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Melittin (MLT), the 26-residue toxic peptide from the European honeybee Apis mellifera, is widely used for studying the principles of membrane permeabilization by antimicrobial and other host-defense peptides. A striking property of MLT is that its ability to permeabilize zwitterionic phospholipid vesicles is dramatically reduced upon the addition of anionic lipids. Because the mechanism of permeabilization may be fundamentally different for the two types of lipids, we examined MLT-induced release of entrapped fluorescent dextran markers of two different molecular masses (4 and 50 kDa) from anionic palmitoyloleoylphosphatidylglycerol (POPG) vesicles. Unlike release from palmitoyloleoylphosphatidylcholine (POPC) vesicles, which is highly selective for the 4 kDa marker, implying release through pores of about 25 A diameter [Ladokhin et al., Biophys. J. 72 (1997) 1762], release from POPG vesicles was found to be non-selective, i.e., 'detergent-like'. Oriented circular dichroism measurements of MLT in oriented POPG and POPC multilayers disclosed that alpha-helical MLT can be induced to adopt a transbilayer orientation in POPC multilayers, but not in POPG multilayers. The apparent inhibition of MLT permeabilization by anionic membranes may thus be due to suppression of translocation ability.

Constitutive ␣-helical membrane proteins (MPs) 1 are assembled in membranes by means of a translocation/insertion process that involves the translocon complex (1). After release into the membrane's bilayer fabric, a MP resides stably in a thermodynamic free energy minimum (evidence reviewed in Refs. 2 and 3). This means that the prediction of MP structure from the amino acid sequence is fundamentally a problem of physical chemistry, albeit a complex one. Physical influences that shape MP structure include interactions of the polypeptide chains with water, each other, the bilayer hydrocarbon core, the bilayer interfaces, and cofactors (Fig. 1). Two recent reviews (3, 4) provide extensive discussions of the evolution, structure, and thermodynamic stability of MPs. Here we provide a distilled (and updated) overview that addresses four broad questions.

Many toxins and antimicrobial peptides permeabilize membrane vesicles by forming multimeric pores. Determination of the size of such pores is an important first step for understanding their structure and the mechanism of their self-assembly. We report a simple method for sizing pores in vesicles based on the differential release of co-encapsulated fluorescently labeled dextran markers of two different sizes. The method was tested using the bee venom peptide melittin, which was found to form pores of 25-30 A diameter in palmitoyloleoylphosphatidylcholine (POPC) vesicles at a lipid-to-peptide ratio of 50. This result is consistent with observations on melittin pore formation in erythrocytes (Katsu, T., C. Ninomiya, M. Kuroko, H. Kobayashi, T. Hirota, and Y. Fujita 1988. Action mechanism of amphipathic peptides gramicidin S and melittin on erythrocyte membrane Biochim. Biophys. Acta. 939:57-63).

The pH-triggered membrane insertion pathway of the T-domain of diphtheria toxin was studied using site-selective fluorescence labeling with subsequent application of several spectroscopic techniques (e.g., fluorescence correlation spectroscopy, FRET, lifetime quenching and kinetic fluorescence). FCS measurements indicate that pH-dependent formation of the membrane-competent form depends only slightly on the amount of anionic lipids in the membrane. The subsequent transbilayer insertion, however, is strongly favored by anionic lipids. Kinetic FRET measurements between donor-labeled T-domain and acceptor-labeled lipid vesicles demonstrate rapid membrane association at all pH values for which binding occurs. In contrast, the transmembrane insertion kinetics is significantly slower, and is also both pH-and lipid-dependent. Analysis of kinetic behavior of binding and insertion indicates the presence of several interfacial intermediates on the insertion pathway of the T-domain, from soluble W-state to transmembrane T-state. Intermediate interfacial I-state can be trapped in membranes with low content of anionic lipids (10%). In membranes of greater anionic lipid content, another pH-dependent transition results in the formation of the insertion-competent state and subsequent transmembrane insertion. Comparison of the results of various kinetic and equilibrium experiments suggests that the pH-dependences determining membrane association and transbilayer insertion transitions are different, but staggered. Anionic lipids not only assist in formation of the insertion competent form, but also lower the kinetic barrier for the final insertion. The function of diphtheria toxin T-domain is to translocate the catalytic domain across the lipid bilayer in response to acidification of the endosome, a task this 178-residue protein is able to perform without the help of any other proteins (1). Although the exact mechanism of membrane translocation is not understood, protein refolding in the lipid bilayer environment has to be the central issue. Thus, deciphering the mechanism of pH-triggered DTT insertion is expected to impact not only the field of cellular entry of toxins, many of which also enter the cell via the *To whom correspondence should be addressed: Phone: 913-588-0489 FAX: 913-588-7440 aladokhin@kumc.edu. † This research was supported by NIH GM-069783 and GM-069783-S1. ‡ Permanent address for Drs. Kyrychenko and Posokhov is Institute for Chemistry at V.N. Karazin Kharkiv National University, 4 Svobody Sq., Kharkiv, 61077, Ukraine NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 August 18. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript endosomal pathway (2-4), but would also advance our understanding of general physicochemical principles underlying membrane protein assembly and stability.The crystallographic structure of DTT in the water-soluble form (5) (Fig. 1) provides a starting point for refolding/insertion studies. The protein consists of 9 ...