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1. Thermodynamics of Protein-Surface Binding- the Model Makes all the Difference

   Nicholas C. Fitzkee, Kayla D (February 2020, Biophysical journal)

   null (Ed.)

   Gold nanoparticles (AuNPs) are now being used in such areas as diagnostics, drug delivery, and biological sensing. In these applications, AuNPs are frequently exposed to biological fluids. These fluids contain many different proteins, any of which may interfere with the intended function of the nanoparticle. In this work, we examine the thermodynamic consequences of proteinnanoparticle binding using a combined spectroscopic and calorimetric approach. We monitored binding using UV-Vis spectroscopy, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC). Six proteins were studied based on their differing chemical properties, and both 15 nm and 30 nm citrate-coated AuNPs were investigated. We interpreted the UV-Vis data using two different models: the commonly-used Langmuir isotherm model and a more complex mass transport model. Both models can be used to determine Kd values for the 30 nm AuNP data; however, the mass transport model is more appropriate for 15 nm AuNPs. This is because, when fitting the Langmuir model, it is commonly assumed that most proteins are not surface-associated, and this assumption fails for 15 nm AuNPs. The DSC thermograms show two transitions for a globular protein adsorbed to a 15 nm AuNP: one high-temperature transition that is similar to global protein unfolding (68 C), and one low-temperature transition that may correspond to unfolding at the surface (56 C). Conversely, ITC experiments show no net heat of adsorption for GB3, even at high protein/AuNP concentrations. Together, the spectroscopic and calorimetric data suggest a complex, multi-step process for protein-nanoparticle adsorption. Moreover, for the proteins studied, both AuNP curvature and protein chemistry contribute to protein adsorption, with proteins generally binding more weakly to the larger nanoparticles. In the future, this work may lead to principles for improving the design of AuNPbased therapeutics and sensors. 

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2. Understanding How Staphylococcal Autolysin Domains Interact With Polystyrene Surfaces

   https://doi.org/10.3389/fmicb.2021.658373  

   Somarathne, Radha P.; Chappell, Emily R.; Perera, Y. Randika; Yadav, Rahul; Park, Joo Youn; Fitzkee, Nicholas C. (May 2021, Frontiers in Microbiology)

   null (Ed.)

   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. 

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3. Predicting protein function and orientation on a gold nanoparticle surface using a residue-based affinity scale

   https://doi.org/10.1038/s41467-022-34749-w  

   Xu, Joanna Xiuzhu; Alom, Md. Siddik; Yadav, Rahul; Fitzkee, Nicholas C. (November 2022, Nature Communications)

   Abstract The orientation adopted by proteins on nanoparticle surfaces determines the nanoparticle’s bioactivity and its interactions with living systems. Here, we present a residue-based affinity scale for predicting protein orientation on citrate-gold nanoparticles (AuNPs). Competitive binding between protein variants accounts for thermodynamic and kinetic aspects of adsorption in this scale. For hydrophobic residues, the steric considerations dominate, whereas electrostatic interactions are critical for hydrophilic residues. The scale rationalizes the well-defined binding orientation of the small GB3 protein, and it subsequently predicts the orientation and active site accessibility of two enzymes on AuNPs. Additionally, our approach accounts for the AuNP-bound activity of five out of six additional enzymes from the literature. The model developed here enables high-throughput predictions of protein behavior on nanoparticles, and it enhances our understanding of protein orientation in the biomolecular corona, which should greatly enhance the performance and safety of nanomedicines used in vivo. 

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4. Understanding the Adsorption of Peptides and Proteins onto PEGylated Gold Nanoparticles

   https://doi.org/10.3390/molecules26195788  

   Perera, Yasiru Randika; Xu, Joanna Xiuzhu; Amarasekara, Dhanush L.; Hughes, Alex C.; Abbood, Ibraheem; Fitzkee, Nicholas C. (October 2021, Molecules)

   Polyethylene glycol (PEG) surface conjugations are widely employed to render passivating properties to nanoparticles in biological applications. The benefits of surface passivation by PEG are reduced protein adsorption, diminished non-specific interactions, and improvement in pharmacokinetics. However, the limitations of PEG passivation remain an active area of research, and recent examples from the literature demonstrate how PEG passivation can fail. Here, we study the adsorption amount of biomolecules to PEGylated gold nanoparticles (AuNPs), focusing on how different protein properties influence binding. The AuNPs are PEGylated with three different sizes of conjugated PEG chains, and we examine interactions with proteins of different sizes, charges, and surface cysteine content. The experiments are carried out in vitro at physiologically relevant timescales to obtain the adsorption amounts and rates of each biomolecule on AuNP-PEGs of varying compositions. Our findings are relevant in understanding how protein size and the surface cysteine content affect binding, and our work reveals that cysteine residues can dramatically increase adsorption rates on PEGylated AuNPs. Moreover, shorter chain PEG molecules passivate the AuNP surface more effectively against all protein types. 

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5. Cell-penetrating protein-recognizing polymeric nanoparticles through dynamic covalent chemistry and double imprinting

   https://doi.org/10.1038/s41467-024-48131-5  

   Ghosh, Avijit; Sharma, Mansi; Zhao, Yan (May 2024, Nature Communications)

   Abstract Molecular recognition of proteins is key to their biological functions and processes such as protein–protein interactions (PPIs). The large binding interface involved and an often relatively flat binding surface make the development of selective protein-binding materials extremely challenging. A general method is reported in this work to construct protein-binding polymeric nanoparticles from cross-linked surfactant micelles. Preparation involves first dynamic covalent chemistry that encodes signature surface lysines on a protein template. A double molecular imprinting procedure fixes the binding groups on the nanoparticle for these lysine groups, meanwhile creating a binding interface complementary to the protein in size, shape, and distribution of acidic groups on the surface. These water-soluble nanoparticles possess excellent specificities for target proteins and sufficient affinities to inhibit natural PPIs such as those between cytochrome c (Cytc) and cytochrome c oxidase (CcO). With the ability to enter cells through a combination of energy-dependent and -independent pathways, they intervene apoptosis by inhibiting the PPI between Cytc and the apoptotic protease activating factor-1 (APAF1). Generality of the preparation and the excellent molecular recognition of the materials have the potential to make them powerful tools to probe protein functions in vitro and in cellulo. 

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