Redox-active bis-cysteinyl peptides. II. Comparative study on the sequence-dependent tendency for disulfide loop formation.
Bis(cysteinyl)octapeptides related to the active sites of the oxidoreductases protein disulfide isomerase (PDI), thioredoxin reductase (trr), glutaredoxin (grx), and thioredoxin (trx) were analyzed for their propensity to form the intramolecular 14-membered disulfide ring in oxidation experiments. The rank order of percentage of cyclic monomer formed in aqueous buffer (pH 7.0) at 10(-3) M concentration was found to be very similar, but opposite to that of the Kox and, correspondingly, of the redox potentials of the native enzymes. Attempts to induce intrinsic conformational preferences of the peptides by addition of trifluoroethanol led to enhancements of beta-turn structures as reflected by the CD and Fourier transform ir spectra. The induced secondary structure, instead of aligning the tendencies of the excised fragments for loop formation with those of the intact proteins, was found to suppress the differences by significantly increasing the preference for cyclic monomers (approximately 90%). Similarly, operating under denaturing conditions, i.e., in 6 M guanidinium hydrochloride, only for the trx peptide was the statistical product distribution obtained. For the remaining peptides, again a strong increase of cyclic monomer contents was 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 hydrophobic collapse of the sparingly soluble peptides. The results of the oxidation experiments further support previous findings from thiol disulfide interchange equilibria, which clearly revealed a decisive role of the characteristic thioredoxin structural motif in dictating the redox properties of the enzymes. Point mutations in the active sites of the oxidoreductases allowed us to affect their redox potentials strongly, but apparently only in the constraint form of the three-dimensional structure as similar exchanges in the excised fragments did not produce the expected effect. This observation contrasts with numerous reports that the conformation of short disulfide loops is mainly dictated by the amino acid sequence.
Siedler
F
F
Max-Planck Institute of Biochemistry, Martinsried, Germany.
Quarzago
D
D
Rudolph-Böhner
S
S
Moroder
L
L
D000595:Amino Acid Sequence; D003545:Cysteine; D004220:Disulfides / Q000737:chemistry*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry*; D010084:Oxidation-Reduction; D010456:Peptides, Cyclic / Q000737:chemistry*
D004220:Disulfides; D009842:Oligopeptides; D010456:Peptides, Cyclic; D003545:Cysteine