Residue Specific Analysis of Frustration in Folding Landscape of Repeat Alpha/Beta Protein Apoflavodoxin

Topological frustrated proteins can give rise to complex folding pathways and transition state profiles. To better understand such systems, we combine experimental and computational methods to study Desulfovibrio desulfuricans apoflavodoxin by producing several point mutation variants. By equilibrium unfolding experiments, we first revealed how different secondary-structure elements contribute to overall protein resistance towards heat and urea. Next using stopped-flow mixing coupled to far-UV circular dichroism (CD), we probed how individual residues affect the amount of structure formed in the experimentally-detected burst-phase intermediate. Together with in silico folding route analysis of the same point-mutated variants and computation of the growth in nucleation size during early folding, computer simulations suggested the presence of two competing folding nuclei at opposite sides of the central β-strand 3 (i.e. at β-strands 1 and 4), which cause early topological frustration (i.e., misfolding) in the folding landscape. Particularly, the extent of heterogeneity in the folding nucleigrowth correlates with the in vitro burst phase CD amplitude. In addition, Φ-value analysis (in vitro and in silico) of the overall folding barrier to apoflavodoxin's native state revealed that native-like interactions in most of the β-strands must form in transition state. Our study reveals that an imbalanced competition between the two sides of apoflavodoxin's central β-sheet directs initial misfolding while proper alignment on both sides of β-strand 3 is necessary for productive folding.