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.

Using NMR Spectroscopy To Measure Protein Binding Capacity on Gold Nanoparticles.

A simple one-dimensional 1H NMR experiment that quantifies protein bound to gold nanoparticles has been developed for upper-division biochemistry and physical chemistry students. This laboratory experiment teaches the basics of NMR techniques, which is a highly effective tool in protein studies and supports students to understand the concepts of NMR spectroscopy and nanoparticle-protein interactions. Understanding the interactions of gold nanoparticles (AuNPs) with biological macromolecules is becoming increasingly important as interest in the clinical use of nanoparticles has been on the rise. Applications in drug delivery, biosensing, diagnostics, and enhanced imaging are all tangible possibilities with a better understanding of AuNP-protein interactions. The ability to use AuNPs as biosensors for drug delivery methods in cellular uptake is dependent on the amount of protein that is able to bind to the surface of the nanoparticle. This laboratory experiment solidifies concepts such as quantitative NMR spectroscopy while reinforcing precision laboratory titrations. Students learn how 1H proton NMR spectra can be used to measure free protein in solution and protein bound to AuNPs. A simple formula is used to determine the binding capacity of the nanoparticle. This analysis helps students to understand the impact of nanoparticle-protein interactions, and it allows them to conceptualize macromolecular binding using NMR spectroscopy.

Surface Plasmon Resonance, Formation Mechanism, and Surface Enhanced Raman Spectroscopy of Ag+-Stained Gold Nanoparticles.

A series of recent works have demonstrated the spontaneous Ag+ adsorption onto gold surfaces. However, a mechanistic understanding of the Ag+ interactions with gold has been controversial. Reported herein is a systematic study of the Ag+ binding to AuNPs using several in-situ and ex-situ measurement techniques. The time-resolved UV-vis measurements of the AuNP surface plasmonic resonance revealed that the silver adsorption proceeds through two parallel pseudo-first order processes with a time constant of 16(±2) and 1,000(±35) s, respectively. About 95% of the Ag+ adsorption proceeds through the fast adsorption process. The in-situ zeta potential data indicated that this fast Ag+ adsorption is driven primarily by the long-range electrostatic forces that lead to AuNP charge neutralization, while the time-dependent pH data shows that the slow Ag+ binding process involves proton-releasing reactions that must be driven by near-range interactions. These experimental data, together with the ex-situ XPS measurement indicates that adsorbed silver remains cationic, but not as a charged-neutral silver atom proposed by the anti-galvanic reaction mechanism. The surface-enhanced Raman activities of the Ag+-stained AuNPs are slightly higher than that for AuNPs, but significantly lower than that for the silver nanoparticles (AgNPs). The SERS feature of the ligands on the Ag+-stained AuNPs can differ from that on both AuNPs and AgNPs. Besides the new insights to formation mechanism, properties, and applications of the Ag+-stained AuNPs, the experimental methodology presented in this work can also be important for studying nanoparticle interfacial interactions.

Protein Interactions with Nanoparticle Surfaces: Highlighting Solution NMR Techniques.

In the last decade, nanoparticles (NPs) have become a key tool in medicine and biotechnology as drug delivery systems, biosensors and diagnostic devices. The composition and surface chemistry of NPs vary based on the materials used: typically organic polymers, inorganic materials, or lipids. Nanoparticle classes can be further divided into sub-categories depending on the surface modification and functionalization. These surface properties matter when NPs are introduced into a physiological environment, as they will influence how nucleic acids, lipids, and proteins will interact with the NP surface. While small-molecule interactions are easily probed using NMR spectroscopy, studying protein-NP interactions using NMR introduces several challenges. For example, globular proteins may have a perturbed conformation when attached to a foreign surface, and the size of NP-protein conjugates can lead to excessive line broadening. Many of these challenges have been addressed, and NMR spectroscopy is becoming a mature technique for in situ analysis of NP binding behavior. It is therefore not surprising that NMR has been applied to NP systems and has been used to study biomolecules on NP surfaces. Important considerations include corona composition, protein behavior, and ligand architecture. These features are difficult to resolve using classical surface and material characterization strategies, and NMR provides a complementary avenue of characterization. In this review, we examine how solution NMR can be combined with other analytical techniques to investigate protein behavior on NP surfaces.

Electrostatic Interactions and Protein Competition Reveal a Dynamic Surface in Gold Nanoparticle-Protein Adsorption.

Gold nanoparticle- (AuNP-) protein conjugates are potentially useful in a broad array of diagnostic and therapeutic applications, but the physical basis of the simultaneous adsorption of multiple proteins onto AuNP surfaces remains poorly understood. Here, we investigate the contribution of electrostatic interactions to protein-AuNP binding by studying the pH-dependent binding behavior of two proteins, GB3 and ubiquitin. For both proteins, binding to 15-nm citrate-coated AuNPs closely tracks with the predicted net charge using standard pKa values, and a dramatic reduction in binding is observed when lysine residues are chemically methylated. This suggests that clusters of basic residues are involved in binding, and using this hypothesis, we model the pKa shifts induced by AuNP binding. Then, we employ a novel NMR-based approach to monitor the binding competition between GB3 and ubiquitin in situ at different pH values. In light of our model, the NMR measurements reveal that the net charge, binding association constant, and size of each protein play distinct roles at different stages of protein adsorption. When citrate-coated AuNPs and proteins first interact, net charge appears to dominate. However, as citrate molecules are displaced by protein, the surface chemistry changes, and the energetics of binding becomes far more complex. In this case, we observed that GB3 is able to displace ubiquitin at intermediate time scales, even though it has a lower net charge. The thermodynamic model for binding developed here could be the first step toward predicting the binding behavior in biological fluids, such as blood plasma.

Understanding Protein Structure Deformation on the Surface of Gold Nanoparticles of Varying Size.

Gold nanoparticles (AuNPs) have been of recent interest due to their unique optical properties and their biocompatibility. Biomolecules spontaneously adsorb to their surface, a trait that could potentially be exploited for drug targeting. Currently, it is unclear whether protein-AuNP interactions at the nanoparticle surface are dependent on nanoparticle size. In this work, we investigate whether varying surface curvature can induce protein unfolding and multilayer binding in citrate-coated AuNPs of various sizes. A recently developed NMR-based approach was utilized to determine the adsorption capacity, and protein NMR spectra were compared to determine whether nanoparticle size influences protein interactions at the surface. In addition, transmission electron microscopy (TEM) and dynamic light scattering (DLS) were employed to corroborate the NMR studies. Over a broad range of AuNP sizes (14-86 nm), we show that adsorption capacity can be predicted by assuming that proteins are compact and globular on the nanoparticle surface. Additionally, roughly one layer of proteins is adsorbed regardless of AuNP size. Our results hold for two proteins of significantly different sizes, GB3 (6 kDa) and bovine carbonic anhydrase (BCA, 29 kDa). However, the unstable drkN SH3 domain (ΔG0 ≈ 0, 7 kDa) does not appear to follow the same trend seen for stable, globular proteins. This observation suggests that unstable proteins can deform significantly when bound to AuNP surfaces. Taken together, the results of this work can be used to improve our knowledge of the mechanism of protein-AuNP interactions to optimize their use in the biomedical field.