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Identification of the hydrophobic thickness of a membrane protein using fluorescence spectroscopy: studies with the mechanosensitive channel MscL

Identification of the hydrophobic thickness of a membrane protein using fluorescence spectroscopy: studies with the mechanosensitive channel MscL
Identification of the hydrophobic thickness of a membrane protein using fluorescence spectroscopy: studies with the mechanosensitive channel MscL
The hydrophobic thickness of a membrane protein is an important parameter, defining how the protein sits within the hydrocarbon core of the lipid bilayer that surrounds it in a membrane. Here we show that Trp scanning mutagenesis combined with fluorescence spectroscopy can be used to define the hydrophobic thickness of a membrane protein. The mechanosensitive channel of large conductance (MscL) contains two transmembrane alpha-helices, of which the second (TM2) is lipid-exposed. The region of TM2 that spans the hydrocarbon core of the bilayer when MscL is reconstituted into bilayers of dioleoylphosphatidylcholine runs from Leu-69 to Leu-92, giving a hydrophobic thickness of ca. 25 angstrom. The results obtained using Trp scanning mutagenesis were confirmed using Cys residues labeled with the N-methylamino-7-nitroben-2-oxa-1,3-diazole [NBD] group; both fluorescence emission maxima and fluorescence lifetimes for the NBD group are sensitive to solvent dielectric constant over the range (2-40) thought to span the lipid headgroup region of a lipid bilayer. Changing phospholipid fatty acyl chain lengths from C14 and C24 results in no significant change for the fluorescence of the interfacial residues, suggesting very efficient hydrophobic matching between the protein and the surrounding lipid bilayer.
0006-2960
5713-5721
Powl, Andrew M.
79b77ec3-767b-42ad-991c-d843e29af8e3
Wright, J. Neville
e53ee4b9-10f5-4365-a9f2-5a7b0eacd86d
East, J. Malcolm
9fe7f794-1d89-4935-9a99-b831d786056e
Lee, Anthony G.
0891914c-e0e2-4ee1-b43e-1b70eb072d8e
Powl, Andrew M.
79b77ec3-767b-42ad-991c-d843e29af8e3
Wright, J. Neville
e53ee4b9-10f5-4365-a9f2-5a7b0eacd86d
East, J. Malcolm
9fe7f794-1d89-4935-9a99-b831d786056e
Lee, Anthony G.
0891914c-e0e2-4ee1-b43e-1b70eb072d8e

Powl, Andrew M., Wright, J. Neville, East, J. Malcolm and Lee, Anthony G. (2005) Identification of the hydrophobic thickness of a membrane protein using fluorescence spectroscopy: studies with the mechanosensitive channel MscL. Biochemistry, 44 (15), 5713-5721. (doi:10.1021/bi047338g).

Record type: Article

Abstract

The hydrophobic thickness of a membrane protein is an important parameter, defining how the protein sits within the hydrocarbon core of the lipid bilayer that surrounds it in a membrane. Here we show that Trp scanning mutagenesis combined with fluorescence spectroscopy can be used to define the hydrophobic thickness of a membrane protein. The mechanosensitive channel of large conductance (MscL) contains two transmembrane alpha-helices, of which the second (TM2) is lipid-exposed. The region of TM2 that spans the hydrocarbon core of the bilayer when MscL is reconstituted into bilayers of dioleoylphosphatidylcholine runs from Leu-69 to Leu-92, giving a hydrophobic thickness of ca. 25 angstrom. The results obtained using Trp scanning mutagenesis were confirmed using Cys residues labeled with the N-methylamino-7-nitroben-2-oxa-1,3-diazole [NBD] group; both fluorescence emission maxima and fluorescence lifetimes for the NBD group are sensitive to solvent dielectric constant over the range (2-40) thought to span the lipid headgroup region of a lipid bilayer. Changing phospholipid fatty acyl chain lengths from C14 and C24 results in no significant change for the fluorescence of the interfacial residues, suggesting very efficient hydrophobic matching between the protein and the surrounding lipid bilayer.

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Submitted date: 20 December 2004
Published date: 1 April 2005
Organisations: Biological Sciences

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Local EPrints ID: 35626
URI: https://eprints.soton.ac.uk/id/eprint/35626
ISSN: 0006-2960
PURE UUID: 1b3c43d4-4642-4566-b131-547c1f32a219

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Date deposited: 22 May 2006
Last modified: 30 Jul 2019 17:47

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