FIG. 3.

Zinc release and chaperone activation are associated with structural changes in the zinc-finger fold. (A-D) The zinc-finger folds of PKCα C1b loose their structure upon exposure to phorbol ester. (A) The HSQC spectrum of [U-¹⁵N] PKCαC1b (250 μM) shows a well-structured fold comparable to that of published spectra [14]. (B) After stimulation with PMA (protein:PMA ratio = 1:2.6), some precipitation occurred. However, after removal of aggregates the spectrum of the supernatant showed a disordered structure. (C) Treatment with P13A (1:2.6 molar ratio) caused only minor shifts. (D) Treatment with vehicle (dimethyl sulfoxide) caused no discernible shifts. (E) The chimeric Hsp33/ɛC1b protein was uniformly labeled with [¹⁵N]L-cysteine. The HSQC spectrum of the untreated protein (100 μM) revealed signals from six cysteine residues, and additional lower intensity signals arising from scrambling of the ¹⁵N label. (F) PMA treatment (protein:PMA, 1:1.3) resulted in some precipitation, as in (B). The HSQC spectrum of the supernatant (∼80 μM) showed broadening of signals suggesting loss of structure. (G) The weak phorbol agonist, P13A, while eliciting shifts of some cysteine residues (shown at 1:2.6 molar ratio), possibly due to secondary rearrangement in Hsp33, retained the six core cysteines in the fixed positions, indicating a structurally intact zinc finger.