Conserved Protein–Polymer Interactions across Structurally Diverse Polymers Underlie Alterations to Protein Thermal Unfolding

Abstract

Protein−polymer conjugates are widely used in many clinical and industrial applications, but lack of experimental data relating protein−polymer interactions to improved protein stability prevents their rational design. Advances in synthetic chemistry have expanded the palette of polymer designs, including development of nonlinear architectures, novel monomer chemical scaffolds, and control of hydrophobicity, but more experimental data are needed to transform advances in chemistry into next generation conjugates. Using an integrative biophysical approach, we investigated the molecular basis for polymer-based thermal stabilization of a human galectin protein, Gal3C, conjugated with polymers of linear and nonlinear architectures, different degrees of polymerization, and varying hydrophobicities. Independently varying the degree of polymerization and polymer architecture enabled delineation of specific polymer properties contributing to improved protein stability. Insights from NMR spectroscopy of the polymer-conjugated Gal3C backbone revealed patterns of protein−polymer interactions shared between linear and nonlinear polymer architectures for thermally stabilized conjugates. Despite large differences in polymer chemical scaffolds, protein−polymer interactions resulting in thermal stabilization appear conserved. We observed a clear relation between polymer length and protein− polymer thermal stability shared among chemically different polymers. Our data indicate a wide range of polymers may be useful for engineering conjugate properties and provide conjugate design criteria.

Protein–polymer bioconjugation, immobilization, and encapsulation: a comparative review towards applicability, functionality, activity, and stability

“…Following bioconjugation, purification is essential due to free or unconjugated polymers causing unintended effects that may be unaccounted by researchers. 44 Techniques for purification include chromatography, 38,45,46 dialysis, 47,48 and filtration 49 to remove unreacted polymer. Methods to confirm soluble protein-polymer bioconjugation synthesis primarily rely on observing an increased apparent molecular weight to unmodified proteins.…”

“…Biomaterials Science to map galectin-polymer interactions of linear and nonlinear PEG conjugated to galectin. 45 Stability. Protein stability is intrinsically linked to tertiary and secondary structure and therefore function.…”

“…Pritzlaff and coworkers address this issue through the conjugation of PEG alternative polymers to the human galectin protein Gal (Gal3C). 45 This work found that linear and nonlinear (comb-shaped) polymer architectures can significantly improve protein stability while investigating the impact of polymer length and architecture on enzymatic stability. This work utilized poly-(oligoethylene glycol methacrylate) ( pOEGMA) and poly-(dimethylacrylamide) ( pDMA) due to their ability to decrease anti-PEG antibody formation, 109 improve plasma circulation times, 110 and increased ability to evade detection by the immune system.…”

Tuning Polymer Composition Leads to Activity–Stability Tradeoff in Enzyme-Polymer Conjugates

“…Of particular importance are enzyme–polymer, polymer–polymer, and polymer–solvent interactions, which may be driven by a combination of hydrophobic, hydrogen bonding, electrostatic, and steric effects. For example, previous studies have shown that enzyme–polymer interactions may influence enzyme activity via effectively mediating substrate binding. , Additionally, protein solubility , and stability can also be dramatically impacted by protein–polymer interactions. ,− Interestingly, the addition of soluble random heteropolymers that are chemically diverse has also been shown to dramatically increase enzyme stability through a chaperone-like mechanism . This approach was specifically demonstrated for a broad range of structurally diverse enzymes, including horseradish peroxidase, glucose oxidase, and organophosphorus hydrolase.…”

“…As a result, the understanding of how these properties may be intentionally manipulated to modulate protein function remains poorly understood. Importantly, polymer–polymer interactions may also impact protein behavior ,, as neighboring chains begin to interact with one another, or fold back on themselves, creating a distinctive polymer-defined niche. For example, the formation of collapsed zwitterionic polymer mats through ion pairing can significantly change the polymer hydration layer .…”