Formation of a protein corona around nanoparticles

In biofluids, nanoparticles rapidly become surrounded by a protein corona. This phenomenon started to attract attention 10 years ago. Since then, this subfield of colloid and interface science was among the most rapidly expanding and progressing. Owing to its strong relation to biology and applications, this area is rich in various questions to explore. The reviews of the corresponding experiments are already numerous. Herein, I focus on the related theory including conventional mean-field kinetic models, dynamic density functional theory, Monte Carlo simulations, and molecular dynamics simulations. The key concept here is that the formation of a protein corona depends on the interplay of competition of different proteins for the location near the nanoparticle–solution interface (Vroman effect) and denaturation at this interface. Although this concept is not new, many details of this interplay are still open for debate.     

What induces pocket openings on protein surface patches involved in protein–protein interactions?

We previously showed for the proteins BCL-X_L, IL-2, and MDM2 that transient pockets at their protein–protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein–protein interfaces. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The interaction of peptides and proteins with nanostructures surfaces: a challenge for nanoscience

The impact of nanotechnologies in biomedicine and biotechnology is becoming more and more evident. It imposes practical challenges, for instance, raising specific issues on the biocompatibility of nanostructures. Nanoparticles are characterized by a high surface-to-volume ratio, which makes them reactive to foreign species. Thus, when proteins or peptides approach an inorganic nanoparticle, as well as a flat surface, they are likely to interact with the substrate to some extent. This interaction is crucial for applications in drug delivery, imaging, diagnostics, implants, and other medical devices. Specifically, gold nanoparticles are highly versatile and particularly appealing. It is widely accepted that the surfaces of nanoparticles adsorb proteins either transiently in the soft corona layer or permanently in the hard corona layer. As a consequence, the protein structure and/or function may undergo profound adjustments or remain conserved. Detailing the interaction of different inorganic substrates with proteins and peptides at the atomic level, and designing ways to control the interaction, is the key for biomedical applications of nanoparticles, both from a fundamental viewpoint and for practical implementations. In the last decade, we have addressed protein–nanoparticle interactions, focusing on interfaces of gold surfaces and nanoparticles with amyloidogenic peptides and protein models. We have developed classical force fields, performed advanced molecular dynamics simulations, and compared computational outcomes with data from nuclear magnetic resonance experiments. Protein–gold complexes with differently coated gold nanoparticles have been modeled to explore the effects of charge and size on the protein structure. Our work unravels that a complex interplay between surface properties and characteristics of the biological adsorbate determines whether peptide conformation is influenced and whether protein aggregation is accelerated or inhibited by the presence of the substrate. General guidelines to cope with amyloidogenic proteins could be inferred: these can be essentially summarized with the necessity of balancing the hydrophobic and electrostatic interactions that the amyloidogenic proteins establish with the coating moieties.     

Potential of mean force of water–proton bath and molecular dynamic simulation of proteins at constant pH

An advanced implicit solvent model of water–proton bath for protein simulations at constant pH is presented. The implicit water–proton bath model approximates the potential of mean force of a protein in water solvent in a presence of hydrogen ions. Accurate and fast computational implementation of the implicit water–proton bath model is developed using the continuum electrostatic Poisson equation model for calculation of ionization equilibrium and the corrected MSR6 generalized Born model for calculation of the electrostatic atom–atom interactions and forces. Molecular dynamics (MD) method for protein simulation in the potential of mean force of water–proton bath is developed and tested on three proteins. The model allows to run MD simulations of proteins at constant pH, to calculate pH‐dependent properties and free energies of protein conformations. The obtained results indicate that the developed implicit model of water–proton bath provides an efficient way to study thermodynamics of biomolecular systems as a function of pH, pH‐dependent ionization‐conformation coupling, and proton transfer events. © 2012 Wiley Periodicals, Inc.     

