CHARACTERIZATION OF 2 MEMBRANE-BOUND FORMS OF OMPA

The insertion of the outer membrane protein A (OmpA) into Lipid bilaye rs was studied by Limited proteolysis, polarized Fourier transform inf rared (FTIR) spectroscopy, and fluorescence spectroscopy. In the nativ e state, OmpA is thought to form a barrel of eight antiparallel beta-s trands. For the present study, it was isolated in an,unfolded form, pu rified, and exposed to preformed vesicles of 1-palmitoyl-2-oleoyl-phos phatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), dipalmi toylphosphatidylcholine (DPPC), and three phospholipids that were brom inated in different positions of their sn-2 chains (4,5-BrPC, 9,10-BrP C, and 11,12-BrPC). Limited proteolysis revealed two membrane-bound fo rms of OmpA, namely an ''adsorbed'' (35 kDa) and an ''inserted'' (30 k Da) form [Surrey, T., and Jahnig, F. (1992) Proc. Natl. Acad. Sci. U.S .A. 89, 7457-7461]. Which form was found after membrane binding and re folding depended. on the Lipids used and on the temperature. Polarized attenuated total reflection (ATR)-FTIR spectra were recorded with Omp A bound to germanium-supported bilayers in both forms. The position of the amide I' band indicated quite large fractions of beta-structure o f OmpA in both membrane-bound forms (35-45% in the adsorbed form and 4 5-55% in the inserted form). Measurements of the linear dichroism of t he amide I' bands in the inserted form are consistent with an antipara llel beta-barrel in which the strands are inclined at about 36 degrees from the membrane normal. The average angle of the beta-strands to th e bilayer normal is likely larger in the 35 kDa form than in the inser ted form. The extent of quenching of the tryptophan fluorescence in th e presence of brominated lipids depended on the position of the bromin es in the fatty acyl chain. In the inserted form, 4,5-BrPC quenched th e fluorescence most efficiently, which indicates an average location o f the tryptophan residues close to the bilayer surface. In the 35 kDa form, tryptophan fluorescence was most efficiently quenched by 9,10-Br PC, which indicates a partial insertion of these residues in the hydro phobic part of the bilayer. Fluorescence spectroscopy in the presence of the soluble quencher acrylamide showed that after binding to the me mbrane the five tryptophan residues of OmpA are protected from the aqu eous environment to a similar extent in both forms.