In vitro refolding and unfolding of subunits of electron-transferring flavoprotein: characterization of the folding intermediates and the effects of FAD and AMP on the folding reaction.
Electron-transferring flavoprotein (ETF) from pig kidney is composed of two subunits (alpha and beta, molecular weights of 33,000 and 29,000) and two small molecules, FAD and AMP. In this study, in vitro refolding and unfolding of the subunits of ETF were carried out with urea as the denaturing reagent. The refolding reaction of alpha and beta was revealed to proceed kinetically in two steps: D in equilibrium with I-->N, where D,I, and N denote the denatured, intermediate, and native forms, respectively. The features of the I forms of alpha and beta, described below, are consistent with the concept of the so-called "molten globule state," which is frequently observed in protein refolding. (i) The conversion between D and I was very rapid. (ii) The I form showed as much secondary structure as the N form as judged from the far-UV circular dichroism. (iii) The solvent accessibility of the I form, estimated by the analysis of equilibrium unfolding experiments, was intermediate between those of the D and N forms. (iv) The standard free energy of the I form is almost the same as that of the D form. The refolding reaction progressed more slowly and the environment of the tryptophan chromophore was changed more drastically in beta refolding that in alpha refolding. We previously reported that the reconstitution of holoETF from denatured subunits is speeded up by increasing the AMP concentration. In this study, the effects of AMP, FAD, and the other subunit on the single subunit folding were examined, but no effect was detected. This result suggests that AMP plays a role in a later process, namely, assembly of the four components (refolded alpha and beta, FAD, and AMP).
Department of Physiology, Kumamoto University School of Medicine.
K
K
Sato
Y
Y
Nishina
K
K
Shiga
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D044927:Electron-Transferring Flavoproteins; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D005420:Flavoproteins / Q000737:chemistry* / Q000187:drug effects; D066298:In Vitro Techniques; D007668:Kidney / Q000737:chemistry; D007700:Kinetics; D015394:Molecular Structure; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D013552:Swine; D013816:Thermodynamics
D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; D000249:Adenosine Monophosphate