J. Opoku et Ss. Simons, ABSENCE OF INTRAMOLECULAR DISULFIDES IN THE STRUCTURE AND FUNCTION OFNATIVE RAT GLUCOCORTICOID RECEPTORS, The Journal of biological chemistry, 269(1), 1994, pp. 503-510
The presence of intramolecular disulfides in different functional stat
es of the native glucocorticoid receptor in the absence of added oxida
nts has been examined on nonreducing SDS-polyacrylamide gels. Possible
disulfides were trapped by the reaction of thiols in crude receptor s
olutions with methyl methanethiolsulfonate or iodoacetamide. The prese
nce of diffuse bands at lower molecular weights than either the fully
reduced or the thiol-blocked species for both the intact 98-kDa recept
or and the 42-kDa chymotryptic fragment was diagnostic of an intramole
cular disulfide(s) that had undergone thiol-disulfide rearrangements.
However, both the rearrangements and the formation of intramolecular d
isulfides were found to occur only with denatured receptors during gel
analysis. It appears that the thiols normally complexed with zinc in
the zinc fingers may be recruited for disulfide bond formation. Finall
y, even when a documented intramolecular disulfide was formed in solut
ions of the native protein, thiol-disulfide rearrangements did not occ
ur. The tertiary structure of the receptor is thus constituted in a ma
nner that not only limits the formation of disulfides but also prevent
s the usually facile rearrangements of disulfide bond-containing struc
tures to receptor forms that may have greatly reduced activity. Theref
ore, although intramolecular disulfide bonds may be of transitory impo
rtance, the structural or functional changes of native glucocorticoid
receptors that are associated with steroid binding, activation, and di
ssociation of heat shock protein 90 neither involve nor require the fo
rmation or reduction of stable intramolecular disulfides.