Protein Binding Leads to Reduced Stability and Solvated Disorder in the Polystyrene Nanoparticle Corona.

Understanding the conformation of proteins in the nanoparticle corona has important implications in how organisms respond to nanoparticle-based drugs. These proteins coat the nanoparticle surface, and their properties will influence the nanoparticle's interaction with cell targets and the immune system. While some coronas are thought to be disordered, two key unanswered questions are the degree of disorder and solvent accessibility. Here, a model is developed for protein corona disorder in polystyrene nanoparticles of varying size. For two different proteins, it is found that binding affinity decreases as nanoparticle size increases. The stoichiometry of binding, along with changes in the hydrodynamic size, supports a highly solvated, disordered protein corona anchored at a small number of attachment sites. The scaling of the stoichiometry versus nanoparticle size is consistent with disordered polymer dimensions. Moreover, it is found that proteins are destabilized less in the presence of larger nanoparticles, and hydrophobic exposure decreases at lower curvatures. The observations hold for proteins on flat polystyrene surfaces, which have the lowest hydrophobic exposure. The model provides an explanation for previous observations of increased amyloid fibrillation rates in the presence of larger nanoparticles, and it may rationalize how cell receptors can recognize protein disorder in therapeutic nanoparticles.

What is allosteric regulation? Exploring the exceptions that prove the rule!

"Allosteric" was first introduced to mean the other site (i.e., a site distinct from the active or orthosteric site), an adjective for "regulation" to imply a regulatory outcome resulting from ligand binding at another site. That original idea outlines a system with two ligand-binding events at two distinct locations on a macromolecule (originally a protein system), which defines a four-state energy cycle. An allosteric energy cycle provides a quantifiable allosteric coupling constant and focuses our attention on the unique properties of the four equilibrated protein complexes that constitute the energy cycle. Because many observed phenomena have been referenced as "allosteric regulation" in the literature, the goal of this work is to use literature examples to explore which systems are and are not consistent with the two-ligand thermodynamic energy cycle-based definition of allosteric regulation. We emphasize the need for consistent language so comparisons can be made among the ever-increasing number of allosteric systems. Building on the mutually exclusive natures of an energy cycle definition of allosteric regulation versus classic two-state models, we conclude our discussion by outlining how the often-proposed Rube-Goldberg-like mechanisms are likely inconsistent with an energy cycle definition of allosteric regulation.

Protein-Functionalized Gold Nanospheres with Tunable Photothermal Efficiency for the Near-Infrared Photothermal Ablation of Biofilms.

Temperature-responsive nanostructures with high antimicrobial efficacy are attractive for therapeutic applications against multidrug-resistant bacteria. Here, we report temperature-responsive nanospheres (TRNs) engineered to undergo self-association and agglomeration above a tunable transition temperature (Tt). The temperature-responsive behavior of the nanoparticles is obtained by functionalizing citrate-capped spherical gold nanoparticles (AuNPs) with elastin-like polypeptides (ELPs). Using protein design principles, we achieve a broad range of attainable Tt values and photothermal conversion efficiencies (η). Two approaches were used to adjust this range: First, by altering the position of the cysteine residue used to attach ELP to the AuNP, we attained a Tt range from 34 to 42 °C. Then, by functionalizing the AuNP with an additional small globular protein, we could extend this range to 34-50 °C. Under near-infrared (NIR) light exposure, all TRNs exhibited reversible agglomeration. Moreover, they showed an enhanced photothermal conversion efficiency in their agglomerated state relative to the dispersed state. Despite their spherical shape, TRNs have a photothermal conversion efficiency approaching that of gold nanorods (η = 68 ± 6%), yet unlike nanorods, the synthesis of TRNs requires no cytotoxic compounds. Finally, we tested TRNs for the photothermal ablation of biofilms. Above Tt, NIR irradiation of TRNs resulted in a 10,000-fold improvement in killing efficiency compared to untreated controls (p < 0.0001). Below Tt, no enhanced antibiofilm effect was observed. In conclusion, engineering the interactions between proteins and nanoparticles enables the tunable control of TRNs, resulting in a novel antibiofilm nanomaterial with low cytotoxicity.

