A stable cold folding intermediate of rabbit muscle D-glyceraldehyde 3-phosphate dehydrogenase.
With decreasing temperature the reactivation yield of denatured D-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) upon dilution increases but the reactivation rate decreases. Neither reactivation nor aggregation during refolding can be detected at 4 degrees C in 48 h, and at 3 degrees C even in 6 days. However, the reactivation takes place once the temperature is raised with little decrease of the yield after incubation for 6 days at 3 degrees C. A cold folding intermediate forms in a burst phase of refolding at 4 degrees C as shown by a fast change of the intrinsic fluorescence followed by further conformational adjustment to a stable state in about 1 h. The stable folding intermediate has been characterized to be a dimer of partially folded GAPDH subunit with secondary structure between that of the native and denatured enzymes, a hydrophobic cluster not found in either the native or the denatured state, and an active site similar to but different from that of the native state. Chaperonin 60 (GroEL) binds with all intermediates formed at 4 degrees C, but the intermediates formed at the early folding stage reactivate with higher yield than those formed after conformational adjustment when dissociated from GroEL in the presence of ATP and further folded and assembled into the native tetramer.
Zhang
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NX
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, China.
Wang
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D018834:Chaperonin 60 / Q000378:metabolism; D003080:Cold Temperature; D019281:Dimerization; D004789:Enzyme Activation; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry* / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D009132:Muscles / Q000201:enzymology*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011817:Rabbits; D013696:Temperature
D018834:Chaperonin 60; D000255:Adenosine Triphosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases