Membrane interactions of the hydrophobic segment of diacylglycerol kinase epsilon.
Diacylglycerol kinase epsilon (DGKepsilon) is unique among mammalian DGK isoforms in having a segment of hydrophobic amino acids as a putative membrane anchor. To model the conformation, and stoichiometry of this segment in membrane-mimetic environments, we have prepared a peptide corresponding to this hydrophobic segment of DGKepsilon of sequence KKKKLILWTLCSVLLPVFITFWKKKKK-NH(2). Flanking Lys residues mimic the natural setting of this peptide in DGKepsilon, while facilitating peptide synthesis and characterization. Circular dichroism and fluorescence spectroscopic analysis demonstrated that the peptide has increased helical content and significant blue shifts in the presence of anionic--but not zwitterionic--bilayer membranes. When labeled with fluorophores that can undergo fluorescence resonance energy transfer, the peptide was found to dimerize--a result also observed from migration rates on SDS-PAGE gels under both reducing and non-reducing disulfide bridge conditions. The peptide was shown to preferentially interact with cholesterol in lipid films comprised of homogeneous mixtures of cholesterol and phosphatidylcholine, yet the presence of cholesterol in hydrated vesicle bilayers decreases its helical content. The peptide was also able to inhibit the activity of DGKepsilon protein in vitro. Our overall findings suggest that the peptide ultimately cannot leave the bulk water for attachment/insertion into the outer leaflet of an erythrocyte-like bilayer, yet its core sequence is sufficiently hydrophobic to insert into membrane core regions when membrane attachment is promoted by electrostatic attraction to anionic lipid head groups of the inner leaflet of an erythrocyte-like bilayer.
Glukhov
Evgenia
E
Division of Molecular Structure & Function, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
Shulga
Yulia V
YV
Epand
Raquel F
RF
Dicu
Armela O
AO
Topham
Matthew K
MK
Deber
Charles M
CM
Epand
Richard M
RM
D000595:Amino Acid Sequence; D002784:Cholesterol / Q000737:chemistry; D019852:Diacylglycerol Kinase / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D057927:Hydrophobic and Hydrophilic Interactions; D007527:Isoenzymes / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
D007527:Isoenzymes; D008565:Membrane Proteins; D002784:Cholesterol; D019852:Diacylglycerol Kinase