Synthesis and membrane interactions of spin-label bifunctional reagents.

Abstract

The design of chemical reagents that interact with biological membranes requires knowledge of the orientation and location of the reagents with respect to the membrane surface and of the partition coefficients of the reagents between membrane and water. These properties are explored here for a class of molecules having the following properties: (1) they have long, flexible chains which are, in some cases, long enough to span the membrane; (2) there is a polar, or functional, group at each end of the chain; (3) the structures are symmetrical about a center atom which is part of a paramagnetic, spin-label ring. Electron paramagnetic resonance (EPR) spectra were used to determine whether molecules with the above features take up an Orientation in a lipid membrane such that the polar or functional groups on each end of the molecule are on the same side of a membrane or are on opposite sides, indicating a membrane-spanning orientation. The only case of a mem- brane-spanning orientation found was for a dicarboxylic acid of total chain length equal to twice that of the fatty acyl chains of the lipid. When amide linkages are substituted for two methylenes in the structure that spans the membrane, a dif- ferent orientation is observed. All of the molecules extended by amide linkages take up an orientation in a membrane in which both polar ends of the molecule are on the same side of the membrane, giving EPR signals in oriented membranes similar to those of bifunctional molecules that are too short to span the membrane. The EPR spectra do not provide unambiguous proof of whether the flexible molecules with both functional groups on one side of a membrane are extended on the surface or are bent and intercalated into the membrane hydrophobic interior, so two other tests of these possibilities In the presence of a membrane, an amphiphilic molecule in aqueous solution partitions between the membrane and water. This sort of process is involved in membrane binding of am- phiphilic drugs (Seeman, 1972), of fluorescent and para- magnetic probes (Radda, 1975; Berliner, 1976, 1979), of chemical reagents directed to membrane components (Peters & Richards, 1977), and, probably, also of nascent polrpeptide chains (Inouye et al., 1982), among others. Our own interest in binding of amphiphilic molecules to membranes is based on a desire to design protein modification reagents with spe- cificity for particular cellular components. We have syn- thesized a series of long, flexible bifunctional molecules. In the following paper (Willingham & Gaffney, 1983) we will show that variations in these structures result in specificities