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 A. The results obtained using Trp scanning mutagenesis were confirmed using Cys residues labeled with the N-methyl-amino-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.
Powl
Andrew M
AM
School of Biological Sciences, University of Southampton, UK.
Wright
J Neville
JN
East
J Malcolm
JM
Lee
Anthony G
AG
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry; D040542:Mechanotransduction, Cellular; D008958:Models, Molecular; D016296:Mutagenesis; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; C116377:Tb-MscL protein, Mycobacterium tuberculosis; D014364:Tryptophan