CHARACTERIZATION OF THE THIOL DISULFIDE CHEMISTRY OF PEPTIDES CORRESPONDING TO THE 603-609-DISULFIDE LOOP OF THE HUMAN-IMMUNODEFICIENCY-VIRUS (HIV) ENVELOPE GLYCOPROTEIN GP41/

The redox chemistry of two synthetic model peptides for the 603-609 di sulfide loop found in envelope glycoprotein gp41 of the human immunode ficiency virus type 1 (HIV-1) are reported. The two peptides: Ac-Trp-G ly-Cys-Ser-Gly-Lys-Leu-Ile-Cys-Thr-Thr-NH2 (I) and Ac-Trp-Gly-Cys-Ser- Gly-Arg-His-Ile-Cys-Thr-Thr-NH2 (II) were synthesized by the solid pha se method. Peptide I corresponds to amino acids 601-611 of gp41 of the North American/European strain of HIV-1. Peptide II incorporates amin o acid replacements frequent in African HIV-1 isolates. The redox chem istry of the disulfide bonds in the two peptides was characterized in aqueous and aqueous/urea solution by studying their thiol-disulfide ex change reactions with the tripeptide glutathione (GSH). GSH reacts wit h the disulfide bonds to form mixed disulfides, which in turn react wi th another molecule of GSH to give the dithiol form of the peptide and GSSG. Equilibrium constants were determined for each step and for the overall reduction reactions. Redox potentials of -0.246V and -0.241V were calculated from the equilibrium constants for the disulfide bonds in peptides I and II in aqueous solution at 25 degrees C and pH 7.0. The overall equilibrium constants are less in 8 M urea solution, which indicates a stabilization of the reduced, dithiol form of both peptid es by secondary structure which can be denatured by urea. This conclus ion is supported by nuclear Overhauser enhancement data obtained from 2D-ROESY NMR spectra which provide evidence of elements of secondary s tructure for the reduced forms of both peptides. The results are discu ssed in terms of a protein disulfide isomerase catalyzed reduction of the disulfide bond in gp41. (C) Munksgaard 1998.