Insights on the Structure, Molecular Weight and Activity of an Antibacterial Protein–Polymer Hybrid

Abstract

Protein-polymer conjugates are attractive biomaterials which combine the functions of both proteins and polymers. The bioactivity of these hybrid materials, however, is often reduced upon conjugation. It is important to determine and monitor the protein structure and active site availability in order to optimize the polymer composition, attachment point, and abundance. The challenges in probing these insights are the large size and high complexity in the conjugates. Herein, we overcome the challenges by combining electron paramagnetic resonance (EPR) spectroscopy and atomic force microscopy (AFM) and characterize the structure of antibacterial hybrids formed by polyethylene glycol (PEG) and an antibacterial protein. We discovered that the primary reasons for activity loss were PEG blocking the substrate access pathway and/or altering protein surface charges. Our data indicated that the polymers tended to stay away from the protein surface and form a coiled conformation. The structural insights are meaningful for and applicable to the rational design of future hybrids.

Size-Tunable Metal–Organic Framework-Coated Magnetic Nanoparticles for Enzyme Encapsulation and Large-Substrate Biocatalysis

“…Degradation or conversion of large-size biological substrates is essential for research in sustainable materials, new energy sources, , and material processing, , which find wide applications in agricultural engineering, , biomass conversion, , and antimicrobial treatment. ,, Enzymes are optimal candidates to carry out these reactions due to their high efficiency, selectivity, and biocompatibility. , Immobilizing enzymes on the surfaces of nanoparticles (NPs) improves the cost-efficiency and reactivity of biocatalysis (due to the increased surface-to-volume ratio) − but is practically challenged when the substrates are large in size (μm or larger) due to the difficulty in isolating the enzyme@NP composites from the large (unreacted) substrates or products via gravimetric separation, limiting reusability. Hosting enzymes on magnetic NPs (MNPs) improves the separation; ,− however, enzyme@MNP composites can be challenged by leaching if the enzyme is adsorbed physically − or chemical perturbation if covalently linked. , Also, the complete exposure of enzymes may lead to enzyme damage under harsher reaction conditions necessary/requested for the catalysis. , …”

Inversion of Polymeric Micelles Probed by Spin Labeled Peptide Incorporation and Electron Paramagnetic Resonance

“…While Nuclear Magnetic Resonance (NMR) spectroscopy has been used to probe the “host–guest” interaction in IMAs, the background signals of micelles limit the selective determination of peptide dynamics. Electron Paramagnetic Resonance (EPR) in combination with Site-Directed Spin Labeling (SDSL) has been proved to be powerful in determining otherwise inaccessible structural information in complex biological systems. − SDSL relies on attaching an EPR active spin label to the target biomacromolecules, especially a peptide/protein, at a residue that reacts specifically with the labeling compound. − Typical information from EPR is the site-specific backbone dynamics of the labeled site, which is dependent on and reports the local environment (crowding, polarity) of macromolecular systems. ,− …”

Effect of Polymer Chain Density on Protein–Polymer Conjugate Conformation

“…Despite the huge number of studied protein–polymer conjugates and the large body of literature available on their biochemical properties, very few studies have been conducted on the protein structural dynamics after conjugation to polymers. ,, However, on a more fundamental level, it is vital to gather an understanding of the molecular level of the underlying molecular processes involved in modulating biological function in polymer-conjugated proteins. On the molecular scale, the question arises as to how the polymer structurally organizes itself around the protein and how the polymer chains affect the protein 3D structure.…”