Mapping the folding pathway of an immunoglobulin domain: Structural detailfrom phi value analysis and movement of the transition state

Background: Do proteins that have the same structure fold by the same pathw ay even when they are unrelated in sequence? To address this question, we a re comparing the folding of a number of different immunoglobulinlike protei ns. Here, we present a detailed protein engineering phi value analysis of t he folding pathway of TI 127, an immunoglobulin domain from human cardiac t itin. Results: TI 127 folds rapidly via a kinetic intermediate that is destabiliz ed by most mutations. The transition state for folding is remarkably native -like in terms of solvent accessibility. We use phi value analysis to map t his transition state and show that it is highly structured; only a few resi dues close to the N-terminal region of the protein remain completely unfold ed. Interestingly, most mutations cause the transition state to become less native-like. This anti-Hammond behavior can be used as a novel means of ob taining additional structural information about the transition state. Conclusions: The residues that are involved in nucleating the folding of TI 127 are structurally equivalent to the residues that form the folding nucl eus in an evolutionary unrelated fibronectin type III protein. These residu es form part of the common structural core of Ig-like domains. The data sup port the hypothesis that interactions essential for defining the structure of these beta sandwich proteins are also important in nucleation of folding .