, we suggest that when developing such nanoparticle conjugates the peptides should be designed to contain significant secondary structure in solution.
We note that since the current study uses a single peptide the results should be considered most relevant to NP conjugates with a low density of peptides.If the conjugate has a high density of peptides on the surface then peptide-peptide interactions could be important for determining peptide structure and dynamics.

Conclusions
We performed molecular dynamics simulation of six peptides to study the effect of gold NP conjugation on peptide structure and dynamics.For each peptide we tested two cases: a single unbound peptide in water, and a single peptide conjugated to a gold NP in water.Results show that, consistent with our hypothesis, the presence of gold NP does alter both the peptide structures and dynamics, and that the magnitude of the effect depends on the peptide sequence.Conjugated peptides typically have decreased conformational flexibility, and the amount of decrease depends on the amino acid sequence.However, it is possible for conjugation to increase the flexibility, as was the case with one of the peptides in this study.Conjugating the peptides to a gold NP can also result in more extended structures or more compact structures, depending on the amino acid sequence of the peptide.
Finally, we suggest that if one wishes to design peptides for nanoparticle conjugates for drug delivery the peptides should contain significant secondary structure in solution.This is because our results show that peptides with little to no secondary structure in solution tend to adsorb to the nanoparticle surface, potentially losing their ability specifically interact with cellular media.
Figure cases f (bars) were e