The role of tryptophan in the membrane protein diacylglycerol kinase
The role of tryptophan in the membrane protein diacylglycerol kinase
The preference of tryptophan residues for the lipid-water interface in integral membrane proteins is believed to be important for anchoring transmembrane α-helices into the lipid bilayer. The membrane protein diacylglycerol kinase (DAGK) from E. coli contains 5 Trip residues. The role of the Trp residues in DAGK was investigated by the construction of several Trp mutants. Expression levels of mutants containing a single Trp were very low with the exception of that containing Trp-112 as the only Trp residue (W112), which was expressed at levels comparable to wild-type (WT). W112 was also the only single Trp mutant to be active. Fluorescence emission spectra from WT and mutant DAGKs in dioleoylphosphatidylcholine [di(C18:1)PC] were all centred at ˜ 327 nm indicating that the Trp residues are all located in the same environment. Quenching of Trp fluorescence by dibromostearoylphosphatidycholine [di(Br2C18:0)PC] confirmed this. The measured separation between Trp residues and bromine atoms in all mutants and WT DAGK was between 7 and 9 Å, locating the Trp residues close to the glycerol backbone of the di(C18:1)PC bilayer. Changes in bilayer thickness did not alter the emission maximum of spectra from WT and mutant DAGKs indicating that the environment of the Trp residues did not change, showing efficient hydrophobic matching of the protein and the bilayer.
Although none of the tryptophan residues are thought to be in the active site, mutation of Trp 112 and Trp 25 had a significant affect on activity. Trp 112, located at the C-terminal of the third transmembrane α-helix, is thought to have a critical role in anchoring this helix into the membrane.
University of Southampton
2003
Clark, Elizabeth Hannah
(2003)
The role of tryptophan in the membrane protein diacylglycerol kinase.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The preference of tryptophan residues for the lipid-water interface in integral membrane proteins is believed to be important for anchoring transmembrane α-helices into the lipid bilayer. The membrane protein diacylglycerol kinase (DAGK) from E. coli contains 5 Trip residues. The role of the Trp residues in DAGK was investigated by the construction of several Trp mutants. Expression levels of mutants containing a single Trp were very low with the exception of that containing Trp-112 as the only Trp residue (W112), which was expressed at levels comparable to wild-type (WT). W112 was also the only single Trp mutant to be active. Fluorescence emission spectra from WT and mutant DAGKs in dioleoylphosphatidylcholine [di(C18:1)PC] were all centred at ˜ 327 nm indicating that the Trp residues are all located in the same environment. Quenching of Trp fluorescence by dibromostearoylphosphatidycholine [di(Br2C18:0)PC] confirmed this. The measured separation between Trp residues and bromine atoms in all mutants and WT DAGK was between 7 and 9 Å, locating the Trp residues close to the glycerol backbone of the di(C18:1)PC bilayer. Changes in bilayer thickness did not alter the emission maximum of spectra from WT and mutant DAGKs indicating that the environment of the Trp residues did not change, showing efficient hydrophobic matching of the protein and the bilayer.
Although none of the tryptophan residues are thought to be in the active site, mutation of Trp 112 and Trp 25 had a significant affect on activity. Trp 112, located at the C-terminal of the third transmembrane α-helix, is thought to have a critical role in anchoring this helix into the membrane.
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Published date: 2003
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Local EPrints ID: 465209
URI: http://eprints.soton.ac.uk/id/eprint/465209
PURE UUID: ee7d4f23-ef78-4005-bb9d-738b164f01af
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Date deposited: 05 Jul 2022 00:29
Last modified: 05 Jul 2022 00:29
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Author:
Elizabeth Hannah Clark
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