Award details

Characterization of the protein ligand binding site on protein disulphide-isomerase (PDI) and its homologues

Reference	BB/D018072/1
Principal Investigator / Supervisor	Dr Richard Williamson
Co-Investigators / Co-Supervisors	Dr Mark Howard
Institution	University of Kent
Department	Sch of Biosciences
Funding type	Research
Value (£)	312,907
Status	Completed
Type	Research Grant
Start date	01/11/2006
End date	31/10/2009
Duration	36 months

Abstract

We aim to define in detail the interaction between protein disulphide-isomerase (PDI) and its protein substrates, in order to picture its mechanism, illuminate its cellular function and inform work on PDI homologues. We will determine the structure of the b' domain of human PDI (the principal ligand binding domain) in various contexts. Our NMR data on PDI constructs including the b' domain indicate conformational heterogeneity within the b' domain that explains previous failures to solve the structure by x-ray and NMR approaches. Our targets will be constructs whose spectra indicate a single defined structure; the b-b'-x construct comprising all the non-catalytic domains of PDI, and mutants of the wild-type b'-x sequence. We will express them in E.coli and purify 15N or 15N/13C or 15N/13C/2H proteins for high resolution heteronuclear NMR studies. We will assign resonances, generate distance and angle constraints and calculate structures using conventional multi-dimensional NMR approaches. We will explore the dynamic properties of the proteins under study, using relaxation and H/D exchange methods, obtaining site-specific data where possible. We will determine the effects of small unstructured peptide ligands on NMR parameters of these proteins in order to define binding sites and impacts of ligand binding on PDI domain dynamics. We will initiate experiments using incompletely-folded protein ligands, focussed on the structural and dynamic properties of the ligand protein and how these reflect binding to PDI. For this work we will express in E.coli mutants of bovine pancreatic trypsin inhibitor (BPTI) which contain Cys-to-Ser mutations to constrain the disulphides that can be formed. These mutants mimic incompletely folded intermediates on the BPTI disulphide-linked folding pathway. We will use 15N relaxation measurements and transferred NOE approaches to explore the structures of these species when bound non- covalently to PDI and the nature of the interaction.

Summary

This application is part of a joint proposal with Prof. Robert Freedman at the University of Warwick. Warwick is the lead Research Organisation and the Joint Reference code is D189108. Please refer to the lead application for the Summary.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/D017807/1 Characterization of ligand-binding domains and the ligand binding site on protein disulphide-isomerase

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