New findings show that poly(N-isopropyl acrylamide) (PNIPAM) increases the thermal stability of proteins adsorbed on PNIPAM thin films above the lower critical solution temperature (LCST) of 32°C. PNIPAM is a thermo responsive polymer that rapidly and reversibly adsorbs protein above the LCST. Despite its widespread use in a range of biotechnology and medical applications, the impact of PNIPAM on protein folding stability had not been established. Here we describe Fast Relaxation Imaging (FReI) measurements of the thermal stability of adsorbed proteins, in situ at sub micron spatial resolution and millisecond time resolution. Studies quantified the melting temperature, Tm and unfolding/folding kinetics of proteins interacting with grafted PNIPAM thin films, as a function of the polymer grafting density. In comparison with proteins in bulk solution or in contact with oligoethylene glycol films, interactions with PNIPAM increased the Tm of adsorbed protein by up to 8.5°C. This significant increase is not due to crowding, which only enhances Tm by ~2-3°C. The stabilization depends non monotonically on the polymer coverage, with the maximum stability at s/2RF ~1.2, where s is the distance between grafted chains and RF is the Flory radius. Unfolding/folding kinetics further revealed that PNIPAM slows unfolding, and a Phi analysis suggests that the polymer stabilizes both the folded protein ground state and the transition state. FReI measurements thus uncovered a new property of PNIPAM that has important implications for current applications as well as the potential to expand uses of this material.

Poly(N-isopropyl acrylamide) thin films stabilize adsorbed protein above the LCST

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