Interactions of phospholipids with the potassium channel KcsA
Interactions of phospholipids with the potassium channel KcsA
The potassium channel KcsA from Streptomyces lividans contains tryptophan residues in two bands around the protein, at the water-lipid interface either side of the membrane, which have been used in fluorescence quenching studies to investigate lipid interactions with KcsA. KcsA was reconstituted into bilayers containing mixtures of brominated and non-brominated phospholipids by the rapid dilution of a solution of lipid and KcsA in cholate into buffer. Fluorescence quenching of KcsA in mixtures of brominated anionic lipids with dioleoylphosphatidylcholine were more marked at low mole fractions of brominated lipid than in mixtures of brominated phophatidylcholine with non-brominated anionic lipids. These results are consistent with a model in which phosphatidylcholine and anionic lipid bind with little selectivity to the bulk (annular) lipid sites around KcsA, but with anionic lipid binding selectively to a small number of additional sites (non-annular sites) on KcsA. The affinity of anionic lipid binding to the non-annular binding site was dependent on phospholipid headgroup structure, with phosphatidylglycerol having the lowest affinity and caridiolipin having the highest affinity. A series of tryptophan knockout experiments showed that zwitterionic lipid was excluded from the non-annular binding site. The affinity for the anionic phospholipid phosphatidylserine decreased at high ionic strength suggesting that electrostatic interactions between the anionic phospholipid headgroup and positively charged residues on KcsA were important for binding at the non-annular site. It is suggested that the non-annular site corresponds to the binding site for phosphatidylglycerol detected at the monomer-monomer interfaces in the crystal structure. By the use of a fluorescence assay it was established that binding of anionic lipid at the non-annular site is essential for the function of the protein.
University of Southampton
2004
Alvis, Simon
(2004)
Interactions of phospholipids with the potassium channel KcsA.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The potassium channel KcsA from Streptomyces lividans contains tryptophan residues in two bands around the protein, at the water-lipid interface either side of the membrane, which have been used in fluorescence quenching studies to investigate lipid interactions with KcsA. KcsA was reconstituted into bilayers containing mixtures of brominated and non-brominated phospholipids by the rapid dilution of a solution of lipid and KcsA in cholate into buffer. Fluorescence quenching of KcsA in mixtures of brominated anionic lipids with dioleoylphosphatidylcholine were more marked at low mole fractions of brominated lipid than in mixtures of brominated phophatidylcholine with non-brominated anionic lipids. These results are consistent with a model in which phosphatidylcholine and anionic lipid bind with little selectivity to the bulk (annular) lipid sites around KcsA, but with anionic lipid binding selectively to a small number of additional sites (non-annular sites) on KcsA. The affinity of anionic lipid binding to the non-annular binding site was dependent on phospholipid headgroup structure, with phosphatidylglycerol having the lowest affinity and caridiolipin having the highest affinity. A series of tryptophan knockout experiments showed that zwitterionic lipid was excluded from the non-annular binding site. The affinity for the anionic phospholipid phosphatidylserine decreased at high ionic strength suggesting that electrostatic interactions between the anionic phospholipid headgroup and positively charged residues on KcsA were important for binding at the non-annular site. It is suggested that the non-annular site corresponds to the binding site for phosphatidylglycerol detected at the monomer-monomer interfaces in the crystal structure. By the use of a fluorescence assay it was established that binding of anionic lipid at the non-annular site is essential for the function of the protein.
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Published date: 2004
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Local EPrints ID: 465460
URI: http://eprints.soton.ac.uk/id/eprint/465460
PURE UUID: f394c24f-2dc7-49d3-b7a5-aba3f7b592d2
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Date deposited: 05 Jul 2022 01:10
Last modified: 05 Jul 2022 01:10
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Author:
Simon Alvis
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