CYS32 PKA MEASUREMENTS OF MUTANTS THIOREDOXINS W/ ALTERED REDOX POTENTIALS

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E. coli thioredoxin (Trx) is a general protein disulfide reductant. The Trx active site, at the N-terminus of an ?-helix, consists of Cys-Gly-Pro-Cys. The enzyme converts between dithiol and disulfide redox states. The first thiol (Cys32) has a depressed pKa relative to a typical protein thiol. This pKa can be measured by observing the titration of the C?H and C?H of Cys32 using a 2QF-COSY experiment. We have generated Trx mutants that have perturbed active site disulfide reduction potentials. pH titration of reduced mutant thioredoxins monitored by fluorescence suggest altered pKa's for Cys32. Two-dimensional 1H-NMR (2QF-CSY) of these mutants will allow accurate determination of Cys32 pKa. Physical chemistry suggests that a lower pKa will correlate with a higher (more positive) reduction potential due to stabilization of the dithiol form of Trx.