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

[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.

[学科分类] 生物化学/生物物理

[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