Self‐Assembly of Differently Shaped Protein–Polymer Conjugates through Modification of the Bioconjugation Site

Abstract

Self-assembly of protein-polymer block copolymers is an attractive route for preparing biocatalytic materials. To clarify the effect of bioconjugate shape on self-assembly without changing the chemical details of either block, four different conjugation sites are selected on the surface of the model globular protein mCherry at residues 3, 108, 131, and 222 to alter the colloidal shape of the bioconjugate. All four mCherry-b-poly(N-isopropylacrylamide) bioconjugates show qualitatively similar phase diagrams, indicating that self-assembly is robust with respect to changes in conjugation site. However, protein orientation has an effect on the location of the order-disorder transition concentration, and the stability of the disordered micellar phase is shown to be different for each conjugate. Differences in domain spacing also suggest changes in protein orientation within the lamellae.

Predicting Protein–Polymer Block Copolymer Self-Assembly from Protein Properties

“…These proteins were then used to synthesize bioconjugates via site-specific bioconjugation chemistry using thiol-maleimide coupling (Scheme 1). In addition to these six conjugates, structural data of conjugates of reduced charged Sso7d (rcSso7d), 45 human heart myoglobin (HMb), 44 human immunoglobulin G (IgG), 43 mCherry, 51 and EGFP 52 from other studies were compared to identify trends in how the properties of each protein affects the quality of ordering and general self-assembly behavior of protein–polymer bioconjugates. For the six bioconjugates in this study as well as the referenced bioconjugates, the weight fraction of protein and polymer was kept roughly symmetric except in the case of IgG, where the mass of the protein was too large to form a symmetric conjugate.…”

“…These C ODT values are considerably higher than for EGFP and mCherry, which have C ODT values as low as 30 wt %. 40,51,52 Additionally, 3 of the proteins exhibit ODT temperatures ( T ODT values) at concentrations of 40 and 45 wt %: HTPI transitions from the disordered phase to a hexagonal phase, and both DFPaseN and BSA transition from disordered to lamellar phases. These transitions are likely due to desolvation of the polymer resulting in phase separation as the water partitions to the protein domains.…”

Topology effects on protein–polymer block copolymer self-assembly

“…Recently, we have reported the topological effects on the self-assembly of globular protein-coil protein fusion proteins 22,39 and protein polymer block copolymers. 40 In this work, changing the elastin-like polypeptide (ELP) tails from the N to C termini of mCherry has no impact on the ability to form well-ordered nanostructures. 39 On the other hand, a bola fusion protein, which has two ELP tails on both N and C termini of mCherry, showed a lower order-disorder transition concentration than the linear fusion protein, which has one EPL tail on the N terminus of mCherry.…”

“…However, differences in domain spacing were observed, suggesting changes in protein orientation within the lamellar phase. 40 The protein-polymer block copolymer approach is a promising approach to expand the capability to obtain topological designs and functional designs. To obtain well-defined bioconjugates, site-specific modification is one of the most important tools.…”

Tunable Polymeric Scaffolds for Enzyme Immobilization

“…Therefore, affinity approaches (biotin-streptavidin binding, cofactor reconstitution) ( Boerakker et al, 2006 ), or site-selective covalent polymer conjugation are used. As usual, the conjugation approach affects the stability of the micelle and the protein orientation ( Huang and Olsen, 2016 ). In a recent example, enzyme-poly( N -(2-hydroxypropyl) methacrylate)) (PHPMA) conjugates were self-assembled through polymerization-induced coassembly (PICA) approach.…”