Two partially unfolded states of torpedo californica acetylcholinesterase

摘要: Chemical modification with sulfhydryl reagents of the single, nonconserved cysteine residue Cys231 in each subunit a disulfide-linked dimer Torpedo californica acetylcholinesterase produces partially unfolded inactive state. Another state can be obtained by exposure enzyme to 1-2 M guanidine hydrochloride. Both these states display several important features molten globule, but differ their spectroscopic (CD, intrinsic fluorescence) and hydrodynamic (Stokes radii) characteristics. With reversal chemical former or removal denaturant from latter, both retain physiochemical Thus, exist two globule states, which are long-lived under physiologic conditions without aggregating, either intraconverting reverting native undergo spontaneous intramolecular thioldisulfide exchange, implying that they flexible. As revealed differential scanning calorimetry, produced lacks any heat capacity peak, presumably due aggregation during scanning, whereas hydrochloride unfolds as single cooperative unit, thermal transition being completely reversible. Sucrose gradient centrifugation reveals reduction interchain disulfide converts it monomers, whereas, after such reduction, subunits remain associated generated hydrochloride, modification. It is suggested novel hydrophobic core, across interfaces, responsible for this noncovalent association. Transition observed only presence denaturant, yielding, on extrapolation zero high free energy barrier (ca. 23.8 kcal/mol) separating flexible, states.