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Heterogeneity in the binding of lipid molecules to the surface of a membrane protein: Hot spots for anionic lipids on the mechanosensitive channel of large conductance MscL and effects on conformation

Heterogeneity in the binding of lipid molecules to the surface of a membrane protein: Hot spots for anionic lipids on the mechanosensitive channel of large conductance MscL and effects on conformation
Heterogeneity in the binding of lipid molecules to the surface of a membrane protein: Hot spots for anionic lipids on the mechanosensitive channel of large conductance MscL and effects on conformation
We have introduced single Trp residues into the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and used fluorescence quenching by brominated phospholipids to detect the presence of a binding site of high affinity for anionic phospholipids. A cluster of three positively charged residues, Arg-98, Lys-99, and Lys-100, is located on the cytoplasmic side of MscL, in a position where they could interact with the headgroup of an anionic phospholipid. Single mutations of these charged residues in the Trp-containing mutant F80W results in a decreased affinity for phosphatidic acid. Single mutations of the charged residues also result in a significant shift in the fluorescence emission spectrum in dioleoylphosphatidylcholine [di(C18:1)PC] but smaller shifts in dioleoylphosphatidic acid [di(C18:1)PA], suggesting that single mutations result in a conformational change for the protein that is reversed by interaction with anionic phospholipids. This is consistent with the observation that single mutations of the charged residues do not result in a gain of function phenotype. In contrast, simultaneous mutation of all three charged residues results in a gain of function phenotype, and a shift in fluorescence emission spectrum in di(C18:1)PC not reversed in di(C18:1)PA. The gain of function mutant F80W:V21K also shows a shifted fluorescence emission spectrum in both di(C18:1)PC and di(C18:1)PA and binds di(C18:1)PC and di(C18:1)PA with equal affinity, suggesting that the conformational change caused by the V21K mutation results in a breakup of the cluster of three positive charges. Experiments with the Trp mutants L69W and Y87W allow us to measure lipid binding constants on the periplasmic and cytoplasmic sides of the membrane, respectively. On both sides of the membrane the affinity for di(C18:1)PC is equal to that for dioleoylphosphatidylethanolamine. On the periplasmic side of the membrane, there is no selectivity for anionic phospholipids. In contrast, quenching data for Y87W provides evidence for the existence of two lipid binding sites on the cytoplasmic side of the membrane close to the Trp residue at position 87, with binding to one of these sites showing a marked preference for anionic lipid over zwitterionic lipid, presumably involving the charged cluster Arg-98, Lys-99, and Lys-100.
0006-2960
5873-5883
Powl, Andrew M.
79b77ec3-767b-42ad-991c-d843e29af8e3
East, J. Malcolm
9fe7f794-1d89-4935-9a99-b831d786056e
Lee, Anthony G.
0891914c-e0e2-4ee1-b43e-1b70eb072d8e
Powl, Andrew M.
79b77ec3-767b-42ad-991c-d843e29af8e3
East, J. Malcolm
9fe7f794-1d89-4935-9a99-b831d786056e
Lee, Anthony G.
0891914c-e0e2-4ee1-b43e-1b70eb072d8e

Powl, Andrew M., East, J. Malcolm and Lee, Anthony G. (2005) Heterogeneity in the binding of lipid molecules to the surface of a membrane protein: Hot spots for anionic lipids on the mechanosensitive channel of large conductance MscL and effects on conformation. Biochemistry, 44 (15), 5873-5883. (doi:10.1021/bi047439e).

Record type: Article

Abstract

We have introduced single Trp residues into the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and used fluorescence quenching by brominated phospholipids to detect the presence of a binding site of high affinity for anionic phospholipids. A cluster of three positively charged residues, Arg-98, Lys-99, and Lys-100, is located on the cytoplasmic side of MscL, in a position where they could interact with the headgroup of an anionic phospholipid. Single mutations of these charged residues in the Trp-containing mutant F80W results in a decreased affinity for phosphatidic acid. Single mutations of the charged residues also result in a significant shift in the fluorescence emission spectrum in dioleoylphosphatidylcholine [di(C18:1)PC] but smaller shifts in dioleoylphosphatidic acid [di(C18:1)PA], suggesting that single mutations result in a conformational change for the protein that is reversed by interaction with anionic phospholipids. This is consistent with the observation that single mutations of the charged residues do not result in a gain of function phenotype. In contrast, simultaneous mutation of all three charged residues results in a gain of function phenotype, and a shift in fluorescence emission spectrum in di(C18:1)PC not reversed in di(C18:1)PA. The gain of function mutant F80W:V21K also shows a shifted fluorescence emission spectrum in both di(C18:1)PC and di(C18:1)PA and binds di(C18:1)PC and di(C18:1)PA with equal affinity, suggesting that the conformational change caused by the V21K mutation results in a breakup of the cluster of three positive charges. Experiments with the Trp mutants L69W and Y87W allow us to measure lipid binding constants on the periplasmic and cytoplasmic sides of the membrane, respectively. On both sides of the membrane the affinity for di(C18:1)PC is equal to that for dioleoylphosphatidylethanolamine. On the periplasmic side of the membrane, there is no selectivity for anionic phospholipids. In contrast, quenching data for Y87W provides evidence for the existence of two lipid binding sites on the cytoplasmic side of the membrane close to the Trp residue at position 87, with binding to one of these sites showing a marked preference for anionic lipid over zwitterionic lipid, presumably involving the charged cluster Arg-98, Lys-99, and Lys-100.

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Submitted date: 7 December 2004
Published date: 1 April 2005
Organisations: Biological Sciences

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Local EPrints ID: 28734
URI: https://eprints.soton.ac.uk/id/eprint/28734
ISSN: 0006-2960
PURE UUID: b336ef18-d6ee-4a7b-9dfb-d2ab9b35ed6d

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Date deposited: 05 May 2006
Last modified: 11 Jul 2017 09:46

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Author: Andrew M. Powl
Author: J. Malcolm East
Author: Anthony G. Lee

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