The redox potential of human Protein Disulphide Isomerase' a' domain, and further characterisation of hPDI's ligand binding behaviour

Protein Disulphide Isomerase (PDI) is a 57 kDa multi-domain protein found within the endoplasmic reticulum. PDI consists of four thioredoxin-like domains (named a and b) each containing five ?-sheets and four ?-helices in the conformation ?????????. The four domains are ordered in the sequence abb'xa'c; with a 19 amino acid x linker between the b' and a' domains, and an acidic tail, c, after the a' domain. PDI acts as an oxidoreductase and chaperone to help fold newly synthesized proteins into their native state through the formation of disulphide bonds via the oxidation of sulfhydryl groups of cysteines, as well as the rearrangement of mispaired disulphides in an isomerisation reaction. The catalytic domains responsible for the thiol/disulphide reactions are a and a', and one of the aims of this work is to determine the redox potential of the a' domain and how it is influenced by the adjacent b', x and c regions. Fragments of human PDI containing the a' domain were expressed in E.coli and their redox potential measured by 15N/1H NMR using mixtures of reduced and oxidised glutathione. The b' domain of PDI which is known to contain the primary binding site for unfolded substrates was also investigated. The interaction of the b'xa'c fragment to the PDI ligand ?-somatostatin fused to the carrier protein GB1 was used to investigate its binding behaviour by NMR in a range of different redox conditions.