2 PARTIALLY UNFOLDED STATES OF TORPEDO-CALIFORNICA ACETYLCHOLINESTERASE

Chemical modification with sulfhydryl reagents of the single, nonconse rved cysteine residue Cys(231) in each subunit of a disulfide-linked d imer of Torpedo californica acetylcholinesterase produces a partially unfolded inactive state. Another partially unfolded state can be obtai ned by exposure of the enzyme to 1-2 M guanidine hydrochloride. Both t hese states display several important features of a molten globule, bu t differ in their spectroscopic (CD, intrinsic fluorescence) and hydro dynamic (Stokes radii) characteristics. With reversal of chemical modi fication of the former state or removal of denaturant from the latter, both states retain their physicochemical characteristics. Thus, acety lcholinesterase can exist in two molten globule states, both of which are long-lived under physiologic conditions without aggregating, and w ithout either intraconverting or reverting to the native state. Both s tates undergo spontaneous intramolecular thiol-disulfide exchange, imp lying that they are flexible. As revealed by differential scanning cal orimetry, the state produced by chemical modification lacks any heat c apacity peak, presumably due to aggregation during scanning, whereas t he state produced by guanidine hydrochloride unfolds as a single coope rative unit, thermal transition being completely reversible. Sucrose g radient centrifugation reveals that reduction of the interchain disulf ide of the native acetylcholinesterase dimer converts it to monomers, whereas, after such reduction, the two subunits remain completely asso ciated in the partially unfolded state generated by guanidine hydrochl oride, and partially associated in that produced by chemical modificat ion. It is suggested that a novel hydrophobic core, generated across t he subunit interfaces, is responsible for this noncovalent association . Transition from the unfolded state generated by chemical modificatio n to that produced by guanidine hydrochloride is observed only in the presence of the denaturant, yielding, on extrapolation to zero guanidi ne hydrochloride, a high free energy barrier (ca. 23.8 kcal/mol) separ ating these two flexible, partially unfolded states.