Expanded monomeric intermediate upon cold and heat unfolding of phosphofructokinase-2 from Escherichia coli.

Folding studies have been focused mainly on small, single-domain proteins or isolated single domains of larger proteins. However, most of the proteins present in biological systems are composed of multiple domains, and to date, the principles that underlie its folding remain elusive. The unfolding of Pfk-2 induced by GdnHCl has been described by highly cooperative three-state equilibrium (N(2)↔2I↔2U). This is characterized by a strong coupling between the subunits' tertiary structure and the integrity of the dimer interface because "I" represents an unstructured and expanded monomeric intermediate. Here we report that cold and heat unfolding of Pfk-2 resembles the N(2)↔2I step of chemically induced unfolding. Moreover, cold unfolding appears to be as cooperative as that induced chemically and even more so than its heat-unfolding counterpart. Because Pfk-2 is a large homodimer of 66 kDa with a complex topology consisting of well-defined domains, these results are somewhat unexpected considering that cold unfolding has been described as a special kind of perturbation that decouples the cooperative unfolding of several proteins.