An In Situ Investigation of the Protein Corona Formation Kinetics of Single Nanomedicine Carriers by Self-Regulated Electrochemiluminescence Microscopy

Protein coronas are present extensively at the bio-nano interface due to the natural adsorption of proteins onto nanomaterials in biological fluids. Aside from the robust property of nanoparticles, the dynamics of the protein corona shell largely define their chemical identity by altering interface properties. However, the soft coronas are normally complex and rapidly changing. To real-time monitor the entire formation, we report here a self-regulated electrochemiluminescence (ECL) microscopy based on the interaction of the Ru(bpy)₃³⁺ with the nanoparticle surface. Thus, the heterogeneity of the protein corona is in situ observed in single nanoparticle “cores” before and after loading drugs in nanomedicine carriers. The label-free, optical stable and dynamic ECL microscopy minimize misinterpretations caused by the variation of nanoparticle size and polydispersity. Accordingly, the synergetic actions of proteins and nanoparticles properties are uncovered by chemically engineered protein corona. After comparing the protein corona formation kinetics in different complex systems and different nanomedicine carriers, the universality and accuracy of this technique were well demonstrated via the protein corona formation kinetics curves regulated by competitive adsorption of Ru(bpy)₃³⁺ and multiple proteins on surface of various carriers. The work is of great significance for studying bio-nano interface in drug delivery and targeted cancer treatment.     

Prediction of protein–protein complexes using replica exchange with repulsive scaling

The realistic prediction of protein–protein complex structures is import to ultimately model the interaction of all proteins in a cell and for the design of new protein–protein interactions. In principle, molecular dynamics (MD) simulations allow one to follow the association process under realistic conditions including full partner flexibility and surrounding solvent. However, due to the many local binding energy minima at the surface of protein partners, MD simulations are frequently trapped for long times in transient association states. We have designed a replica-exchange based scheme employing different levels of a repulsive biasing between partners in each replica simulation. The bias acts only on intermolecular interactions based on an increase in effective pairwise van der Waals radii (repulsive scaling (RS)-REMD) without affecting interactions within each protein or with the solvent. For a set of five protein test cases (out of six) the RS-REMD technique allowed the sampling of near-native complex structures even when starting from the opposide site with respect to the native binding site for one partner. Using the same start structures and same computational demand regular MD simulations sampled near native complex structures only for one case. The method showed also improved results for the refinement of docked structures in the vicinity of the native binding geometry compared to regular MD refinement.     

Impact of the nanoparticle-protein corona on colloidal stability and protein structure

In biological fluids, proteins may associate with nanoparticles (NPs), leading to the formation of a so-called "protein corona" largely defining the biological identity of the particle. Here, we present a novel approach to assess apparent binding affinities for the adsorption/desorption of proteins to silver NPs based on the impact of the corona formation on the agglomeration kinetics of the colloid. Affinities derived from circular dichroism measurements complement these results, simultaneously elucidating structural changes in the adsorbed protein. Employing human serum albumin as a model, apparent affinities in the nanomolar regime resulted from both approaches. Collectively, our findings now allow discrimination between the formation of protein mono- and multilayers on NP surfaces.     

Convergence and sampling efficiency in replica exchange simulations of peptide folding in explicit solvent

Replica exchange methods (REMs) are increasingly used to improve sampling in molecular dynamics (MD) simulations of biomolecular systems. However, despite having been shown to be very effective on model systems, the application of REM in complex systems such as for the simulation of protein and peptide folding in explicit solvent has not been objectively tested in detail. Here we present a comparison of conventional MD and temperature replica exchange MD (T-REMD) simulations of a beta-heptapeptide in explicit solvent. This system has previously been shown to undergo reversible folding on the time scales accessible to MD simulation and thus allows a direct one-to-one comparison of efficiency. The primary properties compared are the free energy of folding and the relative populations of different conformers as a function of temperature. It is found that to achieve a similar degree of precision T-REMD simulations starting from a random set of initial configurations were approximately an order of magnitude more computationally efficient than a single 800 ns conventional MD simulation for this system at the lowest temperature investigated (275 K). However, whereas it was found that T-REMD simulations are more than four times more efficient than multiple independent MD simulations at one temperature (300 K) the actual increase in conformation sampling was only twofold. The overall gain in efficiency using REMD resulted primarily from the ordering of different conformational states over temperature, as opposed to a large increase of conformational sampling. It is also shown that in this system exchanges are accepted primarily based on (random) fluctuations within the solvent and are not strongly correlated with the instantaneous peptide conformation raising questions in regard to the efficiency of T-REMD in larger systems.     

