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An early immunoreactive folding intermediate of the tryptophan synthase β 2 subunit is a ‘molten globule’
Author(s) -
Goldberg Michel E.,
Semisotnov Gennady V.,
Friguet Bertrand,
Kuwajima Kunihiro,
Ptitsyn Oleg B.,
Sugai Shintaro
Publication year - 1990
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(90)80703-l
Subject(s) - molten globule , chemistry , guanidine , circular dichroism , tryptophan synthase , kinetics , native state , protein secondary structure , protein subunit , crystallography , folding (dsp implementation) , biophysics , protein folding , fluorescence , tryptophan , biochemistry , amino acid , biology , physics , quantum mechanics , electrical engineering , gene , engineering
The refolding kinetics of the tryptophan synthase β 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 β 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 β 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 ( t35 s) with the time of formation of the previously characterized [(1988) Biochemistry 27, 7633‐7640] early imuno‐reactive intermediate recognized by a monoclonal antibody ( t12 s), it is shown that this native‐like epitope forms within the ‘molten globule’, before the tight packing of the protein side chains.