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The two main competing models for the structure of discoidal lipoprotein A-I complexes both presume that the protein component is helical and situated around the perimeter of a lipid bilayer disc. However, the more popular "picket fence" model orients the protein helices perpendicular to the surface of the lipid bilayer, while the alternative "belt" model orients them parallel to the bilayer surface. To distinguish between these models, we have investigated the structure of human lipoprotein A-I using a novel form of polarized internal reflection infrared spectroscopy that can characterize the relative orientation of protein and lipid components in the lipoprotein complexes under native conditions. Our results verify lipid bilayer structure in the complexes and point unambiguously to the belt model.

The ability of cecropin A to permeabilize and depolarize the membranes of Escherichia coli ML-35p bacteria has been compared to its bactericidal activity in an extension of earlier studies performed on synthetic lipid vesicle membranes (L. Silvestro, K. Gupta, J. H. Weiser, and P. H. Axelsen, Biochemistry 36:11452-11460, 1997). Our results indicate that differences in the concentration dependences of membrane permeabilization and depolarization seen in synthetic vesicles are not manifested in whole bacteria. The concentration dependences of both phenomena roughly correlate with bactericidal activity, suggesting that the bactericidal mechanism of cecropin A is related to membrane permeabilization.Cecropin A is one of the most extensively studied antimicrobial polypeptides among the many that are produced by insects as components of their host defense systems against bacterial infection (6,7,14). While there is a broad consensus that the site of antibacterial action of these polypeptides is the plasma membrane, the precise mechanism-and the way in which they discriminate between bacterial and host cell membranes-remains unclear.Studies of cecropin A and related peptides have shown that cecropin A is a linear 37-residue polypeptide composed entirely of ordinary L-amino acids (21). It is unstructured in aqueous solution but has the potential to form ␣-helices in partially organic solvent (8,19). Early studies of their bactericidal mechanism suggested that cecropins bind to negatively charged membrane lipids and form a closely packed layer (20) or "carpet" of peptide (5, 15) which renders the membranes permeable. Later studies demonstrated that cecropins form partially selective ion channels (2). Evidence against a mechanism involving specific chiral receptors was provided by studies showing that analogues composed entirely of D-amino acids retained full activity (22). Models have been proposed for the ion channel formed by cecropins (3), and evidence is available indicating that they aggregate and assume a transbilayer orientation in membranes (12, 13). We have recently shown that the action of cecropin A on synthetic lipid vesicles is concentration dependent, forming ion channels at low peptide/lipid ratios and "pores" large enough to pass various probe molecules at higher peptide/lipid ratios (16). In that set of experiments we demonstrated that leakage of water-soluble probe molecules was "all or nothing," supporting the idea that the pores through which the probes escape are stable, water-filled transmembrane conduits.One limitation of many studies of antimicrobial peptide action is that they have been performed on synthetic lipid vesicles rather than bacteria. This is necessary in many cases to make particular types of studies feasible or interpretable. However, vesicle studies leave important questions open about the true nature of antimicrobial peptide action in a vastly more complex chemical milieu. Therefore, we have extended our earlier studies of cecropin A action on phospholipid vesicles to whole bacteri...