The voltage-dependence of channel formation by alamethicin and it natural analogues can be described by a dipole flip-flop gating model, based on electric field-induced transbilayer orientational movements of single molecules. These field-induced changes in orientation result from the large permanent dipole moment of alamethicin, which adopts alpha-helical conformation in hydrophobic medium. It was, therefore, supposed that the only structural requirement for voltage-dependent formation of alamethicin-type channels might be a rigid lipophilic helical segment of minimum length. In order to test this hypothesis we synthesized a family of lipophilic polypeptides--Boc-(Ala-Aib-Ala-Aib-Ala)n-OMe, n = 1-4--which adopt alpha-helical conformation for n = 2-4 and studied their interaction with planar lipid bilayers. Surprisingly, despite their large difference in chain length, all four polypeptides showed quantitatively similar behavior. At low field strength of the membrane electric field these polypeptides induce a significant, almost voltage-independent increase of the bilayer conductivity. At high field strength, however, a strongly voltage-dependent conductance increase occurs similar to that observed with alamethicin. It results from the opening of a multitude of ion translocating channels within the membrane phase. The steady-state voltage-dependent conductance depends on the 8th-9th power of polypeptide concentration and involves the transfer of 4-5 formal elementary charges. From the power dependences on polypeptide concentration and applied voltage of the time constants in voltage-jump current-relaxation experiments, it is concluded that channels could be formed from preexisting dodecamer aggregates by the simultaneous reorientation of six formal elementary charges. Channels exhibit large conductance values of several nS, which become larger towards shorter polypeptide chain length. A mean channel diameter of 19 A is estimated corresponding roughly to the lumen diameter of a barrel comprised of 10 alpha-helical staves. Similar to experiments with the N-terminal Boc-derivative of alamethicin we did not observe the burst sequence of nonintegral conductance steps typical of natural (N-terminal Ac-Aib)-alamethicin. Saturation in current/voltage curves as well as current inactivation in voltage-jump current-relaxation experiments are found. This may be understood by assuming that channels are generated as dodecamers but, while reaching the steady state, reduce their size to that of an octamer or nonamer. We conclude that the overall behavior of these synthetic polypeptides is very similar to that of alamethicin.(ABSTRACT TRUNCATED AT 400 WORDS)

No abstract

The Na+-Ca2+ exchanger plays an important role in cardiac contractility by moving Ca2+ across the plasma membrane during excitation-contraction coupling. A 20 amino acid peptide, XIP, synthesized to mimic a region of the exchanger, inhibits exchange activity. We identify here amino acid residues important for inhibitory function. Effects of modified peptides on Na+-Ca2+ exchange activity were determined. Exchange activity was assessed as 45Ca2+ uptake into Na+-loaded cardiac sarcolemmal vesicles. We find that the entire length of XIP is important for maximal potency, though the major inhibitory components are between residues 5 and 16. Basic and aromatic residues are most important for the inhibitory function of XIP. Substitutions of arginine 12 and arginine 14 with alanine or glutamine dramatically decrease the potency of XIP, suggesting that these residues play a key role in possible charge-charge interactions. Substitutions of other basic residues with alanines or glutamines had less effect on the potency of XIP. All aromatic residues participate in binding with the exchanger, probably via hydrophobic interactions as indicated by tryptophan fluorescence. A tyrosine is required at position 6 for maximal inhibition and phenylalanine 5 and tyrosine 8 can only be replaced by other aromatic residues. Tyrosine 10 and tyrosine 13 can be replaced with other bulky residues. A specific conformation of XIP, with structural constrains provided by all parts of the molecule, is required for optimal inhibitory function.

