Stabilization and Kinetics of an Adsorbed Protein Depends on the Poly(N-isopropylacrylamide) Grafting Density

Abstract

The solubility transition at the lower critical solution temperature (LCST, 32 °C) of poly­(N-isopropylacrylamide) (PNIPAM) is widely used as a thermal switch to rapidly and reversibly capture and release proteins and cells. It is generally assumed that proteins adsorbed to PNIPAM above the LCST are unaffected by polymer interactions. Here we show that the folding stability of the enzyme phosphoglycerate kinase (PGK) is increased by interactions with end-grafted PNIPAM films above the LCST. We systematically compare two protein mutants with different stabilities. The stabilization mirrors the degree of protein adsorption under grafting conditions studied previously. Maximum stabilization occurs when proteins adsorb to low density, collapsed polymer “mushrooms”. In the denser polymer “brush” regime, protein stabilization decreases back to a value indistinguishable from the bulk solution, consistent with low protein adsorption on dense, collapsed brushes. The temperature-dependent kinetics measured by Fast Relaxation Imaging reveals that PNIPAM does not affect the overall folding/unfolding mechanism. Based on the different stabilizations of two mutants and the relaxation kinetics, we hypothesize that the polymer acts mainly by increasing the conformational entropy of the folded protein by interacting with the protein surface and less by crowding the unfolded state of PGK.

Protein Stabilization by Alginate Binding and Suppression of Thermal Aggregation

“…In contrast to alginate, weakly attractive interactions with PNIPAM above the transition increased the stability of adsorbed PGK1-FRET, and adsorption is thermally reversible. 51,53 However, in contrast, PNIPAM did not suppress aggregation. 53 The comparison of PGK1-FRET behavior in contact with alginate versus PNIPAM provides context for evaluating how polymer (e.g., alginate) interactions affect protein behavior.…”

“…51,53 However, in contrast, PNIPAM did not suppress aggregation. 53 The comparison of PGK1-FRET behavior in contact with alginate versus PNIPAM provides context for evaluating how polymer (e.g., alginate) interactions affect protein behavior.…”

“…There are several reports that protein–polyelectrolyte coacervates stabilize proteins. − ,, In comparison with similar studies with other water-soluble polymers, the effect of strong, electrostatically driven alginate interactions contrasts with that of neutral, water-soluble poly­( N -isopropylacrylamide) above the lower critical solution temperature of 32 °C. − PNIPAM reversibly adsorbs proteins above 32 °C and rapidly releases them below the solubility transition at 32 °C. In contrast to alginate, weakly attractive interactions with PNIPAM above the transition increased the stability of adsorbed PGK1-FRET, and adsorption is thermally reversible. , However, in contrast, PNIPAM did not suppress aggregation . The comparison of PGK1-FRET behavior in contact with alginate versus PNIPAM provides context for evaluating how polymer (e.g., alginate) interactions affect protein behavior.…”

Colloidal particles interacting with a polymer brush: a self-consistent field theory

“…Hence, our theory explains appearance of maximum in the dependence of absorbed by the brush number of nanoparticles on the brush grafting density, as observed in experiments, which is a manifestation of the aforesaid local minima in the insertion free energy penalty. 40,41 As demonstrated above, the shape of the DF(z) curves can be tuned not only by solvent strength or polymer-particle affinity, but also by the changing surface-to-volume ratio which depends on the size and shape asymmetry of the particles. This allows, for example, separating particles with respect to their shape.…”

“…Hence, our theory explains appearance of maximum in the dependence of absorbed by the brush number of nanoparticles on the brush grafting density, as observed in experiments, which is a manifestation of the aforesaid local minima in the insertion free energy penalty. 40,41…”