DESIGN, SYNTHESIS, AND PROPERTIES OF A PHOTOACTIVATABLE MEMBRANE-SPANNING PHOSPHOLIPIDIC PROBE

We introduce here a new photochemical probe suitable for labeling deep into the hydrophobic core of membranes: bis-phosphatidylethanolamine (trifluoromethyl)phenyldiazirine 19 (DIPETPD). This is a bipolar phosp holipid provided with a covalently bonded chain designed to span the m embrane and equipped with a centrally defined attachment point for the photolabeling group (trifluoromethyl)phenyldiazirine (TPD). This mole cule was designed to enhance the geometrical resolution of photochemic al labeling of membrane proteins by locating the photoreactive functio nality in the center of the bilayer. The remarkable chemical stability of the photoreactive group TPD1 allowed the design of a straightforwa rd and convergent synthetic strategy. The key steps developed for mole cules of this new general kind are (a) the mild and efficient coupling of two moieties of N-tBOC-protected lysophosphatidylethanolamine meth yl ester to the photoreactive symmetric dicarboxylic fatty acid mediat ed by dicyclohexylcarbodiimide and (dimethylamino)pyridine and (b) the smooth deprotection of the phosphate and amino functionalities with s odium iodide and trifluoroacetic acid, respectively, to yield the fina l product. DIPETPD has been successfully incorporated into small and l arge unilamellar vesicles of different lipid composition and prepared by a variety of procedures. The bilayer location of this reagent (tran smembrane vs 'U'-shaped conformations) was assayed by reaction of the amino groups at the polar heads of the bipolar phospholipid with selec ted membrane-impermeable reagents. Photolysis of the probe incorporate d into vesicles occurs readily upon irradiation with UV light (near 36 0 nm). These 'loaded' vesicles show adequate stability and appear unif orm and unilamellar in electron micrographs. They undergo the fusion r eaction with influenza virus as efficiently as reagent-free vesicles. Evidence is presented here that DIPETPD and a reductively methylated f orm efficiently label the peptide ion channel form of gramicidin A (an d a chemical analogue) and the influenza virus hemagglutinin. DIPETPD may help to identify transmembrane regions of integral membrane protei ns and map the lipid-protein interface in a region known to be deep in the membrane. A new radioactive version of this reagent ([H-3]-DIPETP D)2 has been recently used to ascertain that the HA2 subunit of influe nza virus hemagglutinin inserts into the target membrane prior to fusi on.3