Stabilizing effects of 2‐methylalanine residues on β‐turns and α‐helices

Jung, Günther; Bosch, R.; Katz, Ellen B.; Schmitt, H.; Voges, K.-P.; Winter, Werner

doi:10.1002/bip.360220133

Cited by 42 publications

(10 citation statements)

References 19 publications

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“…As most of these residues occur in the N-terminus, this interpretation is in agreement with the data which suggests that the N-terminus of alamethicin is more stable than the C-terminus (Esposito et al, 1987;Yee and O'Neil, 1992;Franklin et al, 1994;Yee et al, 1995) and that Ala(Me) residues can give remarkable conformational stability to helical peptides (Augspurger et al, 1995). The presence of an N-acetyl group may add additional stability to the N-terminus of the helix (Chakrabartty et al, 1993;Jung et al, 1983).…”

Section: Temperature Dependence Of the Carbonyl Chemical Shiftssupporting

confidence: 88%

The Interactions with Solvent, Heat Stability, and ¹³C‐Labelling of Alamethicin, an Ion‐Channel‐Forming Peptide

Yee

Marat

O'Neil

1997

European Journal of Biochemistry

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The peptide alamethicin was labelled with I3C and "N by growing the fungus Trichoderma viride in a medium containing [U-"C]glucose and K"N0,. Spin-echo difference spectroscopy showed that I3C was incorporated to a level of about SO% and "N to about 98%. Incorporation of "C into the peptide provided residue-specific probes of the interactions with solvent and heat stability of this ion-channelforming peptide. All of the carbonyl carbons and the a-carbons of the a-aminoisobutyric acid [Ala(Me)J residues of alamethicin in methanol were assigned using two-dimensional and three-dimensional heteronuclear correlation experiments. Measurements of '.Ic,, revealed hydrogen bonding with solvent at residues 1 and 19 at the ends of the peptide and at Glyl 1 in the middle. The data also support the thesis [see Juranic, N., Ilich, P. K. & Macara, S. (1995) J. Am. that intramolecular hydrogen bonds in proteins and peptides are weaker than hydrogen bonds to solvent. The sensitivity of alamethicin carbonyl and proton chemical shifts to perturbation by dimethyl sulfoxide correlates well with the calculated solvent accessibilities of the carbonyls in the crystal structures and reveals residues in the middle of the peptide and at the C-terminus which interact with solvent. Taken together with the 'JC,, measurements, the data support a model in which hydrogen bonding to solvent at the GlylULeul2 amide could provide a site of hydration in the interior of the alamethicin channel structure. The temperature dependencies of the carbonyl chemical shifts support the suggestion that the peptide is flexible in the regions where solvent interacts with the backbone of the peptide. The linear temperature dependence of the carbonyl chemical shifts and molar ellipticity indicate that, due to steric constraints at the Ala(Me) residues, the peptide foldinghnfolding transition is non-cooperative and that the peptide is remarkably heat stable. Keywords:alamethicin ; "C labelling; dimethyl sulfoxide ; ion channel ; NMR.Alamethicin is a 20-amino-acid antibiotic peptide secreted by the fungus Trichoderma viride. It forms a voltage-gated conductance in lipid bilayers (Eisenberg et al., 1973;Latore et al., 1981) and is one of the most extensively studied models for vertebrate ion channels [for reviews see Sansom (1993) and Cafiso (1994)l. The peptide has eight residues of a-aminoisobutyric acid [Ala(Me)] which have a-methyl in place of the usual a-hydrogen found in the L-amino acids. The mechanism by which alamethicin inserts into membranes, forms ion channels, and is regulated by an applied voltage is unclear. You et al.(1 996) recently showed that covalent dimers of alamethicin form stabilized ion channels with a single predominant conductance state. They suggest that the channels consist of a bundle of six parallel monomers. Evidence for antiparallel interactions between monomers dissolved in methanol was obtained by North et al. (1994). Their measurements of the paramagnetic enhancements of nuclear relaxation also indicated that the peptide backbone c...

