The Transient Collapsed Ensemble: TO of the Folding Pathway

Abstract

The starting point of any protein refolding transition, whether via well defined pathway or by multiple pathways, is initiated in an ensemble of fully disordered polypeptides under folding conditions. The first intramolecular interactions in the disordered polypeptides have major effect of reduction of the search for the native configuration. In order to detect the earliest formed intramolecular contacts in the refolding ensembles by detection of the intramolecular distances, we must first determine the dimensions of the transient collapsed ensemble. Using E. coli Adenylate kinase (AK) as a model protein and microfluidic mixing device combined with time resolved FRET measurements (“the double kinetics” method) we tested the hypothesis that at the initiation of refolding, the dependence of the mean of the segmental end-to-end distance on the segment length, Δn, is weak (Δn>∼30). We measured the transient distributions of segmental end to end distances of seven segments of the AK molecule, with Δn from 45 to 196 residues at 50 μs after initiation of mixing. The means of these distributions range from 45±2 Å, for the short segment, to 70±2 Å for the long segment. The Flory exponent for the Δn dependence of the mean segmental end to end distance is 0.32±0.01. Thus, this transient ensemble is indeed a collection of disordered molecules. We now have a benchmark for the segmental end to end distance distribution of unfolded AK molecules under folding conditions (poor solvent). Any intramolecular mean distance that would be significantly shorter can be considered as an indication of an early formed intramolecular contact. This experiment enables systematic detection of the time sequence of formation of non local contacts in the refolding protein molecules. A step which is essential for deciphering the mechanism of folding of globular proteins.