Acid denaturation and refolding of green fluorescent protein.
Green fluorescent protein from the jellyfish Aequorea victoria can serve as a good model protein to understand protein folding in a complex environment with molecular chaperones and other macromolecules such as those in biological cells, but little is known about the detailed mechanisms of the in vitro folding of green fluorescent protein itself. We therefore investigated the kinetic refolding of a mutant (F99S/M153T/V163A) of green fluorescent protein, which is known to mature more efficiently than the wild-type protein, from the acid-denatured state; refolding was observed by chromophore fluorescence, tryptophan fluorescence, and far-UV CD, using a stopped-flow technique. In this study, we demonstrated that the kinetics of the refolding of the mutant have at least five kinetic phases and involve nonspecific collapse within the dead time of a stopped-flow apparatus and the subsequent formation of an on-pathway intermediate with the characteristics of the molten globule state. We also demonstrated that the slowest phase and a major portion of the second slowest phase were rate-limited by slow prolyl isomerization in the intermediate state, and this rate limitation accounts for a major portion of the observed kinetics in the folding of green fluorescent protein.
Enoki
Sawako
S
Department of Physics, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Saeki
Kimiko
K
Maki
Kosuke
K
Kuwajima
Kunihiro
K
D000818:Animals; D002942:Circular Dichroism; D021984:Cyclophilin A / Q000494:pharmacology; D004229:Dithiothreitol; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D006851:Hydrochloric Acid; D006863:Hydrogen-Ion Concentration; D037402:Hydrozoa / Q000737:chemistry / Q000235:genetics; D007536:Isomerism; D007700:Kinetics; D044367:Phase Transition / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects / Q000235:genetics; D017510:Protein Folding; D012965:Sodium Chloride; D013050:Spectrometry, Fluorescence; D014325:Tromethamine
D005456:Fluorescent Dyes; D014325:Tromethamine; D049452:Green Fluorescent Proteins; D012965:Sodium Chloride; D021984:Cyclophilin A; D006851:Hydrochloric Acid; D004229:Dithiothreitol