Biophysical characterization of protein-nanoparticle interactions

Nanoparticles (NPs) have become a key tool in medicine and biotechnology; as drug delivery systems, biosensors, and diagnostic devices. However, the mechanism of biocorona formation on nanoparticle surfaces and their impact on drug delivery remains speculative. Nevertheless, functionalized nanoparticles have demonstrated major success in medical applications; having been shown to effectively treat disease. The mechanistic details of protein behavior on nanoparticle surfaces remain poorly understood to date; due to difficulty in determining the orientation and structure of protein on NPs. Furthermore, surface crowding, orientation, and degree of disorder have been shown to perturb the efficacy of protein on NPs; dramatically reducing their benefits. NMR and other biophysical tools can be used to characterize the nanoparticle-protein surface interactions; leading to a better understanding of the biocorona structure. This dissertation investigates the structure, orientation, and function of proteins adsorbed on gold nanoparticles (P-AuNPs). Using hydrogen-deuterium exchange and methylation studies on P-AuNPs, we have elucidated the structure and orientation of proteins on AuNP surfaces. We have also designed fusion proteins that can effectively mitigate structural-, orientation-, and activity-perturbations of P-AuNPs. The benefits of our fusion protein approach have been verified via enzymatic assay; which monitored the enzymatic activity of these P-AuNPs. Biofilms are defined as surface-anchored, multi-cellular, three-dimensional, bacterial communities. Biofilms have a serious impact on public health; because of their role in infectious diseases and medical device-related infections. S. epidermidis is the most common biofilmorming bacteria. Therefore, understanding the mechanisms of biofilm formation could lead to novel therapeutics which prevent biofilm formation. One of the most recognized proteins in the biofilm formation mechanism is the S. epidermidis autolysin domain. Therefore, we have studied the structure and behavior of S. epidermidis autolysin repeat domain R2 (R2ab) via solution NMR and other biophysical techniques. This study has provided a deeper understanding of how R2ab interacts with foreign surfaces and blood proteins; which could lead to future methods of biofilm prevention. Over the course of this dissertation, the characterization of protein-surface interactions was achieved via solution NMR and other biophysical tools; providing insightful information to the fields of medicine and therapeutics

A Structural Perspective on Calprotectin as a Ligand of Receptors Mediating Inflammation and Potential Drug Target

Calprotectin, a heterodimer of S100A8 and S100A9 EF-hand calcium-binding proteins, is an integral part of the innate immune response. Calprotectin (CP) serves as a ligand for several pattern recognition cell surface receptors including the receptor for advanced glycation end products (RAGE), toll-like receptor 4 (TLR4), and cluster of differentiation 33 (CD33). The receptors initiate kinase signaling cascades that activate inflammation through the NF-kB pathway. Receptor activation by CP leads to upregulation of both receptor and ligand, a positive feedback loop associated with specific chronic inflammatory syndromes. Hence, CP and its two constituent homodimers have been viewed as potential targets to suppress certain chronic inflammation pathologies. A variety of inhibitors of CP and other S100 proteins have been investigated for more than 30 years, but no candidates have advanced significantly into clinical trials. Here, current knowledge of the interactions of CP with its receptors is reviewed along with recent progress towards the development of CP-directed chemotherapeutics

Understanding the Adsorption of Peptides and Proteins onto PEGylated Gold Nanoparticles

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