Human Protein Disulphide Isomerase (hPDI) is an oxidoreductase and the archetype/lead family member of essential proteins that facilitate correct protein folding in the endoplasmic reticulum (ER). The PDI protein family are pivotal for the production of secreted proteins and is of interest for optimum biopharmaceutical batch production. Furthermore, hPDI is implicated in many pathophysiological processes including cancers and neurodegenerative diseases; driving the need to understand its molecular operation.
hPDI is a 57 kDa protein with a domain architecture: a-b-b’-x-a’ where a, b, b’ and a’ domain each adopt the thioredoxin fold. Domains connect via short 2-3 amino acid linkers, except or b’-a’ where a 20-amino acid ‘x’ linker exists. a and a’ both contain a WCGHC red-ox active site and the b’-domain contains the primary ligand binding site.
hPDI was considered to act as a ‘sum of its parts’ and each domain could be isolated and understood structurally and functionally. However, our recent investigations involving single and multiple domain constructs demonstrate divergent behaviour under oxidising or reducing conditions. Detailed analysis reveals each catalytic site as having distinct reduction potentials that are manipulated further by the presence or absence of additional domains. This suggests hPDI is gestalt: ‘greater than the sum of its parts’.
NMR spectroscopy and biophysical data (e.g. SEC, limited proteolysis, intrinsic fluorescence and chemical denaturation) of single and multiple domain constructs will be presented to investigate and inform on disengaged and gestalt behaviours of hPDI. NMR data provides an informative view of multiple domain proteins up to 53 kDa and includes assignments, dynamics, RDCs and protein reduction potentials using 1H, 13C, 15N and 19F NMR approaches. Our research demonstrates a structure-function complexity for hPDI that has implications across the protein family and influences protein folding applications for cellular biology, biopharma and disease.