How Nanoparticles Modify Adsorbed Proteins: Impact of Silica Nanoparticles on the Hemoglobin Active Site.

The adsorption of proteins on surfaces has been studied for a long time, but the relationship between the structural and functional properties of the adsorbed protein and the adsorption mechanism remains unclear. Using hemoglobin adsorbed on silica nanoparticles, we have previously shown that hemoglobin's affinity towards oxygen increases with adsorption. Nevertheless, it was also shown that there were no significant changes in the quaternary and secondary structures. In order to understand the change in activity, we decided in this work to focus on the active sites of hemoglobin, the heme and its iron. After measuring adsorption isotherms of porcine hemoglobin on Ludox silica nanoparticles, we analyzed the structural modifications of adsorbed hemoglobin by X-ray absorption spectroscopy and circular dichroism spectra in the Soret region. It was found that upon adsorption, there were modifications in the heme pocket environment due to changes in the angles of the heme vinyl functions. These alterations can explain the greater affinity observed.

Electronic Structure and Solvation Effects from Core and Valence Photoelectron Spectroscopy of Serum Albumin.

X-ray photoelectron spectroscopy of bovine serum albumin (BSA) in a liquid jet is used to investigate the electronic structure of a solvated protein, yielding insight into charge transfer mechanisms in biological systems in their natural environment. No structural damage was observed in BSA following X-ray photoelectron spectroscopy in a liquid jet sample environment. Carbon and nitrogen atoms in different chemical environments were resolved in the X-ray photoelectron spectra of both solid and solvated BSA. The calculations of charge distributions demonstrate the difficulty of assigning chemical contributions in complex systems in an aqueous environment. The high-resolution X-ray core electron spectra recorded are unchanged upon solvation. A comparison of the valence bands of BSA in both phases is also presented. These bands display a higher sensitivity to solvation effects. The ionization energy of the solvated BSA is determined at 5.7 ± 0.3 eV. Experimental results are compared with theoretical calculations to distinguish the contributions of various molecular components to the electronic structure. This comparison points towards the role of water in hole delocalization in proteins.

Relationships between RNA topology and nucleocapsid structure in a model icosahedral virus.

The process of genome packaging in most of viruses is poorly understood, notably the role of the genome itself in the nucleocapsid structure. For simple icosahedral single-stranded RNA viruses, the branched topology due to the RNA secondary structure is thought to lower the free energy required to complete a virion. We investigate the structure of nucleocapsids packaging RNA segments with various degrees of compactness by small-angle x-ray scattering and cryotransmission electron microscopy. The structural differences are mild even though compact RNA segments lead on average to better-ordered and more uniform particles across the sample. Numerical calculations confirm that the free energy is lowered for the RNA segments displaying the larger number of branch points. The effect is, however, opposite with synthetic polyelectrolytes, in which a star topology gives rise to more disorder in the capsids than a linear topology. If RNA compactness and size account in part for the proper assembly of the nucleocapsid and the genome selectivity, other factors most likely related to the host cell environment during viral assembly must come into play as well.

From Protein Corona to Colloidal Self-Assembly: The Importance of Protein Size in Protein-Nanoparticle Interactions.

Protein adsorption on nanoparticles is an important field of study, particularly with regard to nanomedicine and nanotoxicology. Many factors can influence the composition and structure of the layer(s) of adsorbed proteins, the so-called protein corona. However, the role of protein size has not been specifically investigated, although some evidence has indicated its potential important role in corona composition and structure. To assess the role of protein size, we studied the interactions of hemoproteins (spanning a large size range) with monodisperse silica nanoparticles. We combined various techniques-adsorption isotherms, isothermal titration calorimetry, circular dichroism, and transmission electron cryomicroscopy-to address this issue. Overall, the results show that small proteins behaved as typical model proteins, forming homogeneous monolayers on the nanoparticle surface (protein corona). Their adsorption is purely enthalpy-driven, with subtle structural changes. In contrast, large proteins interact with nanoparticles via entropy-driven mechanisms. Their structure is completely preserved during adsorption, and any given protein can directly bind to several nanoparticles, forming bridges in these newly formed protein-nanoparticle assemblies. Protein size is clearly an overlooked factor that should be integrated into proteomics and toxicological studies.

Human Serum Albumin in the Presence of AGuIX Nanoagents: Structure Stabilisation without Direct Interaction.

