The kinetics of G-CSF folding.
The folding kinetics of G-CSF were determined by trp-fluorescence and far-UV circular dichroism. Folding and unfolding was achieved by rapid dilution and mixing of the denaturant, GdnHCl. G-CSF is a four-helical bundle protein with two long loops between the first and second helices and between the third and fourth helices. The entire conformational change expected by fluorescence was observed by stopped-flow technology, but due to rapid refolding kinetics only a portion was observed by circular dichroism. G-CSF contains two trp residues, and their contribution to the fluorescent-detected kinetics were deciphered through the use of single-site trp mutants. The trp moieties are probes of the local conformation surrounding their environment. One trp at residue 118 is located within the third helix while the other trp at residue 58 is part of the long loop between the first and second helices. The refolding results were most consistent with the following mechanism: U <--> I(1) <--> I(2) <--> N; where U represents the unfolded protein, I(1) represents intermediate state 1, I(2) represents intermediate state 2, and N represents the native state. I(1) is characterized as having approximately one-half of the native-like helical structure and none of the native-like fluorescence. I(2) has 100% of the native helical structure and most of the trp-118 and little of the trp-58 native-like fluorescence. Thus refolding occurs in distinct stages with half of the helix forming first followed by the remaining half of the helix including the third helix and finally the loop between the first and second helices folds.
Brems
David N
DN
Department of Pharmaceutics, Amgen Inc., Thousand Oaks, California 91320, USA. dbrems@angen.com
D002942:Circular Dichroism; D005453:Fluorescence; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011489:Protein Denaturation / Q000502:physiology; D017510:Protein Folding; D014364:Tryptophan / Q000031:analogs & derivatives / Q000378:metabolism
D016179:Granulocyte Colony-Stimulating Factor; D014364:Tryptophan