Negative activation enthalpies in the kinetics of protein folding.

Abstract

Although the rates of chemical reactions become faster with increasing temperature, the converse may be observed with protein-folding reactions. The rate constant for folding initially increases with temperature, goes through a maximum, and then decreases. The activation enthalpy is thus highly temperature dependent because of a large change in specific heat (delta Cp). Such a delta Cp term is usually presumed to be a consequence of a large decrease in exposure of hydrophobic surfaces to water as the reaction proceeds from the denatured state to the transition state for folding: the hydrophobic side chains are surrounded by "icebergs" of water that melt with increasing temperature, thus making a large contribution to the Cp of the denatured state and a smaller one to the more compact transition state. The rate could also be affected by temperature-induced changes in the conformational population of the ground state: the heat required for the progressive melting of residual structure in the denatured state will contribute to delta Cp. By examining two proteins with different refolding mechanisms, we are able to find both of these two processes; barley chymotrypsin inhibitor 2, which refolds from a highly unfolded state, fits well to a hydrophobic interaction model with a constant delta Cp of activation, whereas barnase, which refolds from a more structured denatured state, deviates from this ideal behavior.

Hydrophobic association of α-helices, steric dewetting, and enthalpic barriers to protein folding

“…Here, Fig. 2 A and F shows that the Ϸϩ60 kJ⅐mol Ϫ1 enthalpic barrier for helix dimerization is comparable and can be even higher than that for protein folding at 300 K. For L 20 , the ⌬C P around the desolvation barrier is also similar to the ⌬C P ‡-D Ϸ Ϫ1.3 kJ⅐mol Ϫ1 ⅐K Ϫ1 reported for CI2 (11). All in all, the desolvation of the L 20 dimer displays thermodynamic signatures that are qualitatively and quantitatively similar to the folding transition states of small two-state proteins.…”

“…However, the relationship between the behaviors of small solutes (18) and larger-length-scale biomolecular assembly is not always straightforward. Notably, whereas the heat capacity of the protein folding transition state is typically lower than that of the unfolded state (11,12), atomic simulations indicate that the heat capacity at the desolvation barrier of a pair of small nonpolar solutes is higher than that when the pair is far apart (19,20). These conflicting trends imply that the folding transition-state heat capacity cannot be understood as a simple summation of pairwise contributions from contacts between small-solute-like chemical groups (3).…”

“…This picture is readily reconcilable with the empirical folding barriers along onedimensional free-energy profiles in traditional macroscopic descriptions (8) if the barriers arose solely from a reduction in conformational entropy during folding, because restriction on conformational freedom appears, by definition, as decrease in multidimensional surface area, rather than as barriers, on the microscopic free-energy landscape (6,9). However, experiments indicate a significant enthalpic component in empirical folding barriers (10)(11)(12)(13)(14). Our interest in high enthalpic folding barriers is motivated by two physical questions: (i) How does the existence of these barriers impact the folding funnel concept?…”

Dynamics, Energetics, and Structure in Protein Folding

“…differences between wild-type and mutant (37)). Furthermore, an Arrhenius analysis of the temperature dependence of the folding rate can be seriously misleading because: i) the barrier and the diffusion coefficient have temperature dependent and temperature independent contributions; ii) the hydrophobic effect results in large enthalpy-entropy compensations (38); iii) glassy dynamics may arise at low temperatures(3).…”

Barrierless evolution of structure during the submillisecond refolding reaction of a small protein

“…In b and c the data are normalized to a value of 1 for the unfolded protein signal in 8 M urea. occurring in the absence of a dominant barrier (5, 7). Although non-Arrhenius kinetics could possibly also arise from the hydrophobic nature of the collapse reaction (36), this is unlikely because the activation enthalpy for the sub-ms folding reaction of cytochrome c was observed to be positive (35) when measured over a similar temperature range.…”