Residual Structure in Urea-Denatured Chaperonin GroEL

Boris M. Gorovits, Jeffrey W. Seale, Paul M. Horowitz

Research output: Contribution to journalArticlepeer-review

29 Scopus citations


The urea denaturation of the chaperonin GroEL has been studied by circular dichroism, intrinsic tyrosine fluorescence and fluorescence of the hydrophobic probe, l,l,-bis(4-anilino)naphthalene-5,5'- disulfonic acid (bisANS). It is shown that GroEL denaturation, monitored by CD and intrinsic fluorescence measurements, can be well described by a two-state transition that is complete by 3-3.1 M urea. The beginning of this transition overlaps the urea concentrations where the oligomeric protein starts to dissociate into individual monomers. Subsequent addition of the denaturant leads to complete unfolding of the monomers. Monomers unfolded at urea concentrations higher than 3.1 M are not competent to form their native conformations under the conditions employed here, and they are not able to reassemble to oligomers upon dilution of urea. In contrast to the CD and intrinsic fluorescence measurements, bisANS bound to GroEL exhibits considerable fluorescence intensity under conditions where the CD and intrinsic fluorescence signals have already reached their minimum values (>3.1 M urea). This binding of bisANS, under conditions where the majority of the secondary structure of GroEL has already unfolded, indicates the existence of hydrophobic residual structure. This structure cannot be detected by CD measurements, but it can be unfolded by raising further the urea concentration. The existence of this structure does not depend on the source or method of the protein preparation. Intrinsic fluorescence and trypsin digestion demonstrate no difference between the bisANS-bound form of GroEL and the free form of the protein, showing that the GroEL structure is not greatly affected by the interaction with bisANS. Analysis of the chymotryptic fragments of GroEL, photolabeled with bisANS, suggests that photoincorporation of the probe at 3.1 M urea occurs within amino acid residues 203-249 in the apical domain, suggesting this portion of GroEL is the region that contains the residual structure. This residual structure may be important as a nucleation site for folding and/or an interactive region that can lead to misfolding under some conditions. The existence and location of this residual structure may facilitate the registration of the regions of the primary sequence that interact to achieve the interesting fold of GroEL, and they may help understanding of reports that preformed chaperonins can assist refolding/reassembly of the fully unfolded chaperonin.

Original languageEnglish (US)
Pages (from-to)13928-13933
Number of pages6
Issue number42
StatePublished - Oct 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry


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