Abstract:
Little is known about how the sequence of structural changes in one chain of a heterodimeric protein is coupled to those in the other chain during protein folding and unfolding reactions, and whether individual secondary structural changes in the two chains occur in one or many coordinated steps. Here, the unfolding mechanism of a small heterodimeric protein, double chain monellin, has been characterized using hydrogen exchange-mass spectrometry. Transient structure opening, which enables HX, was found to be describable by a five state N ↔ I1 ↔ I2 ↔ I3 ↔ U mechanism. Structural changes occur gradually in the first three steps, and cooperatively in the last step. β strands 2, 4 and 5, as well as the α-helix undergo transient unfolding during all three non-cooperative steps, while β1 and the two loops on both sides of the helix undergo transient unfolding during the first two steps. In the absence of GdnHCl, only β3 in chain A of the protein unfolds during the last cooperative step, while in the presence of 1 M GdnHCl, not only β3, but also β2 in chain B unfolds cooperatively. Hence, the extent of cooperative structural change and size of the cooperative unfolding unit increase when the protein is destabilized by denaturant. The naturally evolved two-chain variant of monellin folds and unfolds in a more cooperative manner than does a single chain variant created artificially, suggesting that increasing folding cooperativity, even at the cost of decreasing stability, may be a driving force in the evolution of proteins.