Mutation of a superfolder green fluorescent protein (GFP) was used to design GFP variants with formal net charges of 0, -8, and -21, providing a set of three proteins in which the total charge is varied to tune protein-protein interactions while controlling for the protein size and tertiary structure. After conjugating poly(N-isopropylacrylamide) (PNIPAM) to each of these three GFP variants, the concentrated solution phase behavior of these three block copolymers is studied using a combination of small-angle X-ray scattering (SAXS), depolarized light scattering (DPLS), and turbidimetry to characterize their morphologies. The electrostatic repulsion between supercharged GFP suppresses ordering, increasing the order-disorder transition concentration (CODT) and decreasing the quality of the ordered nanostructures as measured by the full width at half-maximum of the primary scattering peak. By contrast, the charge distribution of the neutrally charged GFP results in its largest dipole moment, calculated about the protein's center of mass, among the three GFP variants and a self-complementary Janus-like electrostatic surface potential that enhances nanostructure formation. The different electrostatic properties result in different protein-protein interactions that affect the high temperature morphologies, including the formation of macrophase separated or homogeneous micellar phases and the smaller hexagonal ordering window of the supercharged GFP. Small improvements in the quality of the ordered nanostructures of GFP(-21)-PNIPAM can be achieved through protein-divalent cation interactions. Therefore, varying protein charge and electrostatics is demonstrated as a method of tuning the magnitude and directionality of protein-protein interactions to control self-assembly.
Protein–polymer
bioconjugate self-assembly has attracted
a great deal of attention as a method to fabricate protein nanomaterials
in solution and the solid state. To identify protein properties that
affect phase behavior in protein–polymer block copolymers,
a library of 15 unique protein-b-poly(N-isopropylacrylamide) (PNIPAM) copolymers comprising 11 different
proteins was compiled and analyzed. Many attributes of phase behavior
are found to be similar among all studied bioconjugates regardless
of protein properties, such as formation of micellar phases at high
temperature and low concentration, lamellar ordering with increasing
temperature, and disordering at high concentration, but several key
protein-dependent trends are also observed. In particular, hexagonal
phases are only observed for proteins within the molar mass range
20–36 kDa, where ordering quality is also significantly enhanced.
While ordering is generally found to improve with increasing molecular
weight outside of this range, most large bioconjugates exhibited weaker
than predicted assembly, which is attributed to chain entanglement
with increasing polymer molecular weight. Additionally, order–disorder
transition boundaries are found to be largely uncorrelated to protein
size and quality of ordering. However, the primary finding is that
bioconjugate ordering can be accurately predicted using only protein
molecular weight and percentage of residues contained within β
sheets. This model provides a basis for designing protein–PNIPAM
bioconjugates that exhibit well-defined self-assembly and a modeling
framework that can generalize to other bioconjugate chemistries.
Contrast variation small angle neutron scattering experiments reveal the nature of interactions between proteins and polymers in semidilute aqueous solution.
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