PMID:7829481
| Citation |
Horowitz, PM, Hua, S and Gibbons, DL (1995) Hydrophobic surfaces that are hidden in chaperonin Cpn60 can be exposed by formation of assembly-competent monomers or by ionic perturbation of the oligomer. J. Biol. Chem. 270:1535-42 |
|---|---|
| Abstract |
The oligomeric form (14-mer) of the chaperonin protein, Cpn60 (GroEL) from Eschericia coli, displays restricted hydrophobic surfaces and binds tightly one to two molecules of the fluorescent hydrophobic reporter, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bisANS). The 14-mer is resistant to proteolysis by chymotrypsin, and none of the three sulfhydryl groups/monomer react with 6-iodoacetamidofluorescein. When monomers of Cpn60 that are folded and competent to participate in protein folding are formed by low concentrations of urea (< 2.5 M), the hydrophobic exposure increases to accommodate approximately 14 molecules of bisANS/14-mer, the binding affinity for bisANS decreases, and 1 sulfhydryl group/monomer reacts with 6-iodoacetamidofluorescein. These monomers display limited proteolysis by chymotrypsin at several points within a hydrophobic sequence centered around residue 250 to produce a relatively stable N-terminal fragment (approximately = to 26 kDa) and a partially overlapping C-terminal fragment (approximately = to 44 kDa). The exposure of hydrophobic surfaces is facilitated by ATPMg. Ions increase hydrophobic exposure more effectively than urea without dissociation of Cpn60. For example, subdenaturing concentrations of guanidinium chloride (< or = 0.75 M) or the stabilizing salt, guanidinium sulfate, as well as NaCl or KCl are effective. The trivalent cation, spermidine, induces maximum exposure at 5 mM. The results suggest that hydrophobic surfaces can be involved in stabilizing the oligomer and/or in binding proteins to be folded, and they are consistent with suggestions that amphiphilic structures, presenting hydrophobic surfaces within a charged context, would be particularly effective in binding to Cpn60. |
| Links | |
| Keywords |
Amino Acid Sequence; Anilino Naphthalenesulfonates; Chaperonin 60/chemistry; Chaperonin 60/metabolism; Chymotrypsin; Escherichia coli; Fluorescent Dyes; Guanidine; Guanidines/pharmacology; Macromolecular Substances; Osmolar Concentration; Peptide Fragments/chemistry; Peptide Fragments/isolation & purification; Protein Binding; Protein Conformation; Protein Denaturation; Protein Folding; Spectrometry, Fluorescence; Surface Properties; Ultracentrifugation; Urea/pharmacology |
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