Award details

Structure-activity relationships and mechanisms of the nucleobase-cation-symport (NCS1) family of membrane transport proteins

Reference	BB/C51725X/1
Principal Investigator / Supervisor	Professor Peter Henderson
Co-Investigators / Co-Supervisors	Professor So Iwata, Dr Nicholas Rutherford
Institution	University of Leeds
Department	Inst of Molecular & Cellular Biology
Funding type	Research
Value (£)	361,657
Status	Completed
Type	Research Grant
Start date	01/10/2005
End date	31/03/2009
Duration	42 months

Abstract

The ultimate aim is to define the structure-activity relationships and establish the molecular mechanism of the nucleobase-cation symporter (NCS1) family of membrane transport proteins, at least one member of which Ahp-1 see below, is of commercial importance. We have already achieved reproducible crystallisation of the native Ahp1 and its SelenoMethionine (SeMet) derivative to yield x-ray diffraction to a resolution of below 4A. Determination of its 3d structure should be completed during 2004 and early 2005. The NCS1 family occurs in various bacterial species, lower eukaryotes and one plant, but not in mammals. In a few cases their function is established, and from these we expect many of the proteins will transport nucleobases, allantoin, hydantoins or thiamine. The objective are: 1. to complete an atomic resolution model of a membrane transport protein Ahp1 (originally Aureobacterium hydantoin permease, though the producing organism is now classified as Microbacterium liquefaciens) determined by x-ray crystallography; 2. to conduct directed mutagenesis, gene recombination and some cysteine scanning mutagenesis to establish which domains and individual residues are involved in ligand recognition [(i) external site, (ii) internal site, (iii) occluded states] and translocation steps; 3. to clone and amplify expression of 20-30 other members of the family, and establish their physiological activities by functional assays; 4. to incorporate a (His)6 tag at the C-terminus (in a few cases a Strep-tag is more successful) for purification, conduct detergent solubilisation trials, and purify each of the proteins; 5. to undertake stability trials of the purified proteins, and for selected proteins undertake 3d crystallisation trials, determine structures, and by comparison with Ahp1 establish the molecular features of each protein responsible for its unique recognition of ligands. Background. The amplified expression, purification and large-scale production of the Ahp1 protein was achieved in Professor Henderson¿s laboratory in Leeds. The strategies for crystallisation and structure determination were then devised in Professor Iwata¿s laboratory at Imperial College London. Members of both laboratories have visited each other and transfer of the technologies has taken place: protein production to London and crystallisation strategy to Leeds. Technical competencies. The strategies for crystallisation and determination of structures of membrane proteins in general, and transport proteins in particular, were devised in Professor Iwata¿s laboratory. He already has access to beam time for x-ray diffraction experiments in France, Switzerland, Japan and UK, and we are expecting to use DIAMOND in UK, when it comes on stream. Using a modified version of plasmid pTTQ18, Professor Henderson and colleagues have achieved the amplified expression of over 40 bacterial membrane proteins; the majority are members of the Major Facilitator Superfamily of membrane transporters, which includes the sub-family NCS-1. Most have been readily purified exploiting an appropriate affinity tag.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Microbiology, 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

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

export PDF file

back to list new search