PubMed · 5005950
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J M Wrigglesworth, L Packer. 1970. pH-dependent changes in mitochondrial membrane structure.. https://doi.org/10.1007/bf01516087
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The urea denaturation of the chaperonin GroEL has been studied by circular dichroism, intrinsic tyrosine fluorescence and fluorescence of the hydrophobic probe, 1,1'-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.(ABSTRACT TRUNCATED AT 250 WORDS)
The fine-control of gene expression in the trp repressor system is achieved through the thermodynamic linkage of multiple equilibria involving the trp repressor protein (TR), tryptophan (L-Trp) and DNA. We have undertaken studies of superrepressor mutants of TR as a means of dissecting the coupled equilibria that contribute to repressor function. Unlike all the other tested super-repressors that exhibit differences from wild-type TR DNA binding affinity or stoichiometry, the AV77 superrepressor (an alanine to valine substitution at position 77: AV77TR) has been indistinguishable from TR in vitro. The present studies using a variety of biophysical measurements comparing TR and AV77TR provide strong evidence that the helix-turn-helix (HTH) region of apoTR exists in a partially folded conformation. Far UV CD spectra of the two proteins reveal a 10% increase in helical content for the apoAV77TR compared to apoTR. Moreover, urea denaturation studies demonstrate that apoAV77TR is more stable to denaturation than apoTR. ApoTR binds large amounts of 1,8-ANS, a hydrophobic fluorescence probe used to detect protein folding intermediates, with high affinity, where apoAV77TR exhibits only marginal binding of this ligand. While the tryptophan affinities of the two proteins as measured by titration calorimetry are quite similar, the thermodynamic signatures are distinct, with a much reduced unfavorable entropic contribution for AV77TR. Finally, the allosteric effect of L-Trp on oligomerization is abolished by the AV77 mutation. Taken together these data support previous calorimetric studies implicating coupling of folding and L-Trp binding for TR. Moreover, they are consistent with NMR observations indicating partial disorder in the HTH region of apoTR. Based upon the distinct biophysical properties of TR and AV77TR, we propose a model in which folding of the HTH region accompanies ligand binding in TR. In this model distinct protein-protein interactions of the apo- and holoTR link this conformational change to apparent operator affinities, thereby modulating TR function in vivo.
The fluorescent probe bis(8-anilino-1-naphthalenesulfonate) (bis-ANS) has been used to monitor the time- and temperature-dependent aging of tubulin, whereby new hydrophobic binding sites of lower affinity are generated on the protein [Prasad, A. R. S., et al. (1986a) Biochemistry 25, 739-742]. We carried out a detailed analysis of this phenomenon and found that, in addition to antimitotic drugs like colchicine or vinblastine, other parameters, viz., low temperature and protein stabilizers (e.g., glycerol and sucrose), inhibit the extent of enhanced binding of bis-ANS. Moreover, the generation of additional bis-ANS binding sites are also suppressed at high concentrations of tubulin. Cleavage of the carboxy-termini of tubulin (bound to bis-ANS) by subtilisin causes a significant reduction in the enhanced fluorescence, but has no effect on the high-affinity binding site of bis-ANS. All of these observations can be explained by the correlation of the presence of additional binding with the dissociation of heterodimeric tubulin into monomers. Enhanced binding of bis-ANS is due to tubulin dimers that have undergone dissociation, resulting in a loosening of its tertiary structure with the generation of a plethora of hydrophobic sites.