Binding of a burst-phase intermediate formed in the folding of denatured D-glyceraldehyde-3-phosphate dehydrogenase by chaperonin 60 and 8-anilino-1-naphthalenesulphonic acid.
Upon dilution, D-glyceraldehyde-3-phosphate dehydrogenase (GADPH) that has been fully inactivated, but only partially unfolded, in dilute guanidine hydrochloride (GuHCl) recovers activity completely. The fully unfolded enzyme, however, is re-activated only to a limited extent after dilution, and refolds rapidly in a burst phase to a partially folded intermediate characterized by increases in both the emission intensity of intrinsic fluorescence and binding to 8-anilino-1-naphthalenesulphonic acid (ANS). This intermediate aggregates with a time lag of a few minutes, and the aggregation can be suppressed completely by chaperonin 60 (GroEL). Stoichiometric analysis of the suppression of GAPDH re-activation by GroEL suggests that the tetradecameric GroEL binds to a dimeric GAPDH folding intermediate. This intermediate can be re-activated by ATP or ATP/chaperonin 10 (GroES) to an extent considerably greater than that obtained on spontaneous re-activation of the fully denatured enzyme upon dilution. Probing with a fluorescent derivative of NAD+ shows that this folding intermediate does not have a native conformation at the active site. The similar profiles of the effects of GroEL and ANS on the re-activation of GAPDH denatured by different concentrations of GuHCl suggest that GroEL and ANS recognize and bind to the same folding intermediate, which is similar to the relatively stable, partially unfolded, state of the enzyme denatured in 0.5-1.0 MGuHCl. However, the complexes of the intermediate with GroEL or ANS appear to be different, in that GroEL, but not ANS, suppresses aggregation and assists folding in the presence of ATP.
Li
X L
XL
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, 15 Datun Road., Beijing 100101, China.
Lei
X D
XD
Cai
H
H
Li
J
J
Yang
S L
SL
Wang
C C
CC
Tsou
C L
CL
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D018834:Chaperonin 60 / Q000378:metabolism* / Q000494:pharmacology; D019281:Dimerization; D004793:Enzyme Reactivators; D005456:Fluorescent Dyes; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry* / Q000378:metabolism*; D019791:Guanidine; D011489:Protein Denaturation; D017510:Protein Folding
D000817:Anilino Naphthalenesulfonates; D018834:Chaperonin 60; D004793:Enzyme Reactivators; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D000255:Adenosine Triphosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D019791:Guanidine