Correlation Between Interchain Cross-Linking And Enzyme Activity Of Protein Disulfide Isomerase

In vitro, protein cross-linking can be accomplished in three concerted steps: (i) a change in protein conformation; (ii) formation of interchain disulfide bonds; and (iii) formation of interchain isopeptide cross-links. To further substantiate the hypothesis and study the correlation between interchain cross-linking and enzyme activity of protein disulfide isomerase, two point mutants on PDI's activity site Cys36/38 1 Ser were generated. In purified preparations of wild-type PDI protein, dimeric bands in addition to the major 55-kDa bands were observed on the SDS-polyacrylamide gel. In contrast, the Cys36/38 1 Ser mutant completely failed to form dimers under the same conditions. The thermal unfolding of wild-type PDI protein shows a high propensity toward dimer formation, whereas the Cys36/38 1 Ser mutant slightly form oligomers under these conditions as revealed by SDS-polyacrylamide gel electrophoresis. The results suggest that the Cys3 6/381 Ser mutant could evidently deprive PDI's enzyme activity and seriously decrease the interchain cross-linking of PDI proteins. The results again substantiate that to form an interchain disulfide bond is critical in promoting subsequent isopeptide bond cross-links. Till now, no contact crystal of PDI has been obtained. One bottleneck lays PDI is prone to form cross-linking, this heterogeneity made crystallization of PDI much difficult. The mutant Cys36/38 1 Ser shows a fine homogeneity in purified preparations, and maybe a promising protein to be crystallized. Secondary structure prediction, sequence alignment of 15 PDI sequences ranging from Aspergillus nige to human, and phylogenetic analysis of these species were also discussed in this paper, expected to achieve a better understanding of PDI's structure and its function.