Characterization of the structure and membrane interaction of NH2-terminal domain of thromboxane A2 synthase.
Thromboxane A2 synthase (TXAS), a member of the cytochrome P450 superfamily, is believed to be anchored to the endoplasmic reticulum membrane by hydrophobic portions of its NH2-terminal domain. Two hydrophobic peptides, corresponding to potential membrane-anchor segments of the NH2-terminal region of TXAS (residues 1-36, designated LP1 and residues 33-60, designated LP2) were synthesized, and their secondary structure and ability to insert in a lipid bilayer were characterized. The conformation of the synthetic peptides were analyzed in organic and membrane (bilayer) environments. Circular dichroism spectroscopy indicated that both segments adopt structures with significant alpha-helical content in a hydrophobic environment. For the LP1 peptide, the helical content was maximal with 60% trifluoroethanol, whereas 20% trifluoroethanol maximized the helix content for the LP2 peptide. The interaction of several NH2-terminal peptides with a lipid bilayer was determined by reconstitution of these peptides into lipid vesicles composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. Both the LP1 and LP2 peptides bound strongly to the defined lipid vesicles, but peptides corresponding to residues 1-15 and 33-36 were not incorporated into the lipid vesicles. The results indicate that TXAS has two distinct membrane-anchor segments, comprising residues 16-33 and 37-60 in its NH2-terminal domain.
Ruan
K H
KH
Vascular Biology Research Center, University of Texas Health Science Center, Houston 77030.
Li
P
P
Kulmacz
R J
RJ
Wu
K K
KK
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D008081:Liposomes; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017433:Protein Structure, Secondary; D013930:Thromboxane-A Synthase / Q000737:chemistry* / Q000378:metabolism*
D008081:Liposomes; D010446:Peptide Fragments; D013930:Thromboxane-A Synthase