RESIDUAL STRUCTURE IN UREA-DENATURED CHAPERONIN GROEL

The urea denaturation of the chaperonin GroEL has been studied by circ ular dichroism, intrinsic tyrosine fluorescence and fluorescence of th e hydrophobic probe, 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic ac id (bisANS). It is shown that GroEL denaturation, monitored by CD and intrinsic fluorescence measurements, can be well described by a two-st ate transition that is complete by 3-3.1 M urea. The beginning of this transition overlaps the urea concentrations where the oligomeric prot ein starts to dissociate into individual monomers. Subsequent addition of the denaturant leads to complete unfolding of the monomers. Monome rs 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 ur ea. In contrast to the CD and intrinsic fluorescence measurements, bis ANS bound to GroEL exhibits considerable fluorescence intensity under conditions where the CD and intrinsic fluorescence signals have alread y reached their minimum values (>3.1 M urea). This binding of bisANS, under conditions where the majority of the secondary structure of GroE L has already unfolded, indicates the existence of hydrophobic residua l structure. This structure cannot be detected by CD measurements, but it can be unfolded by raising further the urea concentration. The exi stence of this structure does not depend on the source or method of th e protein preparation, Intrinsic fluorescence and trypsin digestion de monstrate no difference between the bisANS-bound form of GroEL and the free form of the protein, showing that the GroEL structure is not gre atly affected by the interaction with bisANS. Analysis of the chymotry ptic fragments of GroEL, photolabeled with bisANS, suggests that photo incorporation of the probe at 3.1 M urea occurs within amino acid resi dues 203-249 in the apical domain, suggesting this portion of GroEL is the region that contains the residual structure. This residual struct ure may be important as a nucleation site for folding and/or an intera ctive region that can lead to misfolding under some conditions. The ex istence and location of this residual structure may facilitate the reg istration of the regions of the primary sequence that interact to achi eve the interesting fold of GroEL, and they may help understanding of reports that preformed chaperonins can assist refolding/reassembly of the fully unfolded chaperonin.