Award details

Role of CETS signalling complexes in controlling the development of plant architecture

Reference	BB/C502414/1
Principal Investigator / Supervisor	Professor R Brady
Co-Investigators / Co-Supervisors	Dr Tim Joseph-Horne
Institution	University of Bristol
Department	Biochemistry
Funding type	Research
Value (£)	280,076
Status	Completed
Type	Research Grant
Start date	01/01/2005
End date	31/12/2007
Duration	36 months

Abstract

In order to progress our understanding of the role of CETS proteins in plants, we aim to reconstruct in vitro their biologically active complexes with signalling partners (Objective 1). Five interacting proteins from each of tomato and Arabidopsis have already been identified to form stable complexes with CETS proteins. Recombinant forms of each of these proteins will be produced by expression in a range of Gateway compatible bacterial and insect cell expression vectors, in each case with cleavable tags to facilitate their purification. After purification of the target proteins by affinity chromatography, we will enzymatically remove the affinity tags prior to studying their ability to form stable complexes with one another using gel-filtration, isothermal calorimetry and analytical ultracentrifugation techniques. The influence of phosphorylation on complex formation will also be studied using the intrinsic kinase action of the tomato SPI-1 protein, as well as other non-specific kinases identified from our and other¿s studies of intracellular phosphorylation. The purified recombinant proteins will then be subjected to crystallisation trials using commercially available sparse-matrix crystallisation kits as well as conditions that have frequently proved successful in our local experience. Individual protein components and their complexes with one another or relevant peptides will be included in the crystallisation screens. When crystals are obtained, their structures will be determined after collection of X-ray diffraction data using a synchrotron source, and structure solution by either molecular replacement or anomalous dispersion phasing techniques. (Objective 2). From the refined structures we will examine the composition of the active surfaces of these proteins, and design mutations that will preserve the overall fold but alter the interactions between components. These mutant forms will be made by site-directed mutagenesis and introduced into plants via Agrobacterium-mediated transformation. The cDNAs will be constitutively expressed via the CaMV 35S promoter, both in wild-type Arabidopsis and in various mutant plants. For example, expression in mutants for the same gene will test how well any mutant version can act, while expression in plants carrying mutations in interacting partners will see how it acts alone (Objective 3).

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	X – not assigned to a current Research Topic
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

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search