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Section: Temperature Dependence Of the Carbonyl Chemical Shiftssupporting

confidence: 88%

The Interactions with Solvent, Heat Stability, and ¹³C‐Labelling of Alamethicin, an Ion‐Channel‐Forming Peptide

Yee

Marat

O'Neil

1997

European Journal of Biochemistry

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“…The flip-flop gating mechanism turns out to be the best framework for an understanding of the molecular mechanisms which lead to voltage-dependent formation of this type of channels. A preliminary report on part of the data has previously been presented (Jung et al, 1983b).…”

Section: Discussionmentioning

confidence: 99%

Voltage-dependent channel formation by rods of helical polypeptides

et al. 1986

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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)

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“…For this purpose we used a hydrophobic model of the antibiotic alamethicin [23], the fifteen amino acid polypeptide Boc-(L-Ala-Aib-LAla-Aib-L-Ala)3-OMe (where Boc = t-butyloxycarbonyl, ALIa = alznine, Aib = aminoisobutyric acid and OMe = methoxy), called P15, which was shown to be largely present as a-types helices in lipophilic solutions [24] due to the high aminoisobutyric acid content of the peptide [25]. This polypeptide and several analogues with different number of repeat units have been found to form voltage-dependent ion-conducting channels in black lipid membranes [26].…”

Section: Introductionmentioning

confidence: 99%

Importance of the tryptophans of gramicidin for its lipid structure modulating activity in lysophosphatidylcholine and phosphatidylethanolamine model membranes. A comparative study employing gramicidin analogs and a synthetic α-helical hydrophobic polypeptide

Aranda

Killian

Kruijff

1987

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The importance of the tryptophan residues of gramicidin for the lipid structure modulating activity of this pentadecapeptide was investigated by studying the interaction of gramicidin analogs A, B, C (which have a tryptophan, phenylalanine and tyrosine in position 11, respectively) and tryptophan-N-formylated gramicidin (in which the four tryptophan residues have been formylated) with several phospholipid systems. In addition an a-helical model pentadecapeptide (P15) was studied to further test the specificity of the gramicidin-lipid interaction. DSC experiments showed that all the gramicidin analogs produced a significant decrease in the gel to liquid-crystalline transition enthalpy of dipalmitoylphosphatidylcholine. The P15 peptide was much less effective in this respect. In dielaidoyiphosphatidylethanolamine the gel ~ liquid-crystalline transition enthalpy was much less affected by the incorporation of these molecules. In this lipid system tryptophan-Nformylated gramicidin was found to be the most ineffective. 31p-NMR and small angle X-ray diffraction experiments showed that the ability of the peptides to induce bilayer structures in paimitoyilysophosphatidyicholine and H n phase promotion in dielaidoylphosphatidylethanolamine systems follows the order. gramicidin A' (natural mixture) -~ gramicidin A > gramicidin B = gramicidin C > tryptophan-N-formylated gramicidin > P15. These results support the hypothesis that the shape of gramicidin and its aggregational behaviour, in which the tryptophan residues play an essential role, are major determinants in the unique lipid structure modulating activity of gramicidin.

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Stabilizing effects of 2‐methylalanine residues on β‐turns and α‐helices

Cited by 42 publications

References 19 publications

The Interactions with Solvent, Heat Stability, and ¹³C‐Labelling of Alamethicin, an Ion‐Channel‐Forming Peptide

The Interactions with Solvent, Heat Stability, and ¹³C‐Labelling of Alamethicin, an Ion‐Channel‐Forming Peptide

Voltage-dependent channel formation by rods of helical polypeptides

Importance of the tryptophans of gramicidin for its lipid structure modulating activity in lysophosphatidylcholine and phosphatidylethanolamine model membranes. A comparative study employing gramicidin analogs and a synthetic α-helical hydrophobic polypeptide

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Stabilizing effects of 2‐methylalanine residues on β‐turns and α‐helices

Cited by 42 publications

References 19 publications

The Interactions with Solvent, Heat Stability, and 13C‐Labelling of Alamethicin, an Ion‐Channel‐Forming Peptide

The Interactions with Solvent, Heat Stability, and 13C‐Labelling of Alamethicin, an Ion‐Channel‐Forming Peptide

Voltage-dependent channel formation by rods of helical polypeptides

Importance of the tryptophans of gramicidin for its lipid structure modulating activity in lysophosphatidylcholine and phosphatidylethanolamine model membranes. A comparative study employing gramicidin analogs and a synthetic α-helical hydrophobic polypeptide

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The Interactions with Solvent, Heat Stability, and ¹³C‐Labelling of Alamethicin, an Ion‐Channel‐Forming Peptide

The Interactions with Solvent, Heat Stability, and ¹³C‐Labelling of Alamethicin, an Ion‐Channel‐Forming Peptide