Ab initio folding of helix bundle proteins using molecular dynamics simulations

We have demonstrated that ab initio fast folding simulations at 400 K using a GB implicit solvent model with an all-atom based force field can describe the spontaneous formation of nativelike structures for the 36-residue villin headpiece and the 46-residue fragment B of Staphylococcal protein A. An implicit solvent model combined with high-temperature MD makes it possible to perform direct folding simulations of small- to medium-sized proteins by reducing the computational requirements tremendously. In the early stage of folding of the villin headpiece and protein A, initial hydrophobic collapse and rapid formation of helices were found to play important roles. For protein A, the third helix forms first in the early stage of folding and exhibits higher stability. The free energy profiles calculated from the folding simulations suggested that both of the helix-bundle proteins show a two-state thermodynamic behavior and protein A exhibits rather broad native basins.     

Self-assembling cyclic peptides: molecular dynamics studies of dimers in polar and nonpolar solvents

The self-assembly of cyclic D,L-alpha-peptides into hollow nanotubes is a crucial mechanistic step in their application as antibacterial and drug-delivery agents. To understand this process, molecular dynamics (MD) simulations were performed on dimers of cyclic peptides formed from cyclo [(-L-Trp-D-N-MeLeu-)4-]2 and cyclo [(-L-Trp-D-Leu-)4-]2 subunits in nonpolar (nonane) and polar (water) solvent. The dimers were observed to be stable only in nonpolar solvent over the full 10 ns length of the MD trajectory. The behavior of the dimers in different solvents is rationalized in terms of the intersubunit hydrogen bonding, hydrogen bonding with the solvent, and planarity of the rings. It is shown that the phi and psi dihedral angles of a single uncapped ring in nonane lie in the beta-sheet region of the Ramachandran plot, and the ring stays in a flat conformation. Steered MD (SMD) simulations based on Jarzynski's equality were performed to obtain the potential of mean force as a function of the distance between the two rings of the capped dimer in nonane. It is also shown that a single peptide subunit prefers to reside close to the nonane/water interface rather than in bulk solvent because of the amphiphilic character of the peptide ring. The present MD results build the foundation for using MD simulations to study the mechanism of the formation of cyclic peptide nanotubes in lipid bilayers.     

Molecular dynamics with the united-residue model of polypeptide chains. II. Langevin and Berendsen-bath dynamics and tests on model alpha-helical systems

The implementation of molecular dynamics (MD) with our physics-based protein united-residue (UNRES) force field, described in the accompanying paper, was extended to Langevin dynamics. The equations of motion are integrated by using a simplified stochastic velocity Verlet algorithm. To compare the results to those with all-atom simulations with implicit solvent in which no explicit stochastic and friction forces are present, we alternatively introduced the Berendsen thermostat. Test simulations on the Ala(10) polypeptide demonstrated that the average kinetic energy is stable with about a 5 fs time step. To determine the correspondence between the UNRES time step and the time step of all-atom molecular dynamics, all-atom simulations with the AMBER 99 force field and explicit solvent and also with implicit solvent taken into account within the framework of the generalized Born/surface area (GBSA) model were carried out on the unblocked Ala(10) polypeptide. We found that the UNRES time scale is 4 times longer than that of all-atom MD simulations because the degrees of freedom corresponding to the fastest motions in UNRES are averaged out. When the reduction of the computational cost for evaluation of the UNRES energy function is also taken into account, UNRES (with hydration included implicitly in the side chain-side chain interaction potential) offers about at least a 4000-fold speed up of computations relative to all-atom simulations with explicit solvent and at least a 65-fold speed up relative to all-atom simulations with implicit solvent. To carry out an initial full-blown test of the UNRES/MD approach, we ran Berendsen-bath and Langevin dynamics simulations of the 46-residue B-domain of staphylococcal protein A. We were able to determine the folding temperature at which all trajectories converged to nativelike structures with both approaches. For comparison, we carried out ab initio folding simulations of this protein at the AMBER 99/GBSA level. The average CPU time for folding protein A by UNRES molecular dynamics was 30 min with a single Alpha processor, compared to about 152 h for all-atom simulations with implicit solvent. It can be concluded that the UNRES/MD approach will enable us to carry out microsecond and, possibly, millisecond simulations of protein folding and, consequently, of the folding process of proteins in real time.     

