REDOX-ACTIVE BIS-CYSTEINYL PEPTIDES .2. COMPARATIVE-STUDY ON THE SEQUENCE-DEPENDENT TENDENCY FOR DISULFIDE LOOP FORMATION

Bis(cysteinyl) octapeptides related to the active sites of the oxidore ductases protein disulfide isomerase (PDI), thioredoxin reductase (trr ), glutaredoxin (grx), and thioredoxin (trx) were analyzed for their p ropensity to form the intramolecular 14-membered disulfide ring in oxi dation experiments. The rank order of percentage of cyclic monomer for med in aqueous buffer (pH 7.0) at 10(-3) M concentration was found to be very similar, but opposite to that of the K-ox and, correspondingly , of the redox potentials of the native enzymes. Attempts to induce in trinsic conformational preferences of the peptides by addition of trif luoroethanol led to enhancements of beta-turn structures as reflected by the CD and Fourier transform ir spectre. The induced secondary stru cture, instead of aligning the tendencies of the excised fragments for loop formation with those of the intact proteins, was found to suppre ss the differences by significantly increasing the preference for cycl ic monomers (approximate to 90%). Similarly, operating under denaturin g conditions, i.e., in 6M guanidinium hydrochloride, only for the trx peptide was the statistical product distribution obtained. For the rem aining peptides, again a strong increase of cyclic monomer contents wa s observed that could not be correlated with dissolution of beta-sheet type aggregates. The CD spectra are more consistent with the presence of ordered structure to some extent, possibly resulting from an hydro phobic collapse of the sparingly soluble peptides. The results of the oxidation experiments further support previous findings from thiol dis ulfide interchange equilibria, which clearly revealed a decisive role of the characteristic thioredoxin structural motif in dictating the re dox properties of the enzymes. Point mutations in the active sites of the oxidoreductases allowed us to affect their redox potentials strong ly, but apparently only in the constraint form of the three-dimensiona l structure as similar exchanges in the excised fragments did not prod uce the expected effect. This observation contrasts with numerous repo rts that the conformation of short disulfide loops is mainly dictated by the amino acid sequence. (C) 1994 John Wiley and Sons, Inc.