Dongsook Chang

The Nature of Protein Interactions Governing Globular Protein–Polymer Block Copolymer Self-Assembly

The effects of protein surface potential on the self-assembly of protein-polymer block copolymers are investigated in globular proteins with controlled shape through two approaches: comparison of self-assembly of mCherry-poly(N-isopropylacrylamide) (PNIPAM) bioconjugates with structurally homologous enhanced green fluorescent protein (EGFP)-PNIPAM bioconjugates, and mutants of mCherry with altered electrostatic patchiness. Despite large changes in amino acid sequence, the temperature-concentration phase diagrams of EGFP-PNIPAM and mCherry-PNIPAM conjugates have similar phase transition concentrations. Both materials form identical phases at two different coil fractions below the PNIPAM thermal transition temperature and in the bulk. However, at temperatures above the thermoresponsive transition, mCherry conjugates form hexagonal phases at high concentrations while EGFP conjugates form a disordered micellar phase. At lower concentration, mCherry shows a two-phase region while EGFP forms homogeneous disordered micellar structures, reflecting the effect of changes in micellar stability. Conjugates of four mCherry variants with changes to their electrostatic surface patchiness also showed minimal change in phase behavior, suggesting that surface patchiness has only a small effect on the self-assembly process. Measurements of protein/polymer miscibility, second virial coefficients, and zeta potential show that these coarse-grained interactions are similar between mCherry and EGFP, indicating that coarse-grained interactions largely capture the relevant physics for soluble, monomeric globular protein-polymer conjugate self-assembly.

Effect of polymer chemistry on globular protein–polymer block copolymer self-assembly

Bioconjugates of the model red fluorescent protein mCherry and synthetic polymer blocks with different hydrogen bonding functionalities show that the chemistry of the polymer block has a large effect on both ordering transitions and the type of nanostructures formed during bioconjugate self-assembly. The phase behaviours of mCherry-b-poly(hydroxypropyl acrylate) (PHPA) and mCherry-b-poly(oligoethylene glycol acrylate) (POEGA) in concentrated aqueous solution show that changes in polymer chemistry result in increase in the order–disorder transition concentrations (CODTs) by approximately 10–15 wt% compared to a previously studied globular protein–polymer block copolymer, mCherry-b-poly(N-isopropylacrylamide) (PNIPAM). The CODTs are always minimized for symmetric bioconjugates, consistent with the importance of protein–polymer interactions in self-assembly. Both mCherry-b-PHPA and mCherry-b-POEGA also form phases that have not previously been observed in other globular protein–polymer conjugates: mCherry-b-PHPA forms a cubic phase that can be indexed to Iad and mCherry-b-POEGA displays coexistence of lamellae and a cubic Iad structure over a narrow range of concentration and temperature. Several common behaviours are also revealed by comparison of different polymer blocks. With increasing concentration and temperature, ordered phases always appear in the order lamellar, cubic/PL, and hexagonal, although not all phases are observed in all materials. High concentration solutions (near 80 wt%) also undergo a re-entrant order–disorder transition to form nematic liquid crystalline phases, regardless of the polymer block chemistry.

Self-assembly of protein-zwitterionic polymer bioconjugates into nanostructured materials

The microphase separation of a bioconjugate made of a globular protein and a zwitterionic polymer is studied in order to elucidate the role of charge in the polymer block on the self-assembly of protein–polymer bioconjugates. Zwitterionic polymer surfaces are resistant to nonspecific protein adsoprtion due to strong hydration; however, bioconjugates constructed from a red fluorescent protein, mCherry, and a zwitterionic polymer, PDMAPS, show a relatively narrow range of conditions for self-assembly in concentrated systems. The bioconjugates demonstrate weaker segregation strengths compared to previously studied mCherry–polymer conjugates with non-ionic polymers, as demonstrated by higher order-disorder transition concentrations (CODT) and a narrower range of ordered concentrations in the phase diagram. The results suggest that electrostatic segregation of mCherry is one of the main parameters governing the self-assembly of protein–nonionic polymer bioconjugates, and this driving force is perturbed by the zwitterionic polymer. Disruption of ordering upon addition of NaCl confirms that electrostatics play a critical role in the bioconjugate self-assembly. Order–disorder–order transitions are observed with increasing concentration of a kosmotropic salt, ammonium sulphate, due to the initial salt-in followed by salt-out effect, suggesting that stabilization of protein domains by enhancing attractive interactions between proteins can significantly improve long range ordering.

Kinetic Effects on Self‐Assembly and Function of Protein–Polymer Bioconjugates in Thin Films Prepared by Flow Coating

The self-assembly of nanostructured globular protein arrays in thin films is demonstrated using protein-polymer block copolymers based on a model protein mCherry and the polymer poly(oligoethylene glycol acrylate) (POEGA). Conjugates are flow coated into thin films on a poly(ethylene oxide) grafted Si surface, forming self-assembled cylindrical nanostructures with POEGA domains selectively segregating to the air-film interface. Long-range order and preferential arrangement of parallel cylinders templated by selective surfaces are demonstrated by controlling relative humidity. Long-range order increases with coating speed when the film thicknesses are kept constant, due to reduced nucleation per unit area of drying film. Fluorescence emission spectra of mCherry in films prepared at <25% relative humidity shows a small shift suggesting that proteins are more perturbed at low humidity than high humidity or the solution state.