INSERTION OF DIPHTHERIA-TOXIN IN LIPID BILAYERS STUDIED BY SPIN-LABELESR

The pH dependence of the insertion of diphtheria toxin into bilayers o f dioleoylphosphatidylglycerol (DOPG) has been studied by using ESR sp ectroscopy of spin-labeled phosphatidylglycerol with the reporter grou p at either the 5-position or the 14-position of the sn-2 chain (5-PGS L and 14-PGSL, respectively). At neutral pH, addition of diphtheria to xin has little effect on the ESR spectra of either spin label in large unilamellar vesicles of DOPG. At acidic pH, the outer hyperfine split ting of the 5-PGSL label is increased, and a second component correspo nding to lipids whose chain motion is selectively restricted appears i n the spectra of the 14-PGSL label, in the presence of diphtheria toxi n. The motionally restricted component of 14-PGSL has a large outer hy perfine splitting (2A(max) approximate to 61 G) and corresponds to spi n-labeled lipids the chains of which are in direct contact with the me mbrane-penetrant part of the inserted toxin. This restricted component is present, although to a lesser extent, in vesicles containing 90% o f the zwitterionic lipid dioleoylphosphatidylcholine and displays a li mited selectivity for negatively charged relative to zwitterionic spin -labeled phospholipids. The fraction of lipids which are motionally re stricted by the toxin increases with decreasing pH, titrating in DOPG vesicles with an apparent pK(a) of approximately 6.1. The outer hyperf ine splitting of the 5-PGSL label titrates with an apparent pK(a) of a pproximately 5.5, suggesting that this might be preferentially sensiti ve to a later stage in the insertion of the toxin. At pH 5.0, correspo nding to completion of the titration, the number of motionally restric ted lipids per bound toxin is approximately 40, whereas at pH 6.2 this stoichiometry is reduced to 24, indicating that the diphtheria toxin molecule is inserted in the lipid bilayer to a greater extent at the l ower pH. On the basis of previous hydrophobic photolabeling experiment s, it is suggested that only the translocation domain of the B fragmen t of the toxin is inserted at the higher pH and that the increased num ber of motionally restricted lipids at the lower pH corresponds to ins ertion also of the catalytic A domain.