The gadolinium-based nanoagent named AGuIX® is a unique radiosensitizer and contrast agent which improves the performance of radiotherapy and medical imaging. Currently tested in clinical trials, AGuIX® is administrated to patients via intravenous injection. The presence of nanoparticles in the blood stream may induce harmful effects due to undesired interactions with blood components. Thus, there is an emerging need to understand the impact of these nanoagents when meeting blood proteins. In this work, the influence of nanoagents on the structure and stability of the most abundant blood protein, human serum albumin, is presented. Synchrotron radiation circular dichroism showed that AGuIX® does not bind to the protein, even at the high ratio of 45 nanoparticles per protein at 3 mg/L. However, it increases the stability of the albumin. Isothermal thermodynamic calorimetry and fluorescence emission spectroscopy demonstrated that the effect is due to preferential hydration processes. Thus, this study confirms that intravenous injection of AGuIX® presents limited risks of perturbing the blood stream. In a wider view, the methodology developed in this work may be applied to rapidly evaluate the impact and risk of other nano-products that could come into contact with the bloodstream.

Protein-Nanoparticle Interactions: What Are the Protein-Corona Thickness and Organization?

Protein adsorption on a surface is generally evaluated in terms of the evolution of the proteins' structures and functions. However, when the surface is that of a nanoparticle, the protein corona formed around it possesses a particular supramolecular structure that gives a "biological identity" to the new object. Little is known about the actual shape of the protein corona. Here, the protein corona formed by the adsorption of model proteins (myoglobin and hemoglobin) on silica nanoparticles was studied. Small-angle neutron scattering and oxygenation studies were combined to assess both the structural and functional impacts of the adsorption on proteins. Large differences in the oxygenation properties could be found while no significant global shape changes were seen after adsorption. Moreover, the structural study showed that the adsorbed proteins form an organized yet discontinuous monolayer around the nanoparticles.

Importance of Post-translational Modifications in the Interaction of Proteins with Mineral Surfaces: The Case of Arginine Methylation and Silica surfaces.

Understanding the mechanisms involved in the interaction of proteins with inorganic surfaces is of major interest for both basic research and practical applications involving nanotechnology. From the list of cellular proteins with the highest affinity for silica nanoparticles, we highlighted the group of proteins containing arginine-glycine-glycine (RGG) motifs. Biochemical experiments confirmed that RGG motifs interact strongly with the silica surfaces. The affinity of these motifs is further increased when the R residue is asymmetrically, but not symmetrically, dimethylated. Molecular dynamics simulations show that the asymmetrical dimethylation generates an electrostatic asymmetry in the guanidinium group of the R residue, orientating and stabilizing it on the silica surface. The RGG motifs (methylated or not) systematically target the siloxide groups on the silica surface through an ionic interaction, immediately strengthened by hydrogen bonds with proximal silanol and siloxane groups. Given that, in vivo, RGG motifs are often asymmetrically dimethylated by specific cellular methylases, our data add support to the idea that this type of methylation is a key mechanism for cells to regulate the interaction of the RGG proteins with their cellular partners.

Protein Adsorption and Reorganization on Nanoparticles Probed by the Coffee-Ring Effect: Application to Single Point Mutation Detection.

The coffee-ring effect denotes the accumulation of particles at the edge of an evaporating sessile drop pinned on a substrate. Because it can be detected by simple visual inspection, this ubiquitous phenomenon can be envisioned as a robust and cost-effective diagnostic tool. Toward this direction, here we systematically analyze the deposit morphology of drying drops containing polystyrene particles of different surface properties with various proteins (bovine serum albumin (BSA) and different forms of hemoglobin). We show that deposit patterns reveal information on both the adsorption of proteins onto particles and their reorganization following adsorption. By combining pattern analysis with adsorption isotherm and zeta potential measurements, we show that the suppression of the coffee-ring effect and the formation of a disk-shaped pattern is primarily associated with particle neutralization by protein adsorption. However, our findings also suggest that protein reorganization following adsorption can dramatically invert this tendency. Exposure of hydrophobic (respectively charged) residues can lead to disk (respectively ring) deposit morphologies independently of the global particle charge. Surface tension measurements and microscopic observations of the evaporating drops show that the determinant factor of the deposit morphology is the accumulation of particles at the liquid/gas interface during evaporation. This general behavior opens the possibility to probe protein adsorption and reorganization on particles by the analysis of the deposit patterns, the formation of a disk being the robust signature of particles rendered hydrophobic by protein adsorption. We show that this method is sensitive enough to detect a single point mutation in a protein, as demonstrated here by the distinct patterns formed by human native hemoglobin h-HbA and its mutant form h-HbS, which is responsible for sickle cell anemia.

Human Serum Albumin in the Presence of Small Platinum Nanoparticles.

