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

Abstract

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.

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

“…At 10 °C, the C ODT occurs between 33 and 35 wt% for mChS3CP, mChT108CP, and mChS131CP, and between 37 and 40 wt% for mChS222CP. The transition in mChS131CP at a similar coil fraction has previously been observed at 33 wt% . As concentration is increased to 45 wt%, each bioconjugate transitions to a more ordered birefringent lamellar phase, indicated by a narrowing of SAXS peaks and in some cases a resolvable 3 q * peak.…”

“…Changing the conjugation site also affects micellar stability at concentrations of 40 wt% and lower. At 35 °C, the bioconjugates begin to form micellar structures as previously observed . This transition is accompanied by macrophase separation, indicated by a sudden drop in optical transmission (Figure S8, Supporting Information).…”

“…For example, the phase behavior of globular protein‐coil block copolymers is significantly affected by hydrogen‐bonding motifs in the polymer . Within the protein block, modifying the electrostatic patchiness of the protein by introducing point charge mutations results in very little change in phase behavior, and structurally homologous but chemically dissimilar bioconjugates, using two β‐barrel proteins mCherry and Enhanced Green Fluorescent Protein (EGFP), have very similar phase behavior . In contrast to structurally similar fluorescent proteins, structurally dissimilar myoglobin‐b‐PNIPAM conjugates order to a considerably lesser extent .…”

Expansion of bioorthogonal chemistries towards site-specific polymer–protein conjugation

“…[1][2][3][4][5][6][7][8][9][10] Such coupling partners provide special functions, such as fluorescence and catalytic activity to polymers. The coupling partners of polymers include fluorescent dyes, carbon-based nanomaterials, metallic nanoparticles, proteins, small molecule drugs, and even different types of polymers.…”

Temperature‐responsive “tadpole‐shaped” protein–polymer hybrids and their self‐assembly behavior

“…pNIPAAm was also used to fabricate protein–pNIPAAm hybrids, which were self‐assembled into the highly disordered nanostructures and showed the temperature‐responsiveness in aqueous solutions. For instance, mCherry‐pNIPAAm hybrid was self‐assembled into the highly disordered lamellae or hexagonally perforated lamellae . Vault‐pNIPAAm hybrid was self‐assembled into the nanocapsules …”