Effects of Mutations on Side-Specific Folding Mechanism of a Helix-Turn-Helix Protein

Abstract

Helix-turn-helix motifs are important super-secondary protein structural elements and excellent models for studying the mechanism of protein folding. We have been investigating folding of a de novo designed 38-residue helix-turn-helix motif α-t-α using IR spectroscopy with site-specific 13C isotopic editing. Our preliminary site-specific thermal unfolding data revealed that α-t-α is most stable near the centers of both α-helices, and likely unfolds from the helical termini and the loose turn region. To obtain further insights into the folding mechanism, and to investigate the roles of the individual residue-residue stabilizing interactions, we have begun mutational studies of the α-t-α protein. The mutations were designed to both destabilize and further stabilize the hydrophobic core near the helical centers. Additional mutations were designed to stabilize the helical termini and the turn/loop sequence. The overall thermodynamic stability of the α-t-α was measured using CD and IR spectroscopies. The core mutations appreciably decreased or increased the overall folding stability as intended, however, stabilizing the turn and helical termini proved to be a rather challenging task. Site-specific thermal unfolding of the mutated α-t-α were probed with IR on multiple 13C isotopically labeled variants of each mutant. The effects of the mutations on both the global and, in particular, local site-specific unfolding provide important clues about the stabilization of the helix-turn-helix motif by specific interactions. Although additional mutational studies are underway, thus far all the data are consistent with the proposed folding mechanism.