Locating the nucleation sites for protein encapsulated gold nanoclusters : a molecular dynamics and fluorescence study

Fluorescent gold nanoclusters encapsulated by proteins have attracted considerable attention in recent years for their unique properties as new fluorescence probes for biological sensing and imaging. However, fundamental questions, such as the nucleation sites of gold nanoclusters within proteins and the fluorescence mechanism remain unsolved. Here we present a study of the location of gold nanoclusters within bovine serum albumin (BSA) combining both fully atomistic molecular dynamic (MD) simulations and fluorescence spectroscopic studies. The MD simulations show gold clusters growing close to a number of cysteine sites across all three domains of BSA, although just two major sites in domains IIB and IA were found to accommodate large clusters comprising more than 12 atoms. The dependence of the fluorescence on pH is found to be compatible with possible nucleation sites in domains IIB and IA. Furthermore, the energy transfer between tryptophan and gold nanoclusters reveals a separation of 29.7 Å, further indicating that gold nanoclusters were most likely located in the major nucleation site in domain IIB. The disclosure of the precise location of the gold nanoclusters and their surrounding amino acid residues should help better understanding of their fluorescence mechanism and aid their optimization as fluorescent nanoprobes

Rationalising drug delivery using nanoparticles: a combined simulation and immunology study of GnRH adsorbed to silica nanoparticles

Silica nanoparticles (SiNPs) have been shown to have significant potential for drug delivery and as adjuvants for vaccines. We have simulated the adsorption of GnRH-I (gonadotrophin releasing hormone I) and a cysteine-tagged modification (cys-GnRH-I) to model silica surfaces, as well as its conjugation to the widely-used carrier protein bovine serum albumin (BSA). Our subsequent immunological studies revealed no significant antibody production was caused by the peptide-SiNP systems, indicating that the treatment was not effective. However, the testosterone response with the native peptide-SiNPs indicated a drug effect not found with cys-GnRH-I-SiNPs; this behaviour is explained by the specific orientation of the peptides at the silica surface found in the simulations. With the BSA systems, we found significant testosterone reduction, particularly for the BSA-native conjugates, and an antibody response that was notably higher with the SiNPs acting as an adjuvant; this behaviour again correlates well with the epitope presentation predicted by the simulations. The range of immunological and hormone response can therefore be interpreted and understood by the simulation results and the presentation of the peptides to solution, paving the way for the future rational design of drug delivery and vaccine systems guided by biomolecular simulation

Interaction of β-Sheet Folds with a Gold Surface

The adsorption of proteins on inorganic surfaces is of fundamental biological importance. Further, biomedical and nanotechnological applications increasingly use interfaces between inorganic material and polypeptides. Yet, the underlying adsorption mechanism of polypeptides on surfaces is not well understood and experimentally difficult to analyze. Therefore, we investigate here the interactions of polypeptides with a gold(111) surface using computational molecular dynamics (MD) simulations with a polarizable gold model in explicit water. Our focus in this paper is the investigation of the interaction of polypeptides with β-sheet folds. First, we concentrate on a β-sheet forming model peptide. Second, we investigate the interactions of two domains with high β-sheet content of the biologically important extracellular matrix protein fibronectin (FN). We find that adsorption occurs in a stepwise mechanism both for the model peptide and the protein. The positively charged amino acid Arg facilitates the initial contact formation between protein and gold surface. Our results suggest that an effective gold-binding surface patch is overall uncharged, but contains Arg for contact initiation. The polypeptides do not unfold on the gold surface within the simulation time. However, for the two FN domains, the relative domain-domain orientation changes. The observation of a very fast and strong adsorption indicates that in a biological matrix, no bare gold surfaces will be present. Hence, the bioactivity of gold surfaces (like bare gold nanoparticles) will critically depend on the history of particle administration and the proteins present during initial contact between gold and biological material. Further, gold particles may act as seeds for protein aggregation. Structural re-organization and protein aggregation are potentially of immunological importance

Spontaneous membrane-translocating peptide adsorption at silica surfaces : a molecular dynamics study

