Residue-specific analysis of frustration in the folding landscape of repeat beta/alpha protein apoflavodoxin.
Flavodoxin adopts the common repeat beta/alpha topology and folds in a complex kinetic reaction with intermediates. To better understand this reaction, we analyzed a set of Desulfovibrio desulfuricans apoflavodoxin variants with point mutations in most secondary structure elements by in vitro and in silico methods. By equilibrium unfolding experiments, we first revealed how different secondary structure elements contribute to overall protein resistance to heat and urea. Next, using stopped-flow mixing coupled with far-UV circular dichroism, 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 growth in nucleation size during early folding, computer simulations suggested the presence of two competing folding nuclei at opposite sides of the central beta-strand 3 (i.e., at beta-strands 1 and 4), which cause early topological frustration (i.e., misfolding) in the folding landscape. Particularly, the extent of heterogeneity in folding nuclei growth correlates with the in vitro burst-phase circular dichroism amplitude. In addition, phi-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 beta-strands must form in transition state. Our study reveals that an imbalanced competition between the two sides of apoflavodoxin's central beta-sheet directs initial misfolding, while proper alignment on both sides of beta-strand 3 is necessary for productive folding.
Stagg
Loren
L
Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251, USA.
Samiotakis
Antonios
A
Homouz
Dirar
D
Cheung
Margaret S
MS
Wittung-Stafshede
Pernilla
P
D000596:Amino Acids / Q000737:chemistry*; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D019295:Computational Biology; D045285:Desulfovibrio desulfuricans / Q000737:chemistry*; D005418:Flavodoxin / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics; D009154:Mutation / Q000235:genetics; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D020449:Repetitive Sequences, Amino Acid; D013696:Temperature
D000596:Amino Acids; D001059:Apoproteins; D005418:Flavodoxin; D050505:Mutant Proteins; C026932:apoflavodoxin