The helix-turn-helix motif as an ultrafast independently folding domain: the pathway of folding of Engrailed homeodomain.
Helices 2 and 3 of Engrailed homeodomain (EnHD) form a helix-turn-helix (HTH) motif. This common motif is believed not to fold independently, which is the characteristic feature of a motif rather than a domain. But we found that the EnHD HTH motif is monomeric and folded in solution, having essentially the same structure as in full-length protein. It had a sigmoidal thermal denaturation transition. Both native backbone and local tertiary interactions were formed concurrently at 4 x 10(5) s(-1) at 25 degrees C, monitored by IR and fluorescence T-jump kinetics, respectively, the same rate constant as for the fast phase in the folding of EnHD. The HTH motif, thus, is an ultrafast-folding, natural protein domain. Its independent stability and appropriate folding kinetics account for the stepwise folding of EnHD, satisfy fully the criteria for an on-pathway intermediate, and explain the changes in mechanism of folding across the homeodomain family. Experiments on mutated and engineered fragments of the parent protein with different probes allowed the assignment of the observed kinetic phases to specific events to show that EnHD is not an example of one-state downhill folding.
Religa
Tomasz L
TL
Medical Research Council Centre for Protein Engineering, Hills Road, Cambridge, United Kingdom.
Johnson
Christopher M
CM
Vu
Dung M
DM
Brewer
Scott H
SH
Dyer
R Brian
RB
Fersht
Alan R
AR
D018360:Crystallography, X-Ray; D019077:Helix-Turn-Helix Motifs; D007700:Kinetics; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D009994:Osmolar Concentration; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013055:Spectrophotometry, Infrared; D013997:Time Factors
D011506:Proteins; D007930:Leucine