We present a detailed structural characterization of the intermediate state populated during the folding and unfolding of the bacterial immunity protein Im7. We achieve this result by incorporating a variety of experimental data available for this species in molecular dynamics simulations. First, we define the structure of the exchange-competent intermediate state of Im7 by using equilibrium hydrogen-exchange protection factors. Second, we use this ensemble to predict ⌽-values and compare the results with the experimentally determined ⌽-values of the kinetic refolding intermediate. Third, we predict chemical-shift measurements and compare them with the measured chemical shifts of a mutational variant of Im7 for which the kinetic folding intermediate is the most stable state populated at equilibrium. Remarkably, we found that the properties of the latter two species are predicted with high accuracy from the exchange-competent intermediate that we determined, suggesting that these three states are characterized by a similar architecture in which helices I, II, and IV are aligned in a native-like, but reorganized, manner. Furthermore, the structural ensemble that we obtained enabled us to rationalize the results of tryptophan fluorescence experiments in the WT protein and a series of mutational variants. The results show that the integration of diverse sets of experimental data at relatively low structural resolution is a powerful approach that can provide insights into the structural organization of this conformationally heterogeneous three-helix intermediate with unprecedented detail and highlight the importance of both native and non-native interactions in stabilizing its structure.hydrogen exchange ͉ protein folding ͉ protein intermediates I ntermediate states have been identified in the folding and unfolding processes of many proteins (1-11). Owing to their transient nature and conformational heterogeneity, however, the experimental determination of their structure is an enormously challenging task (6, 7, 9-11).The bacterial immunity protein Im7 is a four-helical protein that has been shown to fold through an on-pathway kinetic intermediate (12). The structural properties of this kinetic intermediate state (KIS) have been characterized experimentally by ⌽-value analysis (4) and shown to comprise three of the four native helices (I, II, and IV), stabilized by both native and non-native interactions. In addition, equilibrium hydrogen-exchange experiments (7) on the WT protein and the variant I72V showed that amides in these three helices, but not in helix III, exchange slowly with a free energy similar to that associated with global unfolding, suggesting that they are substantially formed in the exchange-competent intermediate state (EIS), whereas helix III is not significantly protected from hydrogen exchange in this state. Furthermore, the double-mutant L53AI54A (13) has been designed to trap the kinetic intermediate as a stable intermediate state (SIS) at equilibrium. Various biophysical methods suggested tha...

No abstract