The structure of the major transition state for folding of an FF domain from experiment and simulation.
We have analysed the transition state of folding of the four-helix FF domain from HYPA/FBP11 by high-resolution experiment and simulation as part of a continuing effort to understand the principles of folding and the refinement of predictive methods. The major transition state for folding was subjected to a Phi-value analysis utilising 50 mutants. The transition state contained a nucleus for folding centred around the end of helix 1 (H1) and the beginning of helix 2 (H2). Secondary structure in this region was fully formed (PhiF=0.9-1) and tertiary interactions were well developed. Interactions in the distal part of the native structure were weak (PhiF=0-0.2). The hydrophobic core and other parts of the protein displayed intermediate Phi-values, suggesting that interactions coalesce as the end of H1 and beginning of H2 are in the process of being formed. The distribution of Phi-values resembled that of barnase, which folds via an intermediate, rather than that of CI2 which folds by a concerted nucleation-condensation mechanism. The overall picture of the transition state structure identified in molecular dynamics simulations is in qualitative agreement, with the turn connecting H1 and H2 being formed, a loosened core, and H4 partially unfolded and detached from the core. There are some differences in the details and interpretation of specific Phi-values.
Jemth
Per
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MRC Centre for Protein Engineering, Hills Road, CB2 2QH Cambridge, UK.
Day
Ryan
R
Gianni
Stefano
S
Khan
Faaizah
F
Allen
Mark
M
Daggett
Valerie
V
Fersht
Alan R
AR
D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D002352:Carrier Proteins; C491404:PRPF40A protein, human; D005998:Glycine