Exposure of hydrophobic surfaces on the chaperonin GroEL oligomer by protonation or modification of His-401.
Hydrophobic exposure on the chaperonin GroEL is increased 6-10-fold after the protein is treated with the His-reactive reagent diethyl pyrocarbonate (DEP), or the solution pH is lowered to 5.5. The induced hydrophobic surfaces have the same 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) binding characteristics as unperturbed GroEL: a Kd approximately equal to 3.5 microM, a maximum intensity at approximately 500 nm, and an average fluorescence lifetime of approximately 8.0 ns. The pKa for the pH-induced transition is 6.6, most likely attributable to the only histidine in GroEL, His-401, located in the intermediate domain. The modification of one histidine residue per monomer upon DEP treatment is supported by the correlation between the change in the absorbance at 242 nm for the N-carbethoxyhistidyl derivative and the increase in bis-ANS fluorescence. GroEL at pH 5.5 is tetradecameric and can capture urea-denatured rhodanese and release it as active enzyme. The GroEL-rhodanese and release it as active enzyme. The GroEL-rhodanese complex is more stable to dissociation by 2.25 M urea than the complex formed at pH 7.8. We propose that His-401 is in a conformationally sensitive region such that protonation or modification can lead to increased exposure of hydrophobic surfaces capable of binding folding intermediates.
Gibbons
D L
DL
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Horowitz
P M
PM
D000817:Anilino Naphthalenesulfonates; D018834:Chaperonin 60 / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D005456:Fluorescent Dyes; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D014508:Urea
D000817:Anilino Naphthalenesulfonates; D018834:Chaperonin 60; D005456:Fluorescent Dyes; D006639:Histidine; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D014508:Urea; D013884:Thiosulfate Sulfurtransferase; D004047:Diethyl Pyrocarbonate