Spontaneous membrane-translocating peptides (SMTPs) have recently been shown to directly penetrate cell membranes. Adsorption of a SMTP, and some engineered extensions, at model silica surfaces is studied herein using fully atomistic molecular dynamics simulations in order to assess their potential to construct novel drug delivery systems. The simulations are designed to reproduce the electric fields above single, siloxide-rich charged surfaces, and the trajectories indicate that the main driving force for adsorption is electrostatic. An increase in the salt concentration slows down but does not prevent adsorption of the SMTP to the surface; it also does not result in peptide desorption, suggesting additional binding via hydrophobic forces. The results are used to design extensions to the peptide sequence which we find enhance adsorption but do not affect the adsorbed conformation. We also investigate the effect of surface hydroxylation on the peptide adsorption. In all cases, the final adsorbed conformations are with the peptide flattened to the surface with arginine residues, which are key to the peptide's function, anchoring it to the surface so that they are not exposed to solution. This conformation could impact their role in membrane translocation and thus has important implications for the design of future drug delivery vehicles

Multiprotein interactions during surface adsorption : a molecular dynamics study of lysozyme aggregation at a charged solid surface

Multiprotein adsorption of hen egg white lysozyme at a model charged ionic surface is studied using fully atomistic molecular dynamics simulations. Simulations with two, three, and five proteins, in various orientations with respect the surface, are performed over a 100 ns time scale. Mutated proteins with point mutations at the major (Arg128 and Arg125) and minor (Arg68) surface adsorption sites are also studied. The 100 ns time scale used is sufficient to observe protein translations, rotations, adsorption, and aggregation. Two competing processes of particular interest are observed, namely surface adsorption and protein-protein aggregation. At low protein concentration, the proteins first adsorb in isolation and can then reorientate on the surface to aggregate. At high concentration, the proteins aggregate in the solution and then adsorb in nonspecific ways. This work demonstrates the role of protein concentration in adsorption, indicates the residues involved in both types of interaction (protein-protein and protein-surface), and gives an insight into processes to be considered in the development of new functionalized material systems

Adsorption of Fibronectin Fragment on Surfaces Using Fully Atomistic Molecular Dynamics Simulations

The effect of surface chemistry on the adsorption characteristics of a fibronectin fragment (FNIII8–10) was investigated using fully atomistic molecular dynamics simulations. Model surfaces were constructed to replicate self-assembled monolayers terminated with methyl, hydroxyl, amine, and carboxyl moieties. It was found that adsorption of FNIII8–10 on charged surfaces is rapid, specific, and driven by electrostatic interactions, and that the anchoring residues are either polar uncharged or of opposing charge to that of the targeted surfaces. On charged surfaces the presence of a strongly bound layer of water molecules and ions hinders FNIII8–10 adsorption. In contrast, adsorption kinetics on uncharged surfaces are slow and non-specific, as they are driven by van der Waals interactions, and the anchoring residues are polar uncharged. Due to existence of a positively charged area around its cell-binding region, FNIII8–10 is available for subsequent cell binding when adsorbed on a positively charged surface, but not when adsorbed on a negatively charged surface. On uncharged surfaces, the availability of the fibronectin fragment’s cell-binding region is not clearly distinguished because adsorption is much less specific. View Full-Tex

Protein diffusion and long-term adsorption states at charged solid surfaces

The diffusion pathways of lysozyme adsorbed to a model charged ionic surface are studied using fully atomistic steered molecular dynamics simulation. The simulations start from existing protein adsorption trajectories, where it has been found that one particular residue, Arg128 at the N,C-terminal face, plays a crucial role in anchoring the lysozyme to the surface [ Langmuir 2010 , 26 , 15954 - 15965 ]. We first investigate the desorption pathway for the protein by pulling the Arg128 side chain away from the surface in the normal direction, and its subsequent readsorption, before studying diffusion pathways by pulling the Arg128 side chain parallel to the surface. We find that the orientation of this side chain plays a decisive role in the diffusion process. Initially, it is oriented normal to the surface, aligning in the electrostatic field of the surface during the adsorption process, but after resorption it lies parallel to the surface, being unable to return to its original orientation due to geometric constraints arising from structured water layers at the surface. Diffusion from this alternative adsorption state has a lower energy barrier of ∼0.9 eV, associated with breaking hydrogen bonds along the pathway, in reasonable agreement with the barrier inferred from previous experimental observation of lysozyme surface clustering. These results show the importance of studying protein diffusion alongside adsorption to gain full insight into the formation of protein clusters and films, essential steps in the future development of functionalized surfaces