Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491928
Title: Structural studies on protein disulphide isomerase
Author: Sidhu, Ateesh
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2008
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Abstract:
Protein disulphide isomerase (POI; EC 5.3.4.1) is a multifunctional enzyme which resides in the lumen of the endoplasmic reticulum (ER). It is approximated that over one-third of all human proteins fold in the ER. POI is one of the main folding catalysts, specifically facilitating native disulphide bond formation. POI has four domains, three of which have been solved and all possessing a thioredoxin-type fold consisting of the J3-a-J3-a-J3-a-J3-J3-a structure. Due to the intransigent nature of the b' domain of POI, it remains structurally unelucidated. The b' domain is vitally important as it holds the principal binding site and is essential for POI activity. The investigation of the two species which were produced during expression of most b' domain containing constructs led to the further biophysical analysis and identification of the monomer and dimer species. It was found that fractionation of the monomer and dimer species was vital in obtaining high resolution NMR data. An elaborate method using assignments from the b domain and b'x were used, and proved very effective in achieving the goal of full backbone assignment of the large 27.5 kOa bb'x protein molecule. The fruit of this labour is that it allowed further probing dynamic and more detailed molecular NMR analysis. NMR analysis identified evidence that the bb'x monomer species can exist in two forms, the closed form where the x-region binds to the b' domain and open form where the x-region is unbound. Chemical shift analysis revealed several key residues involved in protein flexibility and chemical shift mapping revealed the interface between the b domain and b'x. Defining the interface described here offers a method to model the full length POI domain structural organisation; which can be used to more clearly define POI function and how the domains of POI are coordinated in protein function. More detailed NMR analysis of relaxation dynamics (T1 and T2) revealed significantly differing motions attributed to the b, b' domains and x-region in the bb'x construct. The slower b' domain motion is believed to be related to the substrate binding function, where the b' domain appears to be gently flexing, in search for a substrate molecule.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.491928  DOI: Not available
Keywords: QP Physiology
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