How to Characterize the Protein Structure and Polymer Conformation in Protein‐Polymer Conjugates – a Perspective

Abstract

Proteins, having miscellaneous properties perfected by nature over millions of years, are attractive for many biomedical and industrial applications. The exploitation of proteins offers great opportunities to increase, among others, the selectivity, biocompatibility, and sustainability of artificial materials. However, the stability of proteins is limited and exposure to conditions like heat or organic solvents often leads to denaturation or aggregation. The modification with synthetic polymers is a powerful possibility to increase the stability of proteins. Throughout the last few years, various methods have been established to characterize obtained conjugates regarding the conjugation efficiency, protein stability, their self-assembly behavior, and beyond. Nevertheless, it is still very challenging to determine if the protein structure is unaltered in the biohybrid materials and how the polymer chains arrange around the protein surface. Here, an overview of different analysis techniques is presented, their applicability and feasibility are discussed, and current literature examples are provided. The focus of this Perspective lies on nuclear magnetic resonance spectroscopy, size exclusion chromatography coupled with multi-angle laser light scattering, analytical ultracentrifugation, and small-angle scattering techniques as these methods shed light into the structure of proteins in conjugates and the polymer conformation around proteins.

FhuA: From Iron-Transporting Transmembrane Protein to Versatile Scaffolds through Protein Engineering

“…Protein–polymer conjugates have a broad range of applications (e.g., medicine (PEGylated protein , ) or introducing antifouling when polymer brushes are grown from immobilized proteins . There are, in principle, two strategies to combine a polymer and a protein: a grafting-to or a grafting-from technique. , In the grafting-to approach, already synthesized polymers are equipped with functional groups for protein conjugation. In the grafting-from approach, the polymer is formed from the protein.…”

“…20 There are, in principle, two strategies to combine a polymer and a protein: a grafting-to or a grafting-from technique. 21,22 In the grafting-to approach, already synthesized polymers are equipped with functional groups for protein conjugation. In the grafting-from approach, the polymer is formed from the protein.…”

Characterization of single synthetic polymers via magnetic tweezers

“…Notably, the results obtained from MT is given by individual single molecules, preventing the influence of molecular weight dispersions and inter-chain interactions, serving as a valuable complement to conventional bulk characterization techniques for polymer conformation, such as gel chromatography and light scattering. 46 In the case of various multi-structural polymers, such as hyperbranched, star-shaped, and brush/bottlebrush polymers, stiffness and internal forces are crucially linked to their physical properties, 47 finding practical applications in areas like lubrication, plasticizing, and polymer gel electrolytes. 48 The research conducted by Saleh group extended the application of single-molecule MT to investigate these attributes in multi-structured functional macromolecules, such as brush polymers.…”

“…Taken together, the parameters including L 0 , P , l , b , and v , as derived from single polymer chain force‐extension correlation, effectively characterized the ideal coil and swollen coil behavior of polymer chains, which is demonstrated as the regime diagram (Figure 5B). Notably, the results obtained from MT is given by individual single molecules, preventing the influence of molecular weight dispersions and inter‐chain interactions, serving as a valuable complement to conventional bulk characterization techniques for polymer conformation, such as gel chromatography and light scattering 46 …”

A Comparative Study of “Grafting to” and “Grafting from” Conjugation Methods for the Preparation of Antibody-Temperature-Responsive Polymer Conjugates

“…In addition to the GT method, there is another “grafting-from” (GF) method, which is also used for introducing polymers into proteins. The GF method is a technique that has been attracting attention in recent years because it introduces polymers directly by polymerizing monomers in solution using functionalized proteins as the starting point for polymerization, thus overcoming the previously mentioned issues of the GT method, including steric hindrance and excess addition of free polymers. , Studies comparing the GT and GF methods of introducing polymers into RNA, cellulose, and graphene have generally concluded that the GF method is the most efficient way to introduce polymers. − GF basically uses living-radical polymerization in water to introduce the polymer. In some reported studies, a chain transfer agent (CTA) was introduced into bovine serum albumin (BSA) and lysozyme by reversible addition–fragmentation chain transfer polymerization (RAFT). − This demonstrated that the protein functionality of the conjugates was retained after the NIPAAm polymerization from proteins and the introduction of PNIPAAm by living radical polymerization.…”