caa a22 4500 397556772 CHVBK 20180308164804.0 cr unu---uuuuu 161202e199608 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021410 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021410 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 [Elektronische Daten] [Kyosuke Sato, Yasuzo Nishina, Kiyoshi Shiga] Electron-transferring flavoprotein (ETF) from pig kidney is composed of two subunits (α and β , 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 α and β was revealed to proceed kinetically in two steps: D⇄I→N, where D, I, and N denote the denatured, intermediate, and native forms, respectively. The features of the I forms of α and β , 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 β refolding than in α 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 α and β, FAD, and AMP). © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence AMP nationallicence electron-transferring flavoprotein nationallicence folding intermediate nationallicence subunit nationallicence urea denaturation nationallicence Sato Kyosuke Department of Physiology, Kumamoto University School of Medicine, Honjo, Kumamoto, Kumamoto 860 aut Nishina Yasuzo Department of Physiology, Kumamoto University School of Medicine, Honjo, Kumamoto, Kumamoto 860 aut Shiga Kiyoshi Department of Physiology, Kumamoto University School of Medicine, Honjo, Kumamoto, Kumamoto 860 aut The Journal of Biochemistry Oxford University Press 120/2(1996-08), 276-285 0021-924X 120:2<276 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021410 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021410 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Sato Kyosuke Department of Physiology, Kumamoto University School of Medicine, Honjo, Kumamoto, Kumamoto 860 aut NATIONALLICENCE 700 1- Nishina Yasuzo Department of Physiology, Kumamoto University School of Medicine, Honjo, Kumamoto, Kumamoto 860 aut NATIONALLICENCE 700 1- Shiga Kiyoshi Department of Physiology, Kumamoto University School of Medicine, Honjo, Kumamoto, Kumamoto 860 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/2(1996-08), 276-285 0021-924X 120:2<276 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford