Mapping the Free Energy Change of Unfolding Versus Temperature for Two Homologous Cytochromes C Adapted to Differing Environmental Temperatures

Abstract

However, extracting those rate constants from complex HXMS behavior requires fitting the experimental HXMS data to modeled HXMS spectra using a numerical simulations approach. Here we build on our success in using numerical simulations to fit intact protein HXMS data and adapt our approach to also fit peptide HXMS data for the first time. CcdB is a bacterial toxin that interferes with DNA gyrase's activity, leading to DNA breakage in plasmids and chromosomes, and has been proposed to fold through parallel pathways. Our results for both the intact protein and peptide data suggest that HX in CcdB occurs through sub-global and global unfolding events. This complements the previous folding mechanism derived from traditional folding kinetics measurements. In addition, these results open the door to more quantitative interpretation of complex HXMS peptide data through the application of our numerical simulations algorithm. 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, Dn, is weak (Dn>30). We measured the transient distributions of segmental end to end distances of seven segments of the AK molecule, with Dn from 45 to 196 residues at 50 ms after initiation of mixing. The means of these distributions range from 4552 Å , for the short segment, to 7052 Å for the long segment. The Flory exponent for the Dn dependence of the mean segmental end to end distance is 0.3250.01. 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.