Award details

The construction of metal sites in proteins: from loop-directed mutagenesis to rational design

Reference	BB/C504519/1
Principal Investigator / Supervisor	Professor Christopher Dennison
Co-Investigators / Co-Supervisors
Institution	Newcastle University
Department	School of Chemistry
Funding type	Research
Value (£)	183,917
Status	Completed
Type	Research Grant
Start date	01/04/2005
End date	31/12/2008
Duration	45 months

Abstract

Metal sites in proteins are commonly fabricated from loop regions which are constructed on a particular protein structure (scaffold). In this proposal work is described which will investigate the relative importance of the length and sequence of the metal-binding loops, and the protein scaffold, for the structure and function of the active site. The proposal focuses on a family of small beta-barrel proteins that bind a single type 1 (T1) copper ion and which catalyse electron transfer (ET) reactions, and are thus called cupredoxins. In these molecules three of the four ligating amino-acid residues are found on a single loop which links the two C-terminal beta-strands. The loop length and structure varies in the different members of this family of proteins and appears to be tailored to the particular beta-barrel structure to which they are attached. We have recently demonstrated that the native loop sequence of a cupredoxin can be replaced with a shorter loop giving a cupredoxin still competent at ET and which has some interesting attributes. Loop-directed mutagenesis will be used to modify the length and the sequence of the copper binding motif in a variety of cupredoxins. The variants will be characterised with an array of spectroscopic techniques (including paramagnetic NMR spectroscopy) and functional studies (electrochemical and ET rate constant measurements) to assess the influence of the mutations. In one approach the same short, naturally occurring, loop sequence will be introduced into five different cupredoxins and a comparison of the properties of the variants to the wild type (WT) proteins will provide important information about the role of the loop. The variation in the features of these loop mutants will also allow the importance of the scaffold to be determined. Experiments in which non-native loop sequences are introduced into one particular cupredoxin will also be undertaken. The characterisation of these loop variants will highlight the minimal loop length required for a T1 copper site and will provide key information about the importance of the loop length alone. In the final part of the proposal we outline studies aimed at using loop-directed mutagenesis to introduce a T1 copper binding loop into a protein which does not naturally bind such a centre. For these experiments a monomeric Cu, Zn superoxide dismutase (Cu, Zn SOD), which also possesses a beta-barrel topology, but whose native copper site is located in a completely different position to the T1 centre of cupredoxins, and has a totally different geometry, will be used. The native copper site of Cu, Zn SOD will be removed and a copper-binding loop introduced between the two C-terminal beta-strands. The copper binding capabilities of this mutated Cu, Zn SOD, and the detailed properties of the site formed, will be investigated. The introduction of a copper site with authentic T1 properties would represent the first rational design of this key biological ET site.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Industrial Biotechnology, 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

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