Boc-L-Ala-Aib-L-Ala-OMe (1) crystallizes in the space group P2, with a = 11.732(1), b = 6.013(1), c = 14.195(2) A, 0 = 91.76(1)', and Z = 2 ( R value for 3089 reflexions: 0.047). The peptide adopts a new type of B-turn with a very wide 4 + 1 hydrogen bond distance of 3.621 A. As for Ac-r-Ala-Aib-L-Ala-Me this distortion can be attributed to strong intermolecular hydrogen bonds forming a two-dimensional network in the bc plane. Temperature and solvent dependent 'H and I3C NMR reveal a hydrogen bond from Boc-CO to NH-3Ala in solution, E / Z isomerism of the Boc urethane bond, and a large magnetic nonequivalence of the two geminal Aib methyl groups. The unusual conformation of 1 is reflected also in its CD spectrum, which differs from most of comparable Aib containing tripeptides. Konformation von Boc-L-Ala-Aib-I.-Ala-OMe im Kristall und in LosungRoc-L-Ala-Aib-L-Ala-OMe (1) kristallisert in der Raumgruppe P2, mit a = 11.732(1), b = 6.013(1), c = 14.195(2) A, P = 91.76(1)' und Z = 2 (R-Faktor fur 3089 Reflexe: 0.047). Das Peptid bildet einen neuen Typ eines P-Turns mit einer sehr langen 4 -+ 1-Wasserstoffbrucke (3.621 A).Diese Aufweitung laOt sich wie bei Ac-~-Ala-Aib-~-Ala-OMe mi: der Ausbildung eines zweidimensionalen Netzes von starken intermolekularen Wasserstoffbrucken in der bc-Ebene erklaren. Temperatur-und L~sungsmittel-abhangige 'H-und 13C-NMR zeigen eine Wasserstoffbriicke von Boc-CO zu NH -'Ala in Lbsung, E/Z-Isomerie der Boc-Urethanbindung und eine grol3e magnetische Nichtaquivalenz der beiden geminalen Aib-Methylgruppen. Die ungewohnliche Konformation von 1 spiegelt sich auch im CD-Spektrum wider, das sich von den meisten Aib-Tripeptiden unterscheidet.The particular role of a-aminoisobutyryl residues for the restriction of conformational mobility of peptide backbones has been discussed mainly in this journal in context with the following almost complete oligopeptide series of X-ray structures: Boc-Aib-OH 'I, Boc-L-Ala-Aib-OH", Boc-Gly-L-Ala-Aib-OMe", Ac-L-Ala-Aib-~-Ala-OMe~), BocAib-~-Ala-Aib-L-Ala-Aib-OMe~.~', and the a-helical Boc-L-Ala-Aib-L-Ala-Aib-L-Ala-~-Glu(OBzl)-L-Ala-Aib-L-Ala-Aib-L-Ala-OMe~~~'. We refer also t o the literature of other groups working i n this field, which is cited in o u r previous papers'-9). Interest in the conformation of 2-methylalanyl residues-containing peptides steadily increases as shown on publications, which appeared recently" 'j).The present study o n the tripeptide Boc-L-Ala-Aib-L-Ala-OMe (1) was undertaken in order t o clarify the following: Is the new type of an unusually widened and flat 0-turn 0 Verlag

Synopsis13C-, 'H-nmr, CD, and x-ray crystallography revealed @-turns of type 111 for BOC-G~Y-LAla-Aib-OMe, Boc-L-Ala-Aib-L-Ala-OMe; the 3lo-helix for Boc-Aib-L-Ala-Aib-L-Ala-AibOMe; and antiparallel arranged a-helices for Boc-L-Ala-Aib-Ala-Aib-Ala-Glu(0Bzl)-AlaAib-Ala-Aib-Ala-OMe. An N-terminal rigid a-helical segment is found in the polypeptide antibiotics alamethicin, suzukacillin, and trichotoxin. The a-helix dipole is essential for their voltage-dependent pore formation in lipid bilayer membranes, which is explained by a flip-flop gating mechanism based on dipole-dipole interactions of parallel and antiparallel arranged a-helices within oligomeric structures. POTENTIAL-DEPENDENT PORE FORMATIONMultilevel voltage-dependent pore formation in lipid membranes can be induced by the natural amphiphilic polypeptides alamethicin F 30150,' trichotoxin A 40/50,2.3 a synthetic n~nadecapeptide~,~, suzukacillin A,6 several chemically modified analogs of these pep tide^,^ and the bee venom constituent melittin (Fig. 1): These experiments indicate that a lipophilic N-terminal a-helical segment is a sufficient prerequisite for voltage-dependent pore f o r m a t i~n~,~ and hemolytic p r o p e r t i e~.~J~ The unusual a-aminoisobutyric acid residues, the N-terminal acyl group, and even the proline and C-terminal amino alcohols (phenylalaninol, valinol-see Fig. 1) are obviously inessential, as shown by the activities of mellitin and the synthetic Aib polypeptides (e.g. the eicosapeptide; Fig. 1).However, a sufficient length of the helical segment5S1lJ2 and, in particular, its intrinsic dipole moment are the only important factors for the pore-forming activities. For example, the synthetic hexadecapeptide Boc-(~-Ala-Aib)~-Pro-Ala-Aib-Aib-Glu(OBzl)-Gln-OMe~~~ and several a-helical undeca-and dodecapeptide fragments isolated from the natural polypeptide antibiotics are i n a~t i v e .~ Minute differences in the structures of the amphiphilic polypeptides reflect also in their pore-forming properties. Thus, in the case of the somewhat shorter peptide trichotoxin A 40150, no stable pore states are recorded, in contrast to the stable, multilevel pore states of the alamethicins and suzukacillins. On the other hand a C-ter-