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A fluorescence method to define transmembrane alpha-helices in membrane proteins: Studies with bacterial diacylglycerol kinase

Record type: Article

Hydropathy plots have problems in identifying the sequences of transmembrane (TM) -helices when they contain charged residues. Here we show that fluorescence spectroscopy can be used to define the ends of TM -helices. Diacylglycerol kinase (DGK) from Escherichia coli contains three transmembrane (TM) -helices per monomer. We have used fluorescence techniques to define the region of the putative first TM helix (TM1) that spans the hydrophobic core of the lipid bilayer surrounding DGK in reconstituted membranes. Single Cys mutants were introduced into TM1 and flanking sites, in a mutant of DGK lacking the two native Cys residues. Introduction of Cys residues into the region between residues 28 and 34 resulted in mutants with low activities, due to a combination of reduced affinities for ATP and diacylglycerol and a reduced maximum rate. Cross-linking experiments showed that the low-activity mutants were present largely in the normal, trimeric form after reconstitution. Fluorescence emission maxima for the Cys mutants labeled with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD) reconstituted into bilayers of dioleoylphosphatidylcholine varied with position, suggesting that the region of TM1 spanning the hydrophobic core of the bilayer runs from Glu-28 on the cytoplasmic side to Asp-49 or Val-50 on the periplasmic side. This locates the charged/polar cluster 32RQE34 within the hydrophobic core of the bilayer. Fluorescence quenching experiments agree with this assignment for TM1, the results showing a periodicity consistent with distinct stripes of amino acid residues along the length of the helix, the stripes facing the lipid bilayer and facing the rest of the protein, respectively. The residues located close to the glycerol backbone region of the bilayer remained the same when the lipid fatty acyl chain length was changed in the range C14 to C22, showing that hydrophobic matching between the protein and the surrounding lipid bilayer is highly efficient.

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Citation

Jittikoon, Jiraphun, East, J. Malcolm and Lee, Anthony G. (2007) A fluorescence method to define transmembrane alpha-helices in membrane proteins: Studies with bacterial diacylglycerol kinase Biochemistry, 46, (38), pp. 10950-10959. (doi:10.1021/bi7008213).

More information

Submitted date: 1 March 2007
Published date: 1 September 2007

Identifiers

Local EPrints ID: 56096
URI: http://eprints.soton.ac.uk/id/eprint/56096
ISSN: 0006-2960
PURE UUID: 1888d8e0-7dfa-4e70-b5ce-0d4d64b71e56

Catalogue record

Date deposited: 06 Aug 2008
Last modified: 11 Jul 2017 09:46

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Contributors

Author: Jiraphun Jittikoon
Author: J. Malcolm East
Author: Anthony G. Lee

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