Differentiating between the sequence of structural events on alternative pathways of folding of a heterodimeric protein.

Distinguishing between competing pathways of folding of a protein, on the basis of how they differ in their progress of structure acquisition, remains an important challenge in protein folding studies. A previous study had shown that the heterodimeric protein, double chain monellin (dcMN) switches between alternative folding pathways upon a change in guanidine hydrochloride (GdnHCl) concentration. In the current study, the folding of dcMN has been characterized by the pulsed hydrogen exchange (HX) labeling methodology used in conjunction with mass spectrometry. Quantification of the extent to which folding intermediates accumulate and then disappear with time of folding at both low and high GdnHCl concentrations, where the folding pathways are known to be different, shows that the folding mechanism is describable by a triangular three-state mechanism. Structural characterization of the productive folding intermediates populated on the alternative pathways has enabled the pathways to be differentiated on the basis of the progress of structure acquisition that occurs on them. The intermediates on the two pathways differ in the extent to which the α-helix and the rest of the ß-sheet have acquired structure that is protective against HX. The major difference is, however, that ß2 has not acquired any protective structure in the intermediate formed on one pathway, but it has acquired significant protective structure in the intermediate formed on the alternative pathway. Hence, the sequence of structural events is different on the two alternative pathways.

Structural Characterization of the Cooperativity of Unfolding of a Heterodimeric Protein using Hydrogen Exchange-Mass Spectrometry.

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 â†” I1 â†” I2 â†” I3 â†” U mechanism. Structural changes occur gradually in the first three steps, and cooperatively in the last step. ß strands 2, 4 and 5, as well as the α-helix undergo transient unfolding during all three non-cooperative steps, while ß1 and the two loops on both sides of the helix undergo transient unfolding during the first two steps. In the absence of GdnHCl, only ß3 in chain A of the protein unfolds during the last cooperative step, while in the presence of 1 M GdnHCl, not only ß3, but also ß2 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.