Studies On Interchain Isopeptide Cross-links Of Protein Disulfide Isomerase

Interchain isopeptide cross-link is a common kind of molecular cross-links in proteins. Researches about it are focus on transglutaminases and Ubiquitin-proteasome pathway. By using lysozyme, RNase A and protein disulfide isomerase (PDI) as model proteins, we present a hypothesis that protein cross-linking can be accomplished in three concerted steps: (1) a change in protein conformation; (2) formation of interchain disulfide bonds; and (3) formation of interchain isopeptide cross-links.Although we have got much evidence for conforming the mechanism, the details of isopeptide bond cross-linking are not well understood. In this dissertation, we have constructed the b' domain deletion PDI mutant and three point mutation PDI mutants. The b' domain deleted mutant completely failed to form dimers on the SDS-polyacrylamide gel, in contrast, dimeric bands were observed in purified preparations of wild-type PDI protein under the same conditions. The thermal unfolding of wild-type PDI protein shows a high propensity toward dimer formation, whereas the b' domain deleted mutant forms no isopeptide bond dimmer under these conditions as revealed by SDS-PAGE. The results suggest that there is at least one of pivotal amino acids which form the isopeptide bond in the deleted b' domain. To identify the amino acid residues involved in isopeptide bond in b' domain, three mutants (PDI Glu324Ser, PDI Glu331Gln/Lys333Val/Lys335Arg, PDI Lys291Gln/Lys292Ala) were made. Unlike wild-type PDI protein, the PDI mutant (Glu324Ser) failed to form isopeptide bond dimmers which revealed by the SDS- polyacrylamide gel after thermal unfolding. The results suggest that Glu324 is one of the pivotal amino acids that form the isopeptide bond.