Abstract
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.
Highlights
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
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
It has been shown that the range of possible substrates for this chaperonin is rather broad, with GroEL capable of binding about half of the soluble proteins from E. coli cell lysate with relatively high affinity [11]
Summary
It has been shown that the range of possible substrates for this chaperonin is rather broad, with GroEL capable of binding about half of the soluble proteins from E. coli cell lysate with relatively high affinity [11]. Such promiscuity brings into question the specific recognition motif, which appears to involve in some cases, but is not necessarily restricted to, an amphipathic helix that may be stabilized or induced by binding GroEL [12,13,14,15,16]. Some proteins require only K+ and MgATP for release of functional enzyme from the binary complex, whereas others require the co-chaperonin GroES [4,5,6, 19]
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