Christopher N. Lam

Phase transitions in concentrated solution self-assembly of globular protein–polymer block copolymers

The phase behaviour of mCherry-b-PNIPAM (mChP) block copolymers with four different PNIPAM coil fractions is investigated in concentrated aqueous solution as a function of both concentration and temperature, demonstrating both order–order transitions (OOTs) and order–disorder transitions (ODTs) in globular protein–polymer block copolymers. Independent of coil volume fraction from 0.25 to 0.70, the temperature–concentration phase diagrams share several common features. At low concentrations, mCherry-b-PNIPAM forms a homogeneous disordered phase, and macrophase separation into an ordered conjugate-rich phase and a solvent-rich phase is observed at temperatures above the PNIPAM thermoresponsive transition temperature. mChP solutions are also observed to undergo a low-temperature ODT driven by increasing concentration. The order–disorder transition concentration (ODTC) behaviour of mChP is minimized for symmetric conjugates, suggesting that repulsive solvent-mediated protein–polymer interactions provide a driving force for self-assembly. Both coil fraction and solvent selectivity have large effects on the morphologies formed—disordered micelles, hexagonally packed cylinders, lamellae, and perforated lamellae are identified with the combination of small-angle X-ray scattering (SAXS), depolarized light scattering (DPLS), turbidimetry, and differential scanning calorimetry (DSC). An OOT is observed upon increasing temperature for three of the studied coil fractions at concentrations of 40–50 wt% due to changing solvent selectivity. SANS contrast-matching experiments show that water is weakly selective for PNIPAM at low temperatures and strongly selective for mCherry at high temperatures.

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.

Fingerprinting Molecular Relaxation in Deformed Polymers

The flow and deformation of macromolecules is ubiquitous in nature and industry, and an understanding of this phenomenon at both macroscopic and microscopic length scales is of fundamental and practical importance. Here we present the formulation of a general mathematical framework, which could be used to extract, from scattering experiments, the molecular relaxation of deformed polymers. By combining and modestly extending several key conceptual ingredients in the literature, we show how the anisotropic single-chain structure factor can be decomposed by spherical harmonics and experimentally reconstructed from its cross sections on the scattering planes. The resulting wavenumber-dependent expansion coefficients constitute a characteristic fingerprint of the macromolecular deformation, permitting detailed examinations of polymer dynamics at the microscopic level. We apply this approach to survey a long-standing problem in polymer physics regarding the molecular relaxation in entangled polymers after a large step deformation. The classical tube theory of Doi and Edwards predicts a fast chain retraction process immediately after the deformation, followed by a slow orientation relaxation through the reptation mechanism. This chain retraction hypothesis, which is the keystone of the tube theory for macromolecular flow and deformation, was critically examined by analyzing the fine features of the two-dimensional anisotropic spectra from small-angle neutron scattering by entangled polystyrenes. It is shown that the unique scattering patterns associated with the chain retraction mechanism were not experimentally observed. This result calls for a fundamental revision of the current theoretical picture for nonlinear rheological behavior of entangled polymeric liquids.