Noble metal materials, especially platinum nanoparticles (Pt NPs), have immense potential in nanomedicine as therapeutic agents on account of their high electron density and their high surface area. Intravenous injection is proposed as the best mode to deliver the product to patients. However, our understanding of the reaction of nanoparticles with blood components, especially proteins, is far behind the explosive development of these agents. Using synchrotron radiation circular dichroism (SRCD), we investigated the structural and stability changes of human serum albumin (HSA) upon interaction with PEG-OH coated Pt NPs at nanomolar concentrations, conditions potentially encountered for intravenous injection. There is no strong complexation found between HSA and Pt NPs. However, for the highest molar ratio of NP:HSA of 1:1, an increase of 18 °C in the thermal unfolding of HSA was observed, which is attributed to increased thermal stability of HSA generated by preferential hydration. This work proposes a new and fast method to probe the potential toxicity of nanoparticles intended for clinical use with intravenous injection.

Phenol-soluble modulins form amyloids in contact with multiple surface chemistries.

Functional amyloids are commonly produced by many microorganisms and their biological functions are numerous. Staphylococcus aureus can secrete a group of peptides named phenol-soluble modulins (PSMs) in their biofilm extracellular matrix. PSMs have been found inside biofilms both in their soluble form and assembled into amyloid structures. Yet, the actual biological function of these amyloids has been highly debated. Here, we assessed the ability of PSMs to form amyloids in contact with different abiotic surfaces to unravel a potential unknown bioadhesive and/or biofilm stabilization function. We combined surface plasmon resonance imaging, fluorescence aggregation kinetics, and FTIR spectroscopy in order to evaluate the PSM adsorption as well as amyloid formation properties in the presence of various surface chemistries. Overall, PSMs adsorb even on low-binding surfaces, making them highly adaptable adsorbants in the context of bioadhesion. Moreover, the PSM aggregation potential to form amyloid aggregates is not impacted by the presence of the surface chemistries tested. This versatility regarding adsorption and amyloid formation may imply a possible role of PSMs in biofilm adhesion and/or structure integrity.

Insulin aggregation starts at dynamic triple interfaces, originating from solution agitation.

The consequences of agitation on protein stability are particularly relevant to therapeutic proteins. However, the precise contribution of the different effects induced by agitation in pathways leading to protein denaturation and aggregation at interfaces is not entirely understood. In particular, the contribution of a moving triple line, induced by the sweeping of a solution meniscus on a container wall upon agitation, has only been rarely assessed. In this article, we therefore designed experimental setups to analyze how mixing, shear stress, and dynamic triple interfaces influence insulin aggregation in physiological conditions. This has been achieved by controlling agitation speed, shear stress, and the extension of triple interfaces in order to shed light on the contribution of different agitation-induced effects on insulin aggregation in physiological conditions. We demonstrate that strong agitation is necessary for the onset of insulin aggregation, while the growth of the aggregates is sustained even under weak agitation. Kinetic insulin aggregation studies in conditions of intermittent wetting show that the aggregation rate correlates with the amount of dynamic triple interfaces that the proteins are exposed to. Finally, we demonstrate that the triple line, where the protein solution, the air, and a hydrophobic surface meet constitutes a preferential early aggregation site.

How a Virus Circumvents Energy Barriers to Form Symmetric Shells.

Previous self-assembly experiments on a model icosahedral plant virus have shown that, under physiological conditions, capsid proteins initially bind to the genome through an en masse mechanism and form nucleoprotein complexes in a disordered state, which raises the question as to how virions are assembled into a highly ordered structure in the host cell. Using small-angle X-ray scattering, we find out that a disorder-order transition occurs under physiological conditions upon an increase in capsid protein concentrations. Our cryo-transmission electron microscopy reveals closed spherical shells containing in vitro transcribed viral RNA even at pH 7.5, in marked contrast with the previous observations. We use Monte Carlo simulations to explain this disorder-order transition and find that, as the shell grows, the structures of disordered intermediates in which the distribution of pentamers does not belong to the icosahedral subgroups become energetically so unfavorable that the caps can easily dissociate and reassemble, overcoming the energy barriers for the formation of perfect icosahedral shells. In addition, we monitor the growth of capsids under the condition that the nucleation and growth is the dominant pathway and show that the key for the disorder-order transition in both en masse and nucleation and growth pathways lies in the strength of elastic energy compared to the other forces in the system including protein-protein interactions and the chemical potential of free subunits. Our findings explain, at least in part, why perfect virions with icosahedral order form under different conditions including physiological ones.

Protein Corona Composition of Silica Nanoparticles in Complex Media: Nanoparticle Size does not Matter.