Exploring Residue-Level Interactions between the Biofilm-Driving R2ab Protein and Polystyrene Nanoparticles.

In biological systems, proteins can bind to nanoparticles to form a "corona" of adsorbed molecules. The nanoparticle corona is of significant interest because it impacts an organism's response to a nanomaterial. Understanding the corona requires knowledge of protein structure, orientation, and dynamics at the surface. A residue-level mapping of protein behavior on nanoparticle surfaces is needed, but this mapping is difficult to obtain with traditional approaches. Here, we have investigated the interaction between R2ab and polystyrene nanoparticles (PSNPs) at the level of individual residues. R2ab is a bacterial surface protein from Staphylococcus epidermidis and is known to interact strongly with polystyrene, leading to biofilm formation. We have used mass spectrometry after lysine methylation and hydrogen-deuterium exchange (HDX) NMR spectroscopy to understand how the R2ab protein interacts with PSNPs of different sizes. Lysine methylation experiments reveal subtle but statistically significant changes in methylation patterns in the presence of PSNPs, indicating altered protein surface accessibility. HDX rates become slower overall in the presence of PSNPs. However, some regions of the R2ab protein exhibit faster than average exchange rates in the presence of PSNPs, while others are slower than the average behavior, suggesting conformational changes upon binding. HDX rates and methylation ratios support a recently proposed "adsorbotope" model for PSNPs, wherein adsorbed proteins consist of unfolded anchor points interspersed with partially structured regions. Our data also highlight the challenges of characterizing complex protein-nanoparticle interactions using these techniques, such as fast exchange rates. While providing insights into how R2ab adsorbs onto PSNP surfaces, this research emphasizes the need for advanced methods to comprehend residue-level interactions in the nanoparticle corona.

Exploring the Residue-Level Interactions between the R2ab Protein and Polystyrene Nanoparticles.

In biological systems, proteins can bind to nanoparticles to form a "corona" of adsorbed molecules. The nanoparticle corona is of high interest because it impacts the organism's response to the nanomaterial. Understanding the corona requires knowledge of protein structure, orientation, and dynamics at the surface. Ultimately, a residue-level mapping of protein behavior on nanoparticle surfaces is needed, but this mapping is difficult to obtain with traditional approaches. Here, we have investigated the interaction between R2ab and polystyrene nanoparticles (PSNPs) at the level of individual residues. R2ab is a bacterial surface protein from Staphylococcus epidermidis and is known to interact strongly with polystyrene, leading to biofilm formation. We have used mass spectrometry after lysine methylation and hydrogen-deuterium exchange (HDX) NMR spectroscopy to understand how the R2ab protein interacts with PSNPs of different sizes. Through lysine methylation, we observe subtle but statistically significant changes in methylation patterns in the presence of PSNPs, indicating altered protein surface accessibility. HDX measurements reveal that certain regions of the R2ab protein undergo faster exchange rates in the presence of PSNPs, suggesting conformational changes upon binding. Both results support a recently proposed "adsorbotope" model, wherein adsorbed proteins consist of unfolded anchor points interspersed with regions of partial structure. Our data also highlight the challenges of characterizing complex protein-nanoparticle interactions using these techniques, such as fast exchange rates. While providing insights into how proteins respond to nanoparticle surfaces, this research emphasizes the need for advanced methods to comprehend these intricate interactions fully at the residue level.

Near-Infrared Photothermal Ablation of Biofilms using ProteinFunctionalized Gold Nanospheres with a Tunable Temperature Response.

