Electron spin resonance study of human transcortin: Thiol groups and binding site topography.
A series of cortisol analogs bearing a nitroxide free radical on C-17 side chains with a variation of distance between the steroid D-ring and the spin label from 7.4 to 17.6 A has been synthesized. These analogs were found to retain a good affinity for the specific corticosteroid binding site of purified human transcortin. The spin-labeled cortisol analogs were used to probe the human transcortin binding site structure by electron spin resonance (ESR) spectroscopy. A total depth of approx. 25 A was estimated for the binding site crevice. Use of sulfhydryl reagents (N-ethylmaleimide, p-chloromercuribenzoate) showed that a maximum of two sulfhydryl groups were titratable after reduction and denaturation of the protein. One of these thiol groups appeared to be involved in the cortisol binding site and could not be detected in the presence of bound steroid. ESR study of its environment, using spin-labeled N-ethylmaleimide reagents of various side-chain lengths, led to the conclusion that this thiol was at a depth of approx. 15 A or more in the binding site cavity. The second sulfhydryl group may be present in an oxidized form in the purified native transcortin, since it became titratable only after reductive treatment of the protein. ESR study showed that this thiol may be located in a crevice at approx. 15 A from the protein surface. These findings are compatible with a structural organization of the transcortin cortisol binding site, taking into account tentative models previously proposed by others.
D001665:Binding Sites; D003345:Corticosterone; D004228:Dithionitrobenzoic Acid; D004578:Electron Spin Resonance Spectroscopy; D005033:Ethylmaleimide; D006801:Humans; D006854:Hydrocortisone / Q000031:analogs & derivatives; D011485:Protein Binding; D011487:Protein Conformation; D013113:Spin Labels; D013438:Sulfhydryl Compounds / Q000032:analysis; D014156:Transcortin
D013113:Spin Labels; D013438:Sulfhydryl Compounds; D014156:Transcortin; D004228:Dithionitrobenzoic Acid; D005033:Ethylmaleimide; D003345:Corticosterone; D006854:Hydrocortisone