A protein fold is defined as a structural arrangement of a secondary structure in a three-dimensional space. It would be interesting to know whether a particular fold can be assigned to certain features of the corresponding folding/unfolding transitions. To understand the underlying principles of the manifold folding transitions in more detail, single-molecule FRET is the method of choice. Taking the two-domain protein phosphoglycerate kinase (PGK) as an example, we investigated denaturant-induced unfolded states of PGK using the above method. For this purpose, different intramolecular distances within the two domains were measured. In addition to the known two-state transition, a transition with a compact folding intermediate was also identified in each of the two domains. Based on the structural homology of the domains (characterized by a Rossmann fold) and the striking similarity in the features of the measured distance changes during unfolding, clear evidence emerged that the underlying domain topology plays an important role in determining the observed structural changes.
Keywords: domain topology; molten globule intermediate state; protein folding; single-molecule Förster resonance energy transfer; unfolding/folding-induced conformational changes.