An early immunoreactive folding intermediate of the tryptophan synthease beta 2 subunit is a 'molten globule'.
The refolding kinetics of the tryptophan synthase beta 2 subunit have been investigated by circular dichroism (CD) and binding of a fluorescent hydrophobic probe (ANS), using the stopped-flow technique. The kinetics of regain of the native far UV CD signal show that, upon refolding of urea denatured beta 2, more than half of the protein secondary structure is formed within the dead time of the CD stopped-flow apparatus (0.013 s). On the other hand, upon refolding of guanidine unfolded beta 2, the fluorescence of ANS passes through a maximum after about 1 s and then 'slowly' decreases. These results show the accumulation, in the 1-10 s time range, of an early transient folding intermediate which has a pronounced secondary structure and a high affinity for ANS. In this time range, the near UV CD remains very low. This transient intermediate thus appears to have all the characteristics of the 'molten globule' state [(1987) FEBS Lett. 224, 9-13]. Moreover, by comparing the intrinsic time of the disappearance of this transient intermediate (t1/2 35 s) with the time of formation of the previously characterized [(1988) Biochemistry 27, 7633-7640] early immunoreactive intermediate recognized by a monoclonal antibody (t1/2 12 s), it is shown that this native-like epitope forms within the 'molten globule', before the tight packing of the protein side chains.
Unité de Biochimie Cellulaire (CNRS URA 1129), Institut Pasteur, Paris, France.
M E
ME
Goldberg
G V
GV
Semisotnov
B
B
Friguet
K
K
Kuwajima
O B
OB
Ptitsyn
S
S
Sugai
D002942:Circular Dichroism; D000939:Epitopes / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014367:Tryptophan Synthase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
D000939:Epitopes; D046911:Macromolecular Substances; D014508:Urea; D014367:Tryptophan Synthase