title: The binding of bis-ANS to the isolated GroEL apical domain fragment induces the formation of a folding intermediate with increased hydrophobic surface not observed in tetradecameric GroEL.
abstract: The extent of hydrophobic exposure upon bis-ANS binding to the functional apical domain fragment of GroEL, or minichaperone (residues 191-345), was investigated and compared with that of the GroEL tetradecamer. Although a total of seven molecules of bis-ANS bind cooperatively to this minichaperone, most of the hydrophobic sites were induced following initial binding of one to two molecules of probe. From the equilibrium and kinetics studies at low bis-ANS concentrations, it is evident that the native apical domain is converted to an intermediate conformation with increased hydrophobic surfaces. This intermediate binds additional bis-ANS molecules. Tyrosine fluorescence detected denaturation demonstrated that bis-ANS can destabilize the apical domain. The results from (i) bis-ANS titrations, (ii) urea denaturation studies in the presence and absence of bis-ANS, and (iii) intrinsic tyrosine fluorescence studies of the apical domain are consistent with a model in which bis-ANS binds tightly to the intermediate state, relatively weakly to the native state, and little to the denatured state. The results suggest that the conformational changes seen in apical domain fragments are not seen in the intact GroEL oligomer due to restrictions imposed by connections of the apical domain to the intermediate domain and suppression of movement due to quaternary structure.
authors: Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900, USA.
authors: A L
authors: AL
authors: Smoot
authors: M
authors: M
authors: Panda
authors: B T
authors: BT
authors: Brazil
authors: A M
authors: AM
authors: Buckle
authors: A R
authors: AR
authors: Fersht
authors: P M
authors: PM
authors: Horowitz
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D001665:Binding Sites; D018834:Chaperonin 60 / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013499:Surface Properties; D014443:Tyrosine / Q000737:chemistry; D014461:Ultracentrifugation; D014508:Urea / Q000737:chemistry
chemical_list: D000817:Anilino Naphthalenesulfonates; D018834:Chaperonin 60; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D014443:Tyrosine; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D014508:Urea