Redox‐active bis‐cysteinyl peptides. II. Comparative study on the sequence‐dependent tendency for disulfide loop formation

Abstract

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.

A 16-Residue Peptide Fragment of Macrophage Migration Inhibitory Factor, MIF-(50–65), Exhibits Redox Activity and Has MIF-like Biological Functions

“…Although NMR studies will ultimately be necessary to obtain detailed information about the conformation stabilized in MIF-(50 -65), our CD data obtained for reduced and oxidized MIF-(50 -65), the bis-serine mutant, and the side chain-to-side chain lactambridged analog together indicate that MIF-(50 -65) contains a ␤-turn-stabilized conformation, likely a ␤-strand-␤-turn-␤-strand conformation. Of note, significant ␤-turn stabilization and conformational ordering via disulfide bridge formation and synthetic constraints has been described previously also for hexa-and octapeptides containing the CXXC active sites of the TPORs PDI, Grx, Trx, and Trr (35)(36)(37).…”

“…Other than MIF-(56 -65), which covered the ␤4-strand only, MIF-(50 -65) was chosen to include additional residues that could serve to stabilize secondary structure interactions. In fact, it has been proposed for the TPORs that formation of a stable ␤-turn structure in the CXXC region is associated with oxidoreductase activity (37). Therefore, MIF-(56 -65) was N-terminally extended up to residue Phe-50 to enable formation of additional intramolecular CXXC structure-stabilizing interactions.…”

Photomodulation of conformational states. III. Water‐soluble bis‐cysteinyl‐peptides with (4‐aminomethyl) phenylazobenzoic acid as backbone constituent*

“…34,35 To further analyze the effect of the different sequence composition of the Cys-Xaa-YaaCys motif on the redox properties, we have previously synthesized related fragments and analyzed their redox potentials. 36,37 Although the bis-cysteinyl-peptides showed different redox potentials and the rank order was found to be similar to that of the enzymes, their values were all close to Ϫ200 mV. This strongly suggested that the intrinsic free energy of formation of the 14-membered disulfide-bridged ring is strongly affected/amplified by the conformational restraints imparted by the protein structure.…”

Maurotoxin Versus Pi1/HsTx1 Scorpion Toxins

“…3), and second, most protons were identified and the resonance frequencies determined for both analogues. The structures of the analogues were determined using (i) 348 NOE-based distance restraints (151 intra-residue, 114 sequential, 29 ]MTX) constraints per residue. The structures were calculated using a hybrid distance geometry simulated annealing protocol (the distance geometry algorithm for NMR (20) (DIANA) and crystallography and NMR system (21) (CNS)).…”