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 <-> I-1 <-> I-2 <-> I-3 <-> U mechanism. Structural changes occur gradually in the first three steps, and cooperatively in the last step. beta strands 2, 4 and 5, as well as the alpha-helix undergo transient unfolding during all three non-cooperative steps, while beta 1 and the two loops on both sides of the helix undergo transient unfolding during the first two steps. In the absence of GdnHCl, only beta 3 in chain A of the protein unfolds during the last cooperative step, while in the presence of 1 M GdnHCl, not only beta 3, but also beta 2 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. (C) 2021 Elsevier Ltd. All rights reserved.

Automated summary

The unfolding mechanism of a small heterodimeric protein has been characterized using hydrogen exchange-mass spectrometry, revealing a five-state mechanism for transient structure opening. Structural changes occur gradually in the first three steps, and cooperatively in the last step.