The structure of an intrinsic membrane kinase in its lipid environment
The structure of an intrinsic membrane kinase in its lipid environment
Diacylglycerol kinase (DGK) of E. coli is the smallest known kinase containing three transmembrane (TM) α-helices and 2 amphipathic helices. The aim of this study is to define the topology of the first TM α-helix of DGK of E. coli using cysteine (Cys) scanning mutagenesis combined with fluorescence spectroscopy. Trp-less mutants of DGK were found to be inactive due to protein misfolding, as confirmed by crosslinking experiments. Therefore, this study focuses on Cys-scanning mutagenesis, since the Cys-less mutant of DGK is fully function. Thirty single Cys mutants were constructed over the region covering TM1 of DGK. The 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) fluorophore was used as a reporter by labelling to the sulfhydryl group of Cys residues. Fortunately, the labelling process had little effect on activity, suggesting that labelling did not result in large conformational changes in the protein. Of the 30 mutants 7 showed low activity; the substrate dependence of activity was studied for these low-activity mutants. For structural studies using fluorescence techniques, the fluorescence emission maxima were determined and fluorescence quenching experiments were performed using the three quenchers, potassium iodide (KI), Tempo, and 16-doxyl stearic acid. The data from these experiments were used to define the topology of TM1 and to define the residues facing the lipid bilayer or facing the rest of the protein site. The data indicate that the residue Ala-41 is in the centre of the bilayer. The residues located close to the glycerol backbone region of the bilayer remained the same when the lipid fatty chain length was changed in the range C14 to C22, suggesting that hydrophobic matching between the protein and the surrounding lipid bilayer is highly efficient.
University of Southampton
2007
Jittikoon, Jiraphun
(2007)
The structure of an intrinsic membrane kinase in its lipid environment.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Diacylglycerol kinase (DGK) of E. coli is the smallest known kinase containing three transmembrane (TM) α-helices and 2 amphipathic helices. The aim of this study is to define the topology of the first TM α-helix of DGK of E. coli using cysteine (Cys) scanning mutagenesis combined with fluorescence spectroscopy. Trp-less mutants of DGK were found to be inactive due to protein misfolding, as confirmed by crosslinking experiments. Therefore, this study focuses on Cys-scanning mutagenesis, since the Cys-less mutant of DGK is fully function. Thirty single Cys mutants were constructed over the region covering TM1 of DGK. The 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) fluorophore was used as a reporter by labelling to the sulfhydryl group of Cys residues. Fortunately, the labelling process had little effect on activity, suggesting that labelling did not result in large conformational changes in the protein. Of the 30 mutants 7 showed low activity; the substrate dependence of activity was studied for these low-activity mutants. For structural studies using fluorescence techniques, the fluorescence emission maxima were determined and fluorescence quenching experiments were performed using the three quenchers, potassium iodide (KI), Tempo, and 16-doxyl stearic acid. The data from these experiments were used to define the topology of TM1 and to define the residues facing the lipid bilayer or facing the rest of the protein site. The data indicate that the residue Ala-41 is in the centre of the bilayer. The residues located close to the glycerol backbone region of the bilayer remained the same when the lipid fatty chain length was changed in the range C14 to C22, suggesting that hydrophobic matching between the protein and the surrounding lipid bilayer is highly efficient.
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Published date: 2007
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Local EPrints ID: 466277
URI: http://eprints.soton.ac.uk/id/eprint/466277
PURE UUID: ba63032e-d536-4819-8162-fcd826dc7510
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Date deposited: 05 Jul 2022 05:01
Last modified: 05 Jul 2022 05:01
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Author:
Jiraphun Jittikoon
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