Defining the Nature of Thermal Intermediate in 3 State Folding Proteins: Apoflavodoxin, a Study Case

The early stages of the thermal unfolding of apoflavodoxin have been determined by using atomistic multi microsecond-scale molecular dynamics (MD) simulations complemented with a variety of experimental techniques. Results strongly suggest that the intermediate is reached very early in the thermal unfolding process and that it has the properties of an “activated” form of the native state, where thermal fluctuations in the loops break loop-loop contacts. The unrestrained loops gain then kinetic energy corrupting short secondary structure elements without corrupting the core of the protein. The MD-derived ensembles agree with experimental observables and draw a picture of the intermediate state inconsistent with a well-defined structure and characteristic of a typical partially disordered protein. Our results allow us to speculate that proteins with a well packed core connected by long loops might behave as partially disordered proteins under native conditions, or alternatively behave as three state folders. Small details in the sequence, easily tunable by evolution, can yield to one or the other type of proteins. Author Summary: A simplistic view of protein structure tends to emphasize the opposition between the native state and the denatured ensemble of unfolded conformations. In addition to these extreme conformations, proteins subjected to a variety of perturbations often populate alternative partly unfolded conformations, some of which are close in energy to the native state and, accordingly, can be populated under native or quasi-native conditions. There is increasing evidence that these “perturbed” conformations participate in protein function or, in some cases, are related to the outcome of folding diseases. We have used the “state of the art” molecular dynamics combined with a variety of experimental techniques to characterize for the first time, to our knowledge, the thermal intermediate of a three-state folding protein (apoflavodoxin). Based on our results we have been able to suggest a general mechanism of thermal unfolding in complex proteins and to determine interesting links between thermal intermediates and partially unfolded proteins.