(17c) Structure-Function-Dynamics Relationships in Next Generation Protein-Polymer Conjugates

Protein-polymer conjugates utilize synthetic polymers to modulate the biological activity of proteins. The physiochemical properties of the polymers are designed so that they impart a desired effect on the protein including stealth from the immune system, stability in harsh environments, or sensing in a response to an external stimuli. The synthesis of protein-polymer conjugates, specifically when using amino reactive initiators and a grafting-from approach, is not well controlled and therefore, it is not well understood how the polymers impart their effect on the protein depending on polymer charge, molecular weight, modification density, and attachment site. Clarifying these structure-function-dynamic type relationships would provide the foundation to create designer protein-polymer conjugates, using polymer-based protein engineering, for a myriad of applications ranging from therapeutics to agrochemicals to food biotechnology.

There are many hypotheses for how polymers stabilize proteins including, but not limited to, polymers creating a stabilizing hydration layer around the protein (similar to osmolytes), polymers providing protection from autolysis, or polymers increasing the structural stability of the protein. The covalently attached polymers are free to move around, can adopt different conformations, and can participate in various noncovalent interactions at the protein-polymer interface. These noncovalent interactions include van der Waals interactions, electrostatic interactions, hydrogen bonds, or a combination of them all, and can lead to bioconjugate stabilization. Here, we sought to develop a mechanistic understanding of bioconjugate structure-function-dynamic relationships through a series of experimental and theoretical studies to determine the driving force for conjugate activity and stability. It is difficult to determine specific interactions in protein-polymer conjugates experimentally at the macroscale level, so we also used molecular dynamic (MD) simulations. MD simulations can be used to study the dynamic motions of biomolecules over time while visualizing specific interactions. MD simulations have conventionally been performed on native proteins, but recent efforts have extended into studying protein-polymer conjugates.

In this presentation, we will first describe how we were able to modulate the activities and pH dependencies of fully modified grafted-from bioconjugates by engineering the polymer charge and chain length. We will also propose a mechanism for how covalently attached polymers stabilize proteins under acidic conditions, depending on the physiochemical properties of the polymer, that challenges the commonly accepted hypotheses. We will further validate the experimental studies with theoretical molecular dynamics simulations. This work will provide a guide for the synthesis and application of next generation protein-polymer conjugates.