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.