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My Research

Expression and Purification: PDIp was previously uncharacterised. We have optimised the recombinant expression of PDIp in E. coli and also its' purification. This work improved our expected yield of PDIp approximately 10 fold.

Structural Studies of PDIp: We have shown by far UV CD that PDI and PDIp are more different structurally than orginally anticipated. Furthermore unlike PDI which undergoes redox-mediated conformational changes (see Wang et al 2011), PDIp does not (shown by CD, limted proteolysis, ANS fluorescence and DLS). This has obvious implications on the activity of PDIp and could mean that it undergoes substrate-binding induced conformational changes instead.

Understanding the Molecular Basis of Movement in PDIp: Recent work (Wang et al 2012) has implicated several amino acids in the redox-mediated conformational changes exhibited by PDI. In particular, an R300 : W396 interaction has been shown by mutagenesis to be expecically important. This interaction acts as a 'molecular tether' holding the molecule in a compact state when it is reduced but is broken in the oxidised state giving rise to a more open conformation. In PDIp this interaction is not conserved. Our current work is involved in determining the implications of this and whether this explains the lack of redox-mediated conformational activity seen in PDIp.

Determining the Activity of PDIp: We have used a classical PDI activity assay to observe the oxidoreductase activity of PDIp and also a variety of mutant and chimera proteins. We have found that PDIp is ~50% as active as PDI but that this is not simply due to a difference in the active site (CTHC instead of CGHC). We have recently (October 2012) been awarded a MRC centenary award grant to determine the pka of each active site cysteine in PDIp in order to try and elucidate it's activity further. This work is to be completed in the laboratory of Lloyd Ruddock, Finland in the early part of 2013.

Main Supervisor:

Prof. Robert Freedman

robert freedman

R.B.Freedman@warwick.ac.uk