Ultrafast Protein Folding through Polar Interactions in Membrane-Mimetic Environments

Abstract

Proteins fold on timescales from hours down to microseconds. While first principles based on protein size, topology and differences in protein sequence have been identified as determinants of the folding speed, an important but often neglected factor in sculpting the folding landscape is the surrounding environment. This is particularly relevant for membrane-interacting proteins that require the highly complex, anisotropic environment of a lipid membrane or a membrane mimetic to fold. The chemically heterogeneous hydrophilic/hydrophobic water-headgroup-hydrocarbon-core interface provided by such a lipidic environment presents a variety of different polar and non-polar molecular interactions. Thus, changes in the chemical composition of this surrounding may serve to modulate the folding landscape and therefore become a determining factor tuning folding barriers and stability. Here we have addressed this question and explored how properties of a membrane-mimetic environment modulate the stability and folding kinetics of a membrane-interacting protein, also in respect to folding in the absence of a hydrophobic phase in aqueous solution. By employing a single-molecule FRET approach to quantify folding and unfolding rates in detergent micelles from equilibrium measurements, we were able to analyze how contributions from polar and nonpolar interactions modulate the folding landscape of Mistic. Our results demonstrate that, although both hydrophobic and polar interactions contribute to the thermodynamic stability of Mistic, they exert opposing effects on the folding free-energy barrier: hydrophobic burial in the aliphatic core stabilizes only the folded state but not the transition state, thus slowing down unfolding without affecting folding; by contrast, polar headgroup interactions further stabilize the folded state and, additionally, lower the free-energy barrier to speed up the folding reaction to achieve ultrafast folding on timescales down to 30 µs.