STRUCTURAL CHARACTERIZATION AND TOPOLOGY OF THE 2ND POTENTIAL MEMBRANE ANCHOR REGION IN THE THROMBOXANE-A(2) SYNTHASE AMINO-TERMINAL DOMAIN

To test this hypothesis, a solution structure in membrane mimetic envi ronments of a synthetic peptide corresponding to the second region of the NH2-terminal domain (TXAS residues 33-60) has been investigated by circular dichroism (CD), 2D nuclear magnetic resonance (NMR) spectros copy, and peptidoliposome reconstitution, CD spectroscopy indicated th at the peptide adopted a structure with significant alpha-helical-cont ent in 30% trifluoroethanol (TFE) or in dodecylphosphocholine (DPC) mi celles, which mimic hydrophobic membrane environment, Through a combin ation of 2D NMR experiments in the presence of TFE or DPC micelles, co mplete H-1 NMR assignments of the peptide have been obtained and the s tructure of the peptide has been determined, NH2-terminal segment of t he peptide takes an a well-defined alpha-helical conformation; the cen ter segment of the peptide, containing three prolines, adopts a bent c onformation, an the C-terminal segment of the peptide exists in a mixt ure of rapidly interconverting conformations, These results provide di rect structural evidence that residues 33-60 of the TXAS NH2-terminal domain contain a second membrane anchor region, with at least residues 35-46 having their helical structure expected for hydrophobic interac tion with the membrane. The orientation of the peptide in DPC micelles was evaluated from the effect of incorporation of a spin-label 12-dox ylstearate into the micelles; The peptide portions, found to be immers ed in the micelles, include the helical segment, the bent segment, and some hydrophobic residues within the C-terminal segment, Two addition al synthetic peptides, one corresponding to the NH2-terminal helical s egment (TXAS residues 33-46) and the other including tile bent and the C-terminal segments (TXAS residues 47-60) were analyzed for their abi lity to incorporate into peptidoliposomes, The helical peptide readily incorporated into liposomes; the other peptide did not. These results support the presence of a second functional membrane anchor region lo calized to the helical segment within TXAS residues 33-46, with passiv e membrane contacts in the bent and the C-terminal segments of the pep tide (TXAS residues 47-60) due to immersion of the helical in tile mem brane.