Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum.

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Serval ID
serval:BIB_6435
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum.
Journal
Nucleic Acids Research
Author(s)
Biondi R.M., Baehler P.J., Reymond C.D., Véron M.
ISSN
0305-1048
Publication state
Published
Issued date
1998
Volume
26
Number
21
Pages
4946-4952
Language
english
Notes
Publication types: Journal Article
Abstract
The green fluorescent protein (GFP) is currently being used for diverse cellular biology approaches, mainly as a protein tag or to monitor gene expression. Recently it has been shown that GFP can also be used to monitor the activation of second messenger pathways by the use of fluorescence resonance energy transfer (FRET) between two different GFP mutants fused to a Ca2+sensor. We show here that GFP fusions can also be used to obtain information on regions essential for protein function. As FRET requires the two GFPs to be very close, N- or C-terminal fusion proteins will not generally produce FRET between two interacting proteins. In order to increase the probability of FRET, we decided to study the effect of random insertion of two GFP mutants into a protein of interest. We describe here a methodology for random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit using a bacterial expression vector. The selection and analysis of 120 green fluorescent colonies revealed that the insertions were distributed throughout the R coding region. 14 R/GFP fusion proteins were partially purified and characterized for cAMP binding, fluorescence and ability to inhibit PKA catalytic activity. This study reveals that GFP insertion only moderately disturbed the overall folding of the protein or the proper folding of another domain of the protein, as tested by cAMP binding capacity. Furthermore, three R subunits out of 14, which harbour a GFP inserted in the cAMP binding site B, inhibit PKA catalytic subunit in a cAMP-dependent manner. Random insertion of GFP within the R subunit sets the path to develop two-component FRET with the C subunit.
Keywords
Animals, Base Sequence, Binding Sites/genetics, Cyclic AMP/metabolism, Cyclic AMP-Dependent Protein Kinases/chemistry, Cyclic AMP-Dependent Protein Kinases/genetics, DNA Primers/genetics, DNA, Protozoan/genetics, DNA, Recombinant/genetics, Dictyostelium/genetics, Dictyostelium/metabolism, Energy Transfer, Green Fluorescent Proteins, Luminescent Proteins/genetics, Luminescent Proteins/metabolism, Models, Molecular, Molecular Sequence Data, Mutagenesis, Insertional, Protein Conformation, Recombinant Fusion Proteins/chemistry, Recombinant Fusion Proteins/genetics, Second Messenger Systems
Pubmed
Web of science
Open Access
Yes
Create date
19/11/2007 13:43
Last modification date
25/09/2019 7:09
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