An early immunoreactive folding intermediate of the tryptophan synthase β2 subunit is a ‘molten globule’

FEBS Letters

Goldberg, Michel E.¹  Friguet, Bertrand¹  Kuwajima, Kunihiro²  Semisotnov, Gennady V.³  Sugai, Shintaro²  Ptitsyn, Oleg B.³ 

[1] Unité de Biochimie Cellulaire (CNRS URA 1129), Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France;Department of Polymer Science, Faculty of Science, Hokkaido University, Kita-Ku, Sapporo, Hokkaido 060, Japan;Institute of Protein Research, Academy of Sciences of the USSR, 142292 Pushchino, Moscow Region, USSR

关键词: Tryptophan synthase β2 subunit;    Immunoreactive kinetic intermediate;    Circular dichroism;    Molten globule;    Stopped-flow technique;    β2;    the β2 subunit of Escherichia colt tryptophan synthase;    GuHCl;    guanidine hydrochloride;    CD;    circular dichroism;    ANS;    anilino-8-naphthalene sulfonate;    t;    half-time of reaction;    k;    rate constant;    mAb 19;    a monoclonal antibody (IgG 19) specific for native β2 EDTA;    ethylene-diamine-tetraacetic acid;   

DOI  :  10.1016/0014-5793(90)80703-L

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The refolding kinetics of the tryptophan synthase β₂ 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 β₂, 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 β₂ 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 (_t 35 s) with the time of formation of the previously characterized [(1988) Biochemistry 27, 7633-7640] early imuno-reactive intermediate recognized by a monoclonal antibody (t 12 s), it is shown that this native-like epitope forms within the ‘molten globule’, before the tight packing of the protein side chains.

【 授权许可】

Unknown