Chaperonin-mediated folding of green fluorescent protein (GFP) was examined by real-time monitoring of recovery of fluorescence and by gel filtration high-performance liquid chromatography. Acid-denatured GFP can fold spontaneously upon dilution into the neutral buffer. When Escherichia coli GroEL/ES was present, folding of GFP was arrested. Folding was resumed by subsequent addition of 100 M or 1 mM ATP, and native GFP was regenerated to 100% yield. When folding was resumed by 10 M ATP (1.4 mol/mol GroEL subunit), about 60% of GFP recovered native structure, and onehalf of them (30%) was found to be still bound to GroEL/ ES, indicating the occurrence of folding in the central cavity of the GroEL ring underneath GroES (cis-folding). Because the overall rates of GroEL/ES-, ATP-mediated GFP folding were all similar to that of spontaneous folding, it was concluded that cis-folding proceeded as fast as spontaneous folding. The GroEL/ES-bound native GFP was observed only when both GroES and ATP (but not ADP) were present in the folding mixture. Holochaperonin from Thermus thermophilus, which was purified as a cpn60/10 complex, exhibited the similar cisfolding. Consistently, ATP-dependent exchange of cpn10 in the holo-chaperonin with free cpn10 was observed.
GroEL encapsulates non-native protein in a folding cage underneath GroES (cis-cavity). Here we report the maximum size of the non-native protein to stay and fold in the cis-cavity. Using total soluble proteins of Escherichia coli in denatured state as binding substrates and protease resistance as the measure of polypeptide held in the cis-cavity, it was estimated that the cis-cavity can accommodate up to ϳ57-kDa non-native proteins. To know if a protein with nearly the maximum size can complete folding in the cis-cavity, we made a 54-kDa protein in which green fluorescent protein (GFP) and its blue fluorescent variant were fused tandem. This fusion protein was captured in the cis-cavity, and folding occurred there. Fluorescence resonance energy transfer proved that both GFP and blue fluorescent protein moieties of the same fused protein were able to fold into native structures in the cis-cavity. Consistently, simulated packing of crystal structures shows that two native GFPs just fit in the cis-cavity. A fusion protein of three GFPs (82 kDa) was also attempted, but, as expected, it was not captured in the cis-cavity.
It has been believed that when GroEL binds to GroES its apical domain moves upward and outward. To inhibit this "opening" movement, its equatorial and apical domains were cross-linked through a disulfide bond between mutationally introduced cysteine residues at the positions of Asp-83 and Lys-327. To avoid possible undesired cross-linking, we at first prepared a mutant GroEL (GroEL NC ; Cys-138 3 Ser, Cys-458 3 Ser, Cys-519 3 Ser) in which all cysteine residues in wild-type GroEL were replaced by serine residues. GroEL NC was fully functional as a chaperonin. We then introduced the above two point mutations into GroEL NC to generate a mutant (GroEL AEX ; Cys-138 3 Ser, Cys-458 3 Ser, Cys-519 3 Ser and Asp-83 3 Cys, Lys-327 3 Cys). Oxidized GroEL AEX , which is locked in a "closed" conformation by an interdomain disulfide bond, can bind 6 -7 mol of ATP, which remain bound without hydrolysis. This ATPbound, oxidized GroEL AEX can bind the stably nonnative substrate protein isopropylmalate dehydrogenase, whereas the nucleotide-free oxidized GroEL AEX binds it with a weaker affinity. However, oxidized GroEL AEX fails to process further reaction steps such as ATP hydrolysis, binding of GroES, dissociation of substrate protein from GroEL, and facilitating protein folding. When disulfide bonds in oxidized GroEL AEX are reduced, Gro-EL AEX exerts the ability to process all the reactions just as GroEL NC and wild-type GroEL. Indications from these results are: hydrolysis of ATP may require opening movement of the apical domain; GroES binds to an open form of GroEL; and substrate polypeptide is released from GroEL coupled with either ATP hydrolysis or opening movement of the apical domain.
To find microorganisms that could reduce phenyl trifluoromethyl ketone (PTK) to (S)-1-phenyltrifluoroethanol [(S)-PTE], styrene-assimilating bacteria (ca. 900 strains) isolated from soil samples were screened. We found that Leifsonia sp. strain S749 was the most suitable strain for the conversion of PTK to (S)-PTE in the presence of 2-propanol as a hydrogen donor. The enzyme corresponding to the reaction was purified homogeneity, characterized and designated Leifsonia alcohol dehydrogenase (LSADH). The purified enzyme had a molecular weight of 110,000 and was composed of four identical subunits (molecular weight, 26,000). LSADH required NADH as a cofactor, showed little activity with NADPH, and reduced a wide variety of aldehydes and ketones. LSADH catalyzed the enantioselective reduction of some ketones with high enantiomeric excesses (e.e.): PTK to (S)-PTE (>99% e.e.), acetophenone to (R)-1-phenylethanol (99% e.e.), and 2-heptanone to (R)-2-heptanol (>99% e.e.) in the presence of 2-propanol without an additional NADH regeneration system. Therefore, it would be a useful biocatalyst.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.