PhD project proposal template 2012-13 by iIz14f3

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									Principal Supervisor name, department, University:            Dr. Claudia Blindauer, Chemistry,
Warwick

Second Supervisor name, department, University:               Prof. David Scanlan, Life
Sciences, Warwick

Where will the student be based? Warwick Chemistry and Life Sciences

PhD project title:

A novel role for bacterial metallothioneins: protection against oxidative stress

Project description:

Metallothioneins (MT), cysteine-rich proteins of low molecular weight, are found in all
                                              2+    +            2+
kingdoms [1], with high affinity towards Zn , Cu (and Cd ). Recent work in our groups
has shown that the expression of several bacterial MTs in the cyanobacterium
Synechococcus sp. CC9311 is strongly up-regulated under high-light stress. We hypothesise
that this up-regulation occurs in response to high levels of oxidative stress, and that bacterial
metallothioneins may be involved in protecting photosynthetic cyanobacteria against this
important stress. Whilst Synechococcus have developed highly efficient ways to deal with
high light and oxidative stress [2], our findings are the first indication for bacterial MTs
contributing to this resilience.

It is thought that the high number of cysteine thiols in MTs offer a significant pool of reducing
equivalents [3], but in the majority of cases, the mechanism for the observed up-regulation is
unknown, and the actual molecular mechanisms that may provide the protective action, are
unclear. Further open questions concern the metallation state (bacterial MTs are usually
zinc- binding) of the expressed proteins, and whether high light stress also causes
misbalances in metal ion distribution.

It is proposed to study the expression of bacterial MTs in several Synechococcus strains
under different light conditions, as well as under conditions of artificially induced oxidative
stress. The response to these stress conditions will be monitored by analysis of growth rates
and yields; these studies will be extended to Synechococcus strains that do not harbour MT
genes to establish whether the presence of MT genes is correlated with reduced sensitivity to
high light and oxidative stress.

In parallel, we will explore the in vitro redox chemistry of selected MTs, starting with those
highly up-regulated in Synechococcus sp. CC9311. Expression constructs for some of these
are already available, although cloning and biophysical characterisation of new MTs will
constitute an integral part of this project. The reactions of MTs with redox-active species, e.g.
hydrogen peroxide, will bemonitored in the presence and absence of bound zinc ions,
employing a range of spectroscopic techniques including NMR, ESI-MS and optical
spectroscopies.

Key experimental skills involved:

Cyanobacterial cell culture, RT-qPCR, cloning, protein expression and purification,
biophysical characterisation/reaction monitoring by NMR, MS and other auxiliary
spectroscopies (ICP-OES, UV- VIS, CD).

References:

. [1] C. A. Blindauer and O. I. Leszczyszyn, Metallothioneins: unparalleled diversity in
       structures and functions for metal ion homeostasis and more. Nat. Prod. Rep., 2010,
       27, 720-741.

. [2] D. Mella-Flores, C. Six, M. Ratin, F. Partensky, C. Boutte, G. Le Corguille, D. Marie, N.
       Blot, P. Gourvil, C. Kolowrat and L. Garczarek, Frontiers in Microbiology, 2012, 3,
       285.

. [3] S. Zeitoun-Ghandour, O. I. Leszczyszyn, C. A. Blindauer, F. M. Geier, J. G. Bundy and
       S. R. Stürzenbaum, Mol. BioSyst., 2011, 7, 2397-240.



Contact details for application enquiries: c.blindauer@warwick.ac.uk

								
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