title: Isolation and characterization of rhodanese intermediates during thermal inactivation and their implications for the mechanism of protein aggregation.
abstract: The initial steps of heat-induced inactivation and aggregation of the enzyme rhodanese have been studied and found to involve the early formation of modified but catalytically active conformations. These intermediates readily form active dimers or small oligomers, as evident from there being only a small increase in light scattering and an increase in fluorescence energy homotransfer from rhodanese labeled with fluorescein. These species are probably not the domain-unfolded form, as they show activity and increased protection of hydrophobic surfaces. Cross-linking with glutaraldehyde and fractionation by gel filtration show the predominant formation of dimer during heat incubation. Comparison between the rates of aggregate formation at 50 degrees C after preincubation at 25 or 40 degrees C gives evidence of product-precursor relationships, and it shows that these dimeric or small oligomeric species are the basis of the irreversible aggregation. The thermally induced species is recognized by and binds to the chaperonin GroEL. The unfoldase activity of GroEL subsequently unfolds rhodanese to produce an inactive conformation and forms a stable, reactivable complex. The release of 80% active rhodanese upon addition of GroES and ATP indicates that the thermal incubation induces an alteration in conformation, rather than any covalent modification, which would lead to formation of irreversibly inactive species. Once oligomeric species are formed from the intermediates, GroEL cannot recognize them. Based on these observations, a model is proposed for rhodanese aggregation that can explain the paradoxical effect in which rhodanese aggregation is reduced at higher protein concentration.
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78229-3900, USA.
authors.forename: Anusri Mitra
authors.initials: AM
authors.lastname: Bhattacharyya
authors.forename: Paul M
authors.initials: PM
authors.lastname: Horowitz
mesh_terms: D000818:Animals; D002417:Cattle; D018835:Chaperonin 10 / Q000378:metabolism; D018834:Chaperonin 60 / Q000378:metabolism; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D005454:Fluorescence Polarization; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D011485:Protein Binding; D012542:Scattering, Radiation; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D018835:Chaperonin 10; D018834:Chaperonin 60; D003432:Cross-Linking Reagents; D013884:Thiosulfate Sulfurtransferase