Lipid-protein interactions studied by introduction of a tryptophan residue: the mechanosensitive channel MscL.
Trp fluorescence spectroscopy is a powerful tool to study the structures of membrane proteins and their interactions with the surrounding lipid bilayer. Many membrane proteins contain more than one Trp residue, making analysis of the fluorescence data more complex. The mechanosensitive channels MscL's of Mycobacterium tuberculosis (TbMscL) and Escherichia coli (EcMscL) contain no Trp residues. We have therefore introduced single Trp residues into the transmembrane regions of TbMscL and EcMscL to give the Trp-containing mutants F80W-TbMscL and F93W-EcMscL, respectively, which we show are highly suitable for measurements of lipid binding constants. In vivo cell viability assays in E. coli show that introduction of the Trp residues does not block function of the channels. The Trp-containing mutants have been reconstituted into lipid bilayers by mixing in cholate followed by dilution to re-form membranes. Cross-linking experiments suggest that the proteins retain their pentameric structures in phosphatidylcholines with chain lengths between C14 and C24, phosphatidylserines, and phosphatidic acid. Quenching of Trp fluorescence by brominated phospholipids suggests that the Trp residue in F80W-TbMscL is more exposed to the lipid bilayer than the Trp residue in F93W-EcMscL. Binding constants for phosphatidylcholines change with changing fatty acyl chain length, the strongest interaction for both TbMscL and EcMscL being observed with a chain of length C16, corresponding to a bilayer of hydrophobic thickness ca. 24 A, compared to a hydrophobic thickness for TbMscL of about 26 A estimated from the crystal structure. Lipid binding constants change by only a factor of 1.5 in the chain length range from C12 to C24, much less than expected from theories of hydrophobic mismatch in which the protein is treated as a rigid body. It is concluded that MscL distorts to match changes in bilayer thickness. The binding constants for dioleoylphosphatidylethanolamine for both TbMscL and EcMscL relative to those for dioleoylphosphatidylcholine are close to 1. Quenching experiments suggest a single class of binding sites for phosphatidylserine, phosphatidylglycerol, and cardiolipin on TbMscL; binding constants are greater than those for phosphatidylcholine and decrease with increasing ionic strength, suggesting that charge interactions are important in binding these anionic phospholipids. Quenching experiments suggest two classes of lipid binding sites on TbMscL for phosphatidic acid, binding of phosphatidic acid being much less dependent on ionic strength than binding of phosphatidylserine.
Powl
Andrew M
AM
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Southampton SO16 7PX, UK.
East
J Malcolm
JM
Lee
Anthony G
AG
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D001966:Bromine / Q000378:metabolism; D002308:Cardiolipins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D040542:Mechanotransduction, Cellular / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D009997:Osmotic Pressure; D010649:Phenylalanine / Q000235:genetics; D010713:Phosphatidylcholines / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
C096381:1-palmitoyl-2-(6,7-dibromostearoyl)phosphatidylcholine; D002308:Cardiolipins; D029968:Escherichia coli Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; C086596:MscL protein, E coli; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D010718:Phosphatidylserines; D010743:Phospholipids; D010649:Phenylalanine; D014364:Tryptophan; D001966:Bromine