Abstract\ud \ud This work reports the results of coarse-grained molecular dynamics simulations of citrate-coated gold nanoparticles (AuNPs) in interaction with insulin and fibrinogen, two of the most abundant proteins in the plasma. The following have been found: (a) The corona of citrate-coated AuNP of 5 nm core diameter is composed by a single layer of proteins comprising a maximum of 20 insulins, whereas only 3 fibrinogens are contemporaneously present. (b) The binding site for insulin is specific and independent from the number of insulins considered in the computational simulations, whereas fibrinogen presents different binding modes, as a function of protein concentration and composition. Moreover, fibrinogen is able to accommodate two citrate-coated AuNPs in independent binding sites localized at the ending nodes. (c) A competitve process for AuNP binding is observed when insulins and fibrinogens are contemporaneously present in the simulations. (d) The overall protein secondary structure is maintained upon binding to a single citrate-coated AuNP, but small changes in helix and sheet percentages are observed for both proteins. (e) A partial unfolding of the α-helix bundle is found for fibrinogen bound to two AuNPs. This may provide a molecular level understanding of the inflammatory response to nanoparticles. © 2015 American Chemical Society

In this study, coarse-grained computational simulations of the ubiquitin corona around gold nanoparticles have been carried out, and the effect of the nanoparticle size (10, 16, 20, and 24 nm diameter) and environment (bare nanoparticle surface, and citrate-coated surface, where citrate are treated with implicit and explicit models) has been analysed. The results showed that the corona is obtained after a slow reorientation step that occurs at the nanoparticle surface in order to optimize the nanoparticle–ubiquitins interaction. The ubiquitin binding modalities depend on the nanoparticle environment, while conformational changes of ubiquitins upon binding and their aggregation propensity slightly depend on nanoparticle size

Strategies for protein detection via surface-enhanced Raman spectroscopy (SERS) currently exploit the formation of randomly generated hot spots at the interfaces of metal colloidal nanoparticles, which are clustered together by intrusive chemical or physical processes in the presence of the target biomolecule. We propose a different approach based on selective and quantitative gathering of protein molecules at regular hot spots generated on the corners of individual silver nanocubes in aqueous medium at physiological pH. Here, the protein, while keeping its native configuration, experiences an intense local E-field, which boosts SERS efficiency and detection sensitivity. Uncontrolled signal fluctuations caused by variable molecular adsorption to different particle areas or inside clustered nanoparticles are circumvented. Advanced electron microscopy analyses and computational simulations outline a strategy relying on a site-selective mechanism with superior Raman signal enhancement, which offers the perspective of highly controlled and reproducible routine SERS detection of proteins.

Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicities and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the development of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) that efficiently label the edges of synthetic, recombinant, and native amyloid fibrils derived from different amyloidogenic proteins. We demonstrate that these NPs represent powerful tools for assessing amyloid morphological polymorphism, using cryogenic transmission electron microscopy (cryo-EM). The NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including transmission electron microscopy with use of the negative stain or cryo-EM imaging. The use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of a patient with Alzheimer’s disease, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain amyloidosis, revealed a high degree of homogeneity across the fibrils derived from human tissue in comparison with fibrils aggregated in vitro. These findings are consistent with, and strongly support, the emerging view that the physiologic milieu is a key determinant of amyloid fibril strains. Together, these advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM, but also pave the way to elucidate the structural basis of amyloid strains and toxicity, and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.

We demonstrate that the designed ankyrin repeat protein (DARPin)_9-29, which specifically targets human epidermal growth factor receptor 2 (HER 2), binds tightly to gold nanoparticles (GNPs). Binding of the protein strongly increases the colloidal stability of the particles. The results of experimental analysis and molecular dynamics simulations show that approximately 35 DARPin_9-29 molecules are bound to the surface of a 5 nm GNP and that the binding does not involve the receptor-binding domain of the protein. The confocal fluorescent microscopy studies show that the DARPin-coated GNP conjugate specifically interacts with the surface of human cancer cells overexpressing epidermal growth factor receptor 2 (HER2) and enters the cells by endocytosis. The high stability under physiological conditions and high affinity to the receptors overexpressed by cancer cells make conjugates of plasmonic gold nanostructures with DARPin molecules promising candidates for cancer therapy.