Quantitative Protein Corona Composition and Dynamics on Carbon Nanotubes in Biological Environments

When nanoparticles enter biological environments, proteins adsorb to form the “protein corona” which alters nanoparticle biodistribution and toxicity. Herein, we measure protein corona formation on DNA-functionalized single-walled carbon nanotubes (ssDNA-SWCNTs), a nanoparticle used widely for sensing and delivery, in blood plasma and cerebrospinal fluid. We characterize corona composition by mass spectrometry, revealing high-abundance corona proteins involved in lipid binding, complement activation, and coagulation. We investigate roles of electrostatic and entropic interactions driving selective corona formation. Lastly, we study real-time protein binding on ssDNA-SWCNTs, obtaining agreement between enriched proteins binding strongly and depleted proteins binding marginally, while highlighting cooperative adsorption mechanisms. Knowledge of protein corona composition, formation mechanisms, and dynamics informs nanoparticle translation from in vitro design to in vivo application.     

Electrostatics of proteins in dielectric solvent continua. II. First applications in molecular dynamics simulations

In the preceding paper by Stork and Tavan, [J. Chem. Phys. 126, 165105 (2007)], the authors have reformulated an electrostatic theory which treats proteins surrounded by dielectric solvent continua and approximately solves the associated Poisson equation [B. Egwolf and P. Tavan, J. Chem. Phys. 118, 2039 (2003)]. The resulting solution comprises analytical expressions for the electrostatic reaction field (RF) and potential, which are generated within the protein by the polarization of the surrounding continuum. Here the field and potential are represented in terms of Gaussian RF dipole densities localized at the protein atoms. Quite like in a polarizable force field, also the RF dipole at a given protein atom is induced by the partial charges and RF dipoles at the other atoms. Based on the reformulated theory, the authors have suggested expressions for the RF forces, which obey Newton's third law. Previous continuum approaches, which were also built on solutions of the Poisson equation, used to violate the reactio principle required by this law, and thus were inapplicable to molecular dynamics (MD) simulations. In this paper, the authors suggest a set of techniques by which one can surmount the few remaining hurdles still hampering the application of the theory to MD simulations of soluble proteins and peptides. These techniques comprise the treatment of the RF dipoles within an extended Lagrangian approach and the optimization of the atomic RF polarizabilities. Using the well-studied conformational dynamics of alanine dipeptide as the simplest example, the authors demonstrate the remarkable accuracy and efficiency of the resulting RF-MD approach.     

Redox entropy of plastocyanin: developing a microscopic view of mesoscopic polar solvation

We report applications of analytical formalisms and molecular dynamics (MD) simulations to the calculation of redox entropy of plastocyanin metalloprotein in aqueous solution. The goal of our analysis is to establish critical components of the theory required to describe polar solvation at the mesoscopic scale. The analytical techniques include a microscopic formalism based on structure factors of the solvent dipolar orientations and density and continuum dielectric theories. The microscopic theory employs the atomistic structure of the protein with force-field atomic charges and solvent structure factors obtained from separate MD simulations of the homogeneous solvent. The MD simulations provide linear response solvation free energies and reorganization energies of electron transfer in the temperature range of 280-310 K. We found that continuum models universally underestimate solvation entropies, and a more favorable agreement is reported between the microscopic calculations and MD simulations. The analysis of simulations also suggests that difficulties of extending standard formalisms to protein solvation are related to the inhomogeneous structure of the solvation shell at the protein-water interface combining islands of highly structured water around ionized residues along with partial dewetting of hydrophobic patches. Quantitative theories of electrostatic protein hydration need to incorporate realistic density profile of water at the protein-water interface.     

Material binding peptides for nanotechnology

Remarkable progress has been made to date in the discovery of material binding peptides and their utilization in nanotechnology, which has brought new challenges and opportunities. Nowadays phage display is a versatile tool, important for the selection of ligands for proteins and peptides. This combinatorial approach has also been adapted over the past decade to select material-specific peptides. Screening and selection of such phage displayed material binding peptides has attracted great interest, in particular because of their use in nanotechnology. Phage display selected peptides are either synthesized independently or expressed on phage coat protein. Selected phage particles are subsequently utilized in the synthesis of nanoparticles, in the assembly of nanostructures on inorganic surfaces, and oriented protein immobilization as fusion partners of proteins. In this paper, we present an overview on the research conducted on this area. In this review we not only focus on the selection process, but also on molecular binding characterization and utilization of peptides as molecular linkers, molecular assemblers and material synthesizers.