The molecular basis of cooperativity in protein folding. Thermodynamic dissection of interdomain interactions in phosphoglycerate kinase

Abstract

In the presence of guanidine hydrochloride, phosphoglycerate kinase from yeast can be reversibly denatured by either heating or cooling the protein solution above or below room temperature [Griko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 244, 276-278]. The heat denaturation of PGK is characterized by the presence of a single peak in the excess heat capacity function obtained by differential scanning calorimetry. The transition curve approaches the two-state mechanism, indicating that the two domains of the molecule display strong cooperative interactions and that partially folded intermediates are not largely populated during the transition. On the contrary, the cold denaturation is characterized by the presence of two peaks in the heat capacity function. Analysis of the data indicates that at low temperatures the two domains behave independently of each other. The crystallographic structure of PGK has been used to identify the nature of the interactions between the two domains. These interactions involve primarily the apposition of two hydrophobic surfaces of approximately 480 A2 and nine hydrogen bonds. This information, in conjunction with experimental thermodynamic values for hydrophobic, hydrogen bonding interactions and statistical thermodynamic analysis, has been used to quantitatively account for the folding/unfolding behavior of PGK. It is shown that this type of analysis accurately predicts the cooperative behavior of the folding/unfolding transition and its dependence on GuHCl concentration.

Thermal stability of the three domains of streptokinase studied by circular dichroism and nuclear magnetic resonance

“…This result is contrary to what is generally observed for the unfolding of proteins with interacting domains. Theoretical models generally account for painvise domain-domain interactions by using a favorable interface free energy that goes to zero when one of the domains unfolds (Brandts et al, 1989;Freire et al, 1992). These models have been successful in explaining the unfolding properties of several proteins with domains communicating by painvise interactions.…”

Conformational kinetics reveals affinities of protein conformational states

“…Often, the conformational ensembles of these macromolecules can be subdivided into kinetically distinguishable conformational subensembles or states in the thermodynamic sense. Thanks in part to the highly cooperative nature of biopolymer conformational changes (2)(3)(4), these conformational states are distinguishable by the kinetic barriers that separate them; conformers in separate states interconvert much more slowly than conformers within the same state. Thus, the reactions of such a system can be described as the interconversion of a small number of conformational states without ignoring the ensemble nature of each state (5,6).…”

An engineered amino‐terminal domain of yeast phosphoglycerate kinase with native‐like structure

“…The role of interdomain contacts in maintaining the thcrmodynamic stability of the individual domains within a multi-domain protein has been examined by a number of investigators (Hu & Sturtevant, 1987;Brandts et al, 1989;Freire et al, 1992). These studies were prompted by thc observation that the unfolding behavior of multi-domain proteins is often highly cooperative (i.e., the domains unfold simultaneously), even though the intrinsic stabilities of each domain differ.…”