Biomolecules, and particularly proteins, bind on nanoparticle (NP) surfaces to form the so-called protein corona. It is accepted that the corona drives the biological distribution and toxicity of NPs. Here, the corona composition and structure were studied using silica nanoparticles (SiNPs) of different sizes interacting with soluble yeast protein extracts. Adsorption isotherms showed that the amount of adsorbed proteins varied greatly upon NP size with large NPs having more adsorbed proteins per surface unit. The protein corona composition was studied using a large-scale label-free proteomic approach, combined with statistical and regression analyses. Most of the proteins adsorbed on the NPs were the same, regardless of the size of the NPs. To go beyond, the protein physicochemical parameters relevant for the adsorption were studied: electrostatic interactions and disordered regions are the main driving forces for the adsorption on SiNPs but polypeptide sequence length seems to be an important factor as well. This article demonstrates that curvature effects exhibited using model proteins are not determining factors for the corona composition on SiNPs, when dealing with complex biological media.

In Situ Analysis of Weakly Bound Proteins Reveals Molecular Basis of Soft Corona Formation.

Few experimental techniques allow the analysis of the protein corona in situ. As a result, little is known on the effects of nanoparticles on weakly bound proteins that form the soft corona. Despite its biological importance, our understanding of the molecular bases driving its formation is limited. Here, we show that hemoglobin can form either a hard or a soft corona on silica nanoparticles depending on the pH conditions. Using cryoTEM and synchrotron-radiation circular dichroism, we show that nanoparticles alter the structure and the stability of weakly bound proteins in situ. Molecular dynamics simulation identified the structural elements driving protein-nanoparticle interaction. Based on thermodynamic analysis, we show that nanoparticles stabilize partially unfolded protein conformations by enthalpy-driven molecular interactions. We suggest that nanoparticles alter weakly bound proteins by shifting the equilibrium toward the unfolded states at physiological temperature. We show that the classical approach based on nanoparticle separation from the biological medium fails to detect destabilization of weakly bound proteins, and therefore cannot be used to fully predict the biological effects of nanomaterials in situ.

Albumin-driven disassembly of lipidic nanoparticles: the specific case of the squalene-adenosine nanodrug.

In the field of nanomedicine, nanostructured nanoparticles (NPs) made of self-assembling prodrugs emerged in the recent years with promising properties. In particular, squalene-based drug nanoparticles have already shown their efficiency through in vivo experiments. However, a complete pattern of their stability and interactions in the blood stream is still lacking. In this work we assess the behavior of squalene-adenosine (SQAd) nanoparticles - whose neuroprotective effect has already been demonstrated in murine models - in the presence of fetal bovine serum (FBS) and of bovine serum albumin (BSA), the main protein of blood plasma. Extensive physicochemical characterizations were performed using Small Angle Neutron Scattering (SANS), cryogenic transmission electron microscopy (Cryo-TEM), circular dichroism (CD), steady-state fluorescence spectroscopy (SSFS) and isothermal titration calorimetry (ITC) as well as in silico by means of ensemble docking simulations with human serum albumin (HSA). Significant changes in the colloidal stability of the nanoparticles in the presence of serum albumin were observed. SANS, CD and SSFS analyses demonstrated an interaction between SQAd and BSA, with a partial disassembly of the nanoparticles in the presence of BSA and the formation of a complex between SQAd and BSA. The interaction free energy of SQAd nanoparticles with BSA derived from ITC experiments, is about -8 kcal mol-1 which is further supported in silico by ensemble docking simulations. Overall, our results show that serum albumin partially disassembles SQAd nanoparticles by extracting individual SQAd monomers from them. As a consequence, the SQAd nanoparticles would act as a circulating reservoir in the blood stream. The approach developed in this study could be extended to other soft organic nanoparticles.

RNA-binding proteins are a major target of silica nanoparticles in cell extracts.

Upon contact with biological fluids, nanoparticles (NPs) are readily coated by cellular compounds, particularly proteins, which are determining factors for the localization and toxicity of NPs in the organism. Here, we improved a methodological approach to identify proteins that adsorb on silica NPs with high affinity. Using large-scale proteomics and mixtures of soluble proteins prepared either from yeast cells or from alveolar human cells, we observed that proteins with large unstructured region(s) are more prone to bind on silica NPs. These disordered regions provide flexibility to proteins, a property that promotes their adsorption. The statistical analyses also pointed to a marked overrepresentation of RNA-binding proteins (RBPs) and of translation initiation factors among the adsorbed proteins. We propose that silica surfaces, which are mainly composed of Si-O- and Si-OH groups, mimic ribose-phosphate molecules (rich in -O- and -OH) and trap the proteins able to interact with ribose-phosphate containing molecules. Finally, using an in vitro assay, we showed that the sequestration of translation initiation factors by silica NPs results in an inhibition of the in vitro translational activity. This result demonstrates that characterizing the protein corona of various NPs would be a relevant approach to predict their potential toxicological effects.