Temperature-responsive nanostructures with high antimicrobial efficacy are attractive for therapeutic applications against multi-drug-resistant bacteria. Here, we report temperature-responsive nanospheres (TRNs) that are engineered to undergo self-association and agglomeration above a tunable transition temperature (Tt). Temperature-responsive behavior of the nanoparticles is obtained by functionalizing citrate-capped, spherical gold nanoparticles (AuNPs) with elastin-like polypeptides (ELPs). Using protein design principles, we achieve a broad range of attainable Tt values and photothermal conversion efficiencies (η). Two approaches were used to adjust this range: First, by altering the position of the cysteine residue used to attach ELP to the AuNP, we attained a Tt range from 34-42 °C. Then, functionalizing the AuNP with an additional small globular protein, we were able to extend this range to 34-50 °C. Under near-infrared (NIR) light exposure, all TRNs exhibited reversible agglomeration. Moreover, they showed enhanced photothermal conversion efficiency in their agglomerated state relative to the dispersed state. Despite their spherical shape, TRNs have a photothermal conversion efficiency approaching that of gold nanorods (η = 68±6%), yet unlike nanorods, the synthesis of TRNs requires no cytotoxic compounds. Finally, we tested TRNs for photothermal ablation of biofilms. Above Tt, NIR irradiation of TRNs resulted in a 10,000-fold improvement in killing efficiency compared to untreated controls (p < 0.0001). Below Tt, no enhanced anti-biofilm effect was observed. In conclusion, engineering the interactions between proteins and nanoparticles enables the tunable control of TRNs, resulting in a novel, anti-biofilm nanomaterial with low cytotoxicity.

Selective Near-Infrared Blood Detection Driven by Ionic Liquid-Dye-Albumin Nanointeractions.

Due to its abundance in blood, a great deal of research has been undertaken to develop efficient biosensors for serum albumin and provide insight into the interactions that take place between these biosensing molecules and the protein. Near-infrared (NIR, >700 nm) organic dyes have been shown to be effective biosensors of serum albumin, but their effectiveness is diminished in whole blood. Herein, it is shown that an NIR sulfonate indolizine-donor-based squaraine dye, SO3SQ, can be strengthened as a biosensor of albumin through the addition of biocompatible ionic liquids (ILs). Specifically, the IL choline glycolate (1:1), at a concentration of 160 mM, results in the enhanced fluorescence emission ("switch-on") of the dye in the presence of blood. The origin of the fluorescence enhancement was investigated via methods, including DLS, ITC, and molecular dynamics. Further, fluorescence measurements were conducted to see the impact the dye-IL system had on the fluorescence of the tryptophan residue of human serum albumin (HSA), as well as to determine its apparent association constants in relation to albumin. Circular dichroism (CD) spectroscopy was used to provide evidence that the dye-IL system does not alter the secondary structures of albumin or DNA. Our results suggest that the enhanced fluorescence of the dye in the presence of IL and blood is due to diversification of binding sites in albumin, controlled by the interaction of the IL-dye-albumin complex.

Protein Binding Leads to Reduced Stability and Solvated Disorder in the Polystyrene Nanoparticle Corona.

Understanding the conformation of proteins in the nanoparticle corona has important implications in how organisms respond to nanoparticle-based drugs. These proteins coat the nanoparticle surface, and their properties will influence the nanoparticle's interaction with cell targets and the immune system. While some coronas are thought to be disordered, two key unanswered questions are the degree of disorder and solvent accessibility. Here, using a comprehensive thermodynamic approach, along with supporting spectroscopic experiments, we develop a model for protein corona disorder in polystyrene nanoparticles of varying size. For two different proteins, we find that binding affinity decreases as nanoparticle size increases. The stoichiometry of binding, along with changes in the hydrodynamic size, support a highly solvated, disordered protein corona anchored at a small number of enthalpically-driven attachment sites. The scaling of the stoichiometry vs. nanoparticle size is consistent disordered polymer dimensions. Moreover, we find that proteins are destabilized less severely in the presence of larger nanoparticles, and this is supported by measurements of hydrophobic exposure, which becomes less pronounced at lower curvatures. Our observations hold for flat polystyrene surfaces, which, when controlled for total surface area, have the lowest hydrophobic exposure of all systems. Our model provides an explanation for previous observations of increased amyloid fibrillation rates in the presence of larger nanoparticles, and it may rationalize how cell receptors can recognize protein disorder in therapeutic nanoparticles.