Fluorescence properties of green fluorescent protein FRET pairs concatenated with the small G protein, Rac, and its interacting domain of the kinase, p21-activated kinase.

Fluorescence properties of green fluorescent protein FRET pairs concatenated with the small G protein, Rac, and its interacting domain of the kinase, p21-activated kinase. Research Abstract Details 

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Fluorescence properties of green fluorescent protein FRET pairs concatenated with the small G protein, Rac, and its interacting domain of the kinase, p21-activated kinase. Abstract Text:

Andie D Collinson,S W Annie Bligh,Debbie L Graham,Helen R Mott,Peter A Chalk,Nadia Korniotis,Peter N Lowe,

Many diseases are caused by aberrant cell signalling controlled by intracellular protein-protein interactions. Inhibitors of such interactions thus have enormous potential as chemotherapeutic agents. It is advantageous to test for such inhibitors using cell-based screens in which modulation of the interaction gives a rapid response. Fluorescence resonance energy transfer (FRET) systems, based on interacting donor and acceptor green fluorescent proteins (GFPs), have potential in such screens. Here, we describe experiments aimed at using a FRET system to monitor the interaction between the small G protein Rac and a region of its binding partner, the Ser/Thr kinase, p21-activated kinase (PAK). Initial attempts to use a previously described construct, enhanced GFP-PAK-enhanced blue fluorescent protein, failed because of the difficulty of obtaining equal and high expression levels of both the fusion protein and Rac in mammalian cells. Here, three proteins in which Rac, PAK, and the two GFPs were concatenated in different combinations on a single protein were expressed and characterised. In each construct, however, intramolecular interaction of PAK and Rac was observed. As this was of extremely high affinity, presumably because of entropy effects from the interacting partners being tethered, these molecules were not suitable for detection of inhibitors of the interaction. Molecular modelling was used to investigate the way in which the concatenated constructs might form intramolecular interactions. As this explained key properties of these proteins, it is likely that this approach could be used to design constructs where the unwanted intramolecular protein-protein interactions are prevented, whilst allowing the desired intermolecular Rac/PAK interaction. This would provide constructs that are useable for drug discovery.

Fluorescence properties of green fluorescent protein FRET pairs concatenated with the small G protein, Rac, and its interacting domain of the kinase, p21-activated kinase. Publishing Authors By Initials

AD Collinson,SW Bligh,DL Graham,HR Mott,PA Chalk,N Korniotis,PN Lowe,

For similar enzymes and coenzymes: enzymes: hydrolases: acid anhydride hydrolases: gtp phosphohydrolases: gtp-binding proteins: monomeric gtp-binding proteins: rho gtp-binding proteins: rac gtp-binding proteins research abstracts see: enzymes and coenzymes: enzymes: hydrolases: acid anhydride hydrolases: gtp phosphohydrolases: gtp-binding proteins: monomeric gtp-binding proteins: rho gtp-binding proteins: rac gtp-binding proteins research

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Fluorescence properties of green fluorescent protein FRET pairs concatenated with the small G protein, Rac, and its interacting domain of the kinase, p21-activated kinase. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Assay and drug development technologies

VOLUME: 2

Page Numbers: 659-73

Journal Abbreviation:

ISSN: 1540-658X

DAY: 19

MONTH: Dec

YEAR: 2004

Fluorescence properties of green fluorescent protein FRET pairs concatenated with the small G protein, Rac, and its interacting domain of the kinase, p21-activated kinase. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 101151468

Fluorescence properties of green fluorescent protein FRET pairs concatenated with the small G protein, Rac, and its interacting domain of the kinase, p21-activated kinase. Keywords Mesh Terms:

KEYWORDS: rac GTP-Binding Proteins

MESH TERMS: chemistry

Chemical & Substance for Abstract: Fluorescence properties of green fluorescent protein FRET pairs concatenated with the small G protein, Rac, and its interacting domain of the kinase, p21-activated kinase. Information

Substance Name: rac GTP-Binding Proteins

Registry Number: EC 3.6.5.2

Grant and Affiliation Information for Fluorescence properties of green fluorescent protein FRET pairs concatenated with the small G protein, Rac, and its interacting domain of the kinase, p21-activated kinase.

AFFILIATION: Structural & Biophysical Sciences, GlaxoSmithKline Medicines Research Centre, Stevenage, Herts, UK.

Country: United States

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MEDLINETA: Assay Drug Dev Technol

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