TY - JOUR
T1 - Hydrophobic surfaces that are hidden in chaperonin Cpn60 can be exposed by formation of assembly-competent monomers or by ionic perturbation of the oligomer
AU - Horowitz, Paul M.
AU - Hua, Su
AU - Gibbons, Don L.
PY - 1995/1/27
Y1 - 1995/1/27
N2 - The oligomeric form (14-mer) of the chaperonin protein, Cpn60 (GroEL) from Escherichia 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 (biaANS). 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 (≃26 kDa) and a partially overlapping C-terminal fragment (≃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 (≤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.
AB - The oligomeric form (14-mer) of the chaperonin protein, Cpn60 (GroEL) from Escherichia 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 (biaANS). 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 (≃26 kDa) and a partially overlapping C-terminal fragment (≃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 (≤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.
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U2 - 10.1074/jbc.270.4.1535
DO - 10.1074/jbc.270.4.1535
M3 - Article
C2 - 7829481
AN - SCOPUS:0028899753
SN - 0021-9258
VL - 270
SP - 1535
EP - 1542
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 4
ER -