title: Folding of the Pit1 homeodomain near the speed limit.
abstract: Current questions in protein folding mechanisms include how fast can a protein fold and are there energy barriers for the folding and unfolding of ultrafast folding proteins? The small 3-helical engrailed homeodomain protein folds in 1.7 μs to form a well-characterized intermediate, which rearranges in 17 μs to native structure. We found that the homologous pituitary-specific transcription factor homeodomain (Pit1) folded in a similar manner, but in two better separated kinetic phases of 2.3 and 46 μs. The greater separation and better fluorescence changes facilitated a detailed kinetic analysis for the ultrafast phase for formation of the intermediate. Its folding rate constant changed little with denaturant concentration or mutation but unfolding was very sensitive to denaturant and energy changes on mutation. The folding rate constant of 3 × 10(5) s(-1) in water decreased with increasing viscosity, and was extrapolated to 4.4 × 10(5) s(-1) at zero viscosity. Thus, the formation of the intermediate was partly rate limited by chain diffusion and partly by an energy barrier to give a very diffuse transition state, which was followed by the formation of structure. Conversely, the unfolding reaction required the near complete disruption of the tertiary structure of the intermediate in a highly cooperative manner, being exquisitely sensitive to individual mutations. The folding is approaching, but has not reached, the downhill-folding scenario of energy landscape theory. Under folding conditions, there is a small energy barrier between the denatured and transition states but a larger barrier between native and transition states.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom.
authors.forename: Wiktor
authors.initials: W
authors.lastname: Banachewicz
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Johnson
authors.forename: Alan R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D000595:Amino Acid Sequence; D001703:Biophysics / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012984:Software; D013696:Temperature; D013816:Thermodynamics; D050817:Transcription Factor Pit-1 / Q000737:chemistry*; D014783:Viscosity
chemical_list: D050817:Transcription Factor Pit-1