Predissociated dimers and molten globule monomers in the equilibrium unfolding of yeast glutathione reductase |
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| Authors: | Louzada Paulo Roberto Sebollela Adriano Scaramello Marcelo E Ferreira Sérgio T |
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| Affiliation: | Paulo Roberto Louzada, Adriano Sebollela, Marcelo E. Scaramello, and Sérgio T. Ferreira |
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| Abstract: | The equilibrium unfolding of dimeric yeast glutathione reductase (GR) by guanidine hydrochloride (GdnHCl) was investigated. Unfolding was monitored by a variety of techniques, including intrinsic fluorescence emission, anisotropy and iodide quenching measurements, far-ultraviolet circular dichroism and thiol reactivity measurements. At 1 M GdnHCl, one thiol group of GR became accessible to modification with 5,5′-dithiobis-(2-nitrobenzoic) acid (DTNB), whereas no changes could be detected in the spectroscopic properties (fluorescence, circular dichroism) of the protein. Between 2 and 3 M GdnHCl, two partially folded intermediate states possessing flexible tertiary structures (revealed by fluorescence data) but compact secondary structures (as indicated by circular dichroism measurements) were identified. The quaternary structure of GR in the presence of GdnHCl was also investigated by size-exclusion liquid chromatography. These results indicated the presence of an expanded predissociated dimer at 2.5 M GdnHCl and partially folded monomers at 3 M GdnHCl. Taken together, these results suggest the existence of two molten-globule-like intermediate species (one dimeric and one monomeric) in the unfolding of GR. The results are discussed in terms of the mechanism of GR folding and dimerization. |
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| Keywords: | CD, circular dichroism DTNB, 5,5′-dithiobis-(2-nitrobenzoic) acid EDTA, ethylenediaminetetracetic acid GdnHCl, guanidine hydrochloride GR, yeast glutathione reductase GSH, reduced glutathione GSSG, oxidized glutathione HEPES, n-(2-hydroxyethyl) piperazine-n′-(2-ethanesulfonic acid) HPLC, high performance liquid chromatography MG, molten globule |
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