Protein-S-S-glutathione mixed disulfides as models of unfolded proteins.
Mixed disulfides between glutathione and the reduced forms of disulfide-bonded proteins were generated and characterized to explore their suitability as models of the unfolded state of newly-synthesized secretory proteins. RNase T1 and alpha-lactalbumin were reduced and converted to mixed disulfide derivatives, named GS-RNase T1 and GS-alpha-lactalbumin, in good yield; the molecular masses of the derivatives were confirmed by electrospray mass spectrometry. The intrinsic fluorescence of the derivatives and the binding of the hydrophobic fluorescent dye ANS were characteristic of fully unfolded proteins. Fluorescence studies and enzyme activity data indicated that GS-RNase T1 could be refolded to a nativelike state at NaCl concentrations greater than 1.5 M, as was previously demonstrated for the reduced, carboxymethylated derivative of this protein. The [NaCl]-dependent folding/unfolding equilibrium for GS-RNase T1 was reversible and could be influenced by urea. Fluorescence studies indicated that GS-alpha-lactalbumin showed a [NaCl]-dependent partial shift toward a more nativelike state, which was enhanced by the presence of Ca2+ ions. Both of the GS derivatives stimulated the ATPase activity of BiP, with apparent affinities in the range 0.1-1.0 mM. The results indicate that these GS-S-protein mixed disulfide derivatives are ideal model unfolded proteins that can be used as substrates for detailed studies on secretory protein folding in vitro and on the interactions between unfolded proteins and facilitators of protein folding.
Ruoppolo
M
M
Research School of Biosciences, University of Kent, Canterbury, U.K.
Freedman
R B
RB
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D000091342:Endoplasmic Reticulum Chaperone BiP; D005456:Fluorescent Dyes; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006360:Heat-Shock Proteins; D007768:Lactalbumin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D013058:Mass Spectrometry; D008958:Models, Molecular; D018832:Molecular Chaperones; D008970:Molecular Weight; D011489:Protein Denaturation; D017510:Protein Folding; D006163:Ribonuclease T1 / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D002352:Carrier Proteins; D004220:Disulfides; D000091342:Endoplasmic Reticulum Chaperone BiP; D005456:Fluorescent Dyes; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D012965:Sodium Chloride; C027132:1-anilino-8-naphthalenesulfonate; D000255:Adenosine Triphosphate; D007768:Lactalbumin; D004492:Edetic Acid; D006163:Ribonuclease T1; D000251:Adenosine Triphosphatases; D005978:Glutathione; D002122:Calcium Chloride