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.

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.

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.

Human disease-associated calmodulin mutations alter calcineurin function through multiple mechanisms.

Calmodulin (CaM) is a ubiquitous, calcium-sensing protein that regulates a multitude of processes throughout the body. In response to changes in [Ca2+], CaM modifies, activates, and deactivates enzymes and ion channels, as well as many other cellular processes. The importance of CaM is highlighted by the conservation of an identical amino acid sequence in all mammals. Alterations to CaM amino acid sequence were once thought to be incompatible with life. During the last decade modifications to the CaM protein sequence have been observed in patients suffering from life-threatening heart disease (calmodulinopathy). Thus far, inadequate or untimely interaction between mutant CaM and several proteins (LTCC, RyR2, and CaMKII) have been identified as mechanisms underlying calmodulinopathy. Given the extensive number of CaM interactions in the body, there are likely many consequences for altering CaM protein sequence. Here, we demonstrate that disease-associated CaM mutations alter the sensitivity and activity of the Ca2+-CaM-enhanced serine/threonine phosphatase calcineurin (CaN). Biophysical characterization by circular dichroism, solution NMR spectroscopy, stopped-flow kinetic measurements, and MD simulations provide mechanistic insight into mutation dysfunction as well as highlight important aspects of CaM Ca2+ signal transduction. We find that individual CaM point mutations (N53I, F89L, D129G, and F141L) impair CaN function, however, the mechanisms are not the same. Specifically, individual point mutations can influence or modify the following properties: CaM binding, Ca2+ binding, and/or Ca2+kinetics. Moreover, structural aspects of the CaNCaM complex can be altered in manners that indicate changes to allosteric transmission of CaM binding to the enzyme active site. Given that loss of CaN function can be fatal, as well as evidence that CaN modifies ion channels already associated with calmodulinopathy, our results raise the possibility that altered CaN function contributes to calmodulinopathy.

Pneumococcal surface adhesion A protein (PsaA) interacts with human Annexin A2 on airway epithelial cells.

Streptococcus pneumoniae (pneumococcus) is a normal colonizer of the human nasopharynx capable of causing serious invasive disease. Since colonization of the nasopharynx is a prerequisite for progression to invasive diseases, the development of future protein-based vaccines requires an understanding of the intimate interaction of bacterial adhesins with host receptors. In this study, we identified that pneumococcal surface adhesin A (PsaA), a highly conserved pneumococcal protein known to play an important role in colonization of pneumococcus, can interact with Annexin A2 (ANXA2) on Detroit 562 nasopharyngeal epithelial cells. Lentiviral expression of ANXA2 in HEK 293 T/17 cells, which normally express minimal ANXA2, significantly increased pneumococcal adhesion. Blocking of ANXA2 with recombinant PsaA negatively impacted pneumococcal adherence to ANXA2-transduced HEK cells. These results suggest that ANXA2 is an important host cellular receptor for pneumococcal colonization.

Understanding How Staphylococcal Autolysin Domains Interact With Polystyrene Surfaces.

Biofilms, when formed on medical devices, can cause malfunctions and reduce the efficiency of these devices, thus complicating treatments and serving as a source of infection. The autolysin protein of Staphylococcus epidermidis contributes to its biofilm forming ability, especially on polystyrene surfaces. R2ab and amidase are autolysin protein domains thought to have high affinity to polystyrene surfaces, and they are involved in initial bacterial attachment in S. epidermidis biofilm formation. However, the structural details of R2ab and amidase binding to surfaces are poorly understood. In this study, we have investigated how R2ab and amidase influence biofilm formation on polystyrene surfaces. We have also studied how these proteins interact with polystyrene nanoparticles (PSNPs) using biophysical techniques. Pretreating polystyrene plates with R2ab and amidase domains inhibits biofilm growth relative to a control protein, indicating that these domains bind tightly to polystyrene surfaces and can block bacterial attachment. Correspondingly, we find that both domains interact strongly with anionic, carboxylate-functionalized as well as neutral, non-functionalized PSNPs, suggesting a similar binding interaction for nanoparticles and macroscopic surfaces. Both anionic and neutral PSNPs induce changes to the secondary structure of both R2ab and amidase as monitored by circular dichroism (CD) spectroscopy. These changes are very similar, though not identical, for both types of PSNPs, suggesting that carboxylate functionalization is only a small perturbation for R2ab and amidase binding. This structural change is also seen in limited proteolysis experiments, which exhibit substantial differences for both proteins when in the presence of carboxylate PSNPs. Overall, our results demonstrate that the R2ab and amidase domains strongly favor adsorption to polystyrene surfaces, and that surface adsorption destabilizes the secondary structure of these domains. Bacterial attachment to polystyrene surfaces during the initial phases of biofilm formation, therefore, may be mediated by aromatic residues, since these residues are known to drive adsorption to PSNPs. Together, these experiments can be used to develop new strategies for biofilm eradication, ensuring the proper long-lived functioning of medical devices.