Date Published: July 29, 2011
Publisher: Public Library of Science
Author(s): M. Luisa Romero-Romero, Alvaro Inglés-Prieto, Beatriz Ibarra-Molero, Jose M. Sanchez-Ruiz, Vladimir N. Uversky. http://doi.org/10.1371/journal.pone.0023050
Abstract: Despite several careful experimental analyses, it is not yet clear whether protein cold-denaturation is just a “mirror image” of heat denaturation or whether it shows unique structural and energetic features. Here we report that, for a well-characterized small protein, heat denaturation and cold denaturation show dramatically different experimental energetic patterns. Specifically, while heat denaturation is endothermic, the cold transition (studied in the folding direction) occurs with negligible heat effect, in a manner seemingly akin to a gradual, second-order-like transition. We show that this highly anomalous energetics is actually an apparent effect associated to a large folding/unfolding free energy barrier and that it ultimately reflects kinetic stability, a naturally-selected trait in many protein systems. Kinetics thus emerges as an important factor linked to differential features of cold denaturation. We speculate that kinetic stabilization against cold denaturation may play a role in cold adaptation of psychrophilic organisms. Furthermore, we suggest that folding-unfolding kinetics should be taken into account when analyzing in vitro cold-denaturation experiments, in particular those carried out in the absence of destabilizing conditions.
Partial Text: The existence of cold denaturation is a straightforward prediction of a widely-accepted phenomenological view of protein folding thermodynamics . The simplest rendering of the prediction is as follows. The enthalpy change for protein unfolding (ΔH) is temperature-dependent (as given by the positive unfolding heat capacity change and the Kirchoff equation) and equals zero at the so-called enthalpy-inversion temperature (TH). At temperatures above TH, ΔH is positive, the unfolding process is endothermic and, hence, it is driven by a temperature increase (heat denaturation). Below TH, ΔH is negative, the unfolding process is exothermic and it is driven by a temperature decrease (cold denaturation).
Figures 1a and 1b show the results of far-UV and near-UV circular dichroism experiments in which E. coli thioredoxin solutions were heated at a constant temperature scanning-rate of 1.5 degrees/min. [Thioredoxin solutions were kept at 2°C for 3 hours prior to the start of the temperature scanning experiment; we checked, nevertheless, that a several-days incubation at 2°C led essentially to the same results]. Protein ellipticity is an average over protein states and, consequently, folding-unfolding transitions are revealed by sigmoidal-like changes in profiles of ellipticity vs. temperature. The profiles for solutions including 2M guanidine reveal two well-defined sigmoidal-like changes obviously corresponding to the cold and heat denaturation processes. A differential scanning calorimetry (DSC) profile for the same thioredoxin solution and using the same temperature protocol is shown in Figure 1c. DSC measures the heat capacity of a protein solution, which is the temperature-derivative of the average enthalpy. Hence, a folding-unfolding transition is usually revealed by a heat capacity “peak” (the derivative of a sigmoidal-like profile), the area under which equals the total enthalpy change for the transition. Then, DSC thermograms for heat-and-cold denaturation typically show two peaks, often loosely resembling the double hump of a Bactrian camel. The experimental calorimetric profile in Figure 1c is, therefore, most unusual, as a peak is observed only for heat denaturation. Cold denaturation in this profile is actually signaled by a gradual increase in heat capacity upon temperature decrease (roughly from the heat capacity level expected for the native protein to the level expected for the unfolded protein). That is, contrary to heat denaturation, cold denaturation appears to occur with negligible heat effect (close to zero transition enthalpy change).