Non-random coil behavior as a consequence of extensive PPII structure in the denatured state

TL;DR

This study reveals that extensive non-native polyproline II structure causes denatured proteins to be more compact than expected, challenging the assumption that residual structure does not influence global size scaling.

Contribution

It demonstrates that non-native PPII structure significantly impacts the size scaling of denatured proteins, using single-molecule fluorescence correlation spectroscopy and polymer models.

Findings

Denatured proteins are more compact than random coil predictions.

Extensive PPII structure localizes to specific chain segments.

Polymer models can accurately describe PPII-induced compaction.

Abstract

Unfolded proteins may contain native or non-native residual structure, which has important implications for the thermodynamics and kinetics of folding as well as for misfolding and aggregation diseases. However, it has been universally accepted that residual structure should not affect the global size scaling of the denatured chain, which obeys the statistics of random coil polymers. Here we use a single-molecule optical technique, fluorescence correlation spectroscopy, to probe the denatured state of set of repeat proteins containing an increasing number of identical domains, from two to twenty. The availability of this set allows us to obtain the scaling law for the unfolded state of these proteins, which turns out to be unusually compact, strongly deviating from random-coil statistics. The origin of this unexpected behavior is traced to the presence of extensive non-native polyproline II helical structure, which we localize to specific segments of the polypeptide chain. We show that the experimentally observed effects of PPII on the size scaling of the denatured state can be well-described by simple polymer models. Our findings suggest an hitherto unforeseen potential of non-native structure to induce significant compaction of denatured proteins, affecting significantly folding pathways and kinetics.