Ultrarapid mixing experiments reveal that Im7 folds via an on-pathway intermediate.
Many proteins populate partially organized structures during folding. Since these intermediates often accumulate within the dead time (2-5 ms) of conventional stopped-flow and quench-flow devices, it has been difficult to determine their role in the formation of the native state. Here we use a microcapillary mixing apparatus, with a time resolution of approximately 150 micros, to directly follow the formation of an intermediate in the folding of a four-helix protein, Im7. Quantitative kinetic modeling of folding and unfolding data acquired over a wide range of urea concentrations demonstrate that this intermediate ensemble lies on a direct path from the unfolded to the native state.
Capaldi
A P
AP
School of Biochemistry and Molecular Biology and the Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Shastry
M C
MC
Kleanthous
C
C
Roder
H
H
Radford
S E
SE
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D003087:Colicins; D007700:Kinetics; D008956:Models, Chemical; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D001426:Bacterial Proteins; D003087:Colicins; C007104:colicin immunity proteins; D014508:Urea