Biochimica et Biophysica Acta, 1069 (1991) 77-86 © 1991 Elsevier Science Publishers B.V. All rights reserved 0005-2736/91/$03.50 ADONIS 000527369100366L 77 BBAMEM 75382 A combined study of aggregation, membrane affinity and pore activity of natural and modified melittin Stefan Stankowski, Michael Pawlak, Elizabet Kaisheva, Charles H. Robert and Gerhard Schwarz Department of Biophysical Chemistry, Biocenter of the University, Basel (Switzerland) (Received 3 April 1991) Key words: Lipid-peptide interaction; Bilayer permeabilization; Melittin, formylated; Melittin, acetylated; Melittin, succinylated; TD~tophan modification The pore activity of mefitfin and several chemically modified derivatives has been investigated using conductance measurements on planar lipid bilayers and marker release from small unilamellar vesicles. The modifications included N-terminal formylation, acetylation, succinylafion and modification of the tryptophan residue. All of the compounds showed bilayer permeabilizing properties, though quantitative differences were evident. These com- prised changes in the voltage dependence of the conductance, in the single-pore kinetics, in the concentration of aqueous peptide required to induce a given pore activity and in the apparent 'molecularity' reflected by the power law of its concentration dependence. A strong tendency for disrupting bilayers was not always correlated with strong pore activity. For a better understanding of these results, measurements of pore activity were complemented by studying the aggregation behavior in solution and the water-membrane partition equilibrium. Modifications of charged residues gave rise to significant changes in the aggregation properties, but had virtually no influence on the partition coefficient. The latter decreased strongly, however, as a result of tryptophan modification. Analysis of the isotherms was consistent with the assumption that the arginine residues in melittin do not contribute very much to charge accumulation at the immediate membrane/water interface. Introduction Honeybee melittin is a small natural peptide (26 amino acids) that has been widely studied as a model for various aspects of protein-lipid interactions (see Ref. 1 for a recent review). It has a pronounced am- phiphilic nature and belongs to a group of peptides that induce voltage-dependent conductance in mem- branes [2] apparently through aggregation of several monomers to form pores. However, very few melittin molecules do ordinarily exist in an open pore state. Abbreviations: DOPC, 1,2-dioleoyl-sn-glyce;~o-3-phosphocholine; POPC, l-paimitoyl-2-oleoyl-sn-glycero-3-phosphocholine; DMPC, 1,2-dimyri~toyl-sn-glycero-3-phosphocholine; NBS, N-bromosuc- cinimide; HNBB, 2-hydroxy-5-nitrobenzylbromide; HPLC, high-per- formance liquid chromatography; CD, circular dichroism; BLM, blaL:k lipid membrane. Correspondence: G. Schwarz~ Biocenter, Department of Biophysical Chemistry, Klingelbergstrasse 70, CH-,1056 Basel, Switzerland. This is much too little for most experimental tech- niques to get a direct insight into the actual channel structure and function. On the other hand, titration of an aqueous peptide solution with lipid vesicles results in a substantial circular dichroism signal indicating that melittin strongly partitions into the lipid bilayer envi- ronment where it assumes a helical structure [3]. How a small part of this apparently non-pore form of the bilayer associated peptide will be turned into a voltage-sensitive porous aggregate is the crucial ques- tion regarding the molecular mechanism of the pore acti~ty. In aqueous solution at high salt concentrations the existence of a tetrameric aggregate is well estab- lished which is composed of amphipathic monomeric helices with their hydrophobic faces pointing to the inside and the opposite hydrophilic faces presenting themselves to the water phase [4]. Vice versa, one may model a melittin pore in the lipid bilayer moiety as a kind of barrel stave collection of membrane spanning helices (not necessarily only four) with the polar faces lining a water-filled pore and the nonpolar faces con- tacting the surrounding lipid.