Biomedical subjects

T C Vogt

Publications and source records attributed to T C Vogt.

8 recordsLinked to original sources

PubMed1999

The interactions of histidine-containing amphipathic helical peptide antibiotics with lipid bilayers. The effects of charges and pH.

The alpha-helix of the designed amphipathic peptide antibiotic LAH(4 )(KKALLALALHHLAHLALHLALALKKA-NH(2)) strongly interacts with phospholipid membranes. The peptide is oriented parallel to the membrane surface under acidic conditions, but transmembrane at physiological pH (Bechinger, B. (1996) J. Mol. Biol. 263, 768-775). LAH(4) exhibits antibiotic activities against Escherichia coli and Bacillus subtilis; the peptide does not, however, lyse human red blood cells at bacteriocidal concentrations. The antibiotic activities of LAH(4) are 2 orders of magnitude more pronounced at pH 5 when compared with pH 7.5. Although peptide association at low pH is reduced when compared with pH 7.5, the release of the fluorophore calcein from large unilamellar 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol vesicles is more pronounced at pH values where LAH(4) adopts an orientation along the membrane surface. The calcein release experiments thereby parallel the results obtained in antibiotic assays. Despite a much higher degree of association, calcein release activity of LAH(4) is significantly decreased for negatively charged membranes. Pronounced differences in the interactions of LAH(4) with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol or 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine membranes also become apparent when the mechanisms of dye release are investigated. The results presented in this paper support models in which antibiotic activity is caused by detergent-like membrane destabilization, rather than pore formation by helical peptides in transmembrane alignments.

Amino Acid Sequence↗

PubMed1999

Peptide structural analysis by solid-state NMR spectroscopy.

Solid-state nmr spectroscopy provides a robust method for investigating polypeptides that have been prepared by chemical synthesis and that are immobilized by strong interactions with solid surfaces or large macroscopic complexes. Solid-state nmr spectroscopy has been widely used to investigate membrane polypeptides or peptide aggregates such as amyloid fibrils. Whereas magic angle spinning solid-state nmr spectroscopy allows one to measure distances and dihedral angles with high accuracy, static membrane samples that are aligned with respect to the magnetic field direction allow one to determine the secondary structure of bound polypeptides and their orientation with respect to the bilayer normal. Peptide dynamics and the effect of polypeptides on the macroscopic phase preference of phospholipid membranes have been investigated in nonoriented samples. Investigations of the structure and topology of membrane channels, peptide antibiotics, signal sequences as well as model systems that allow one to dissect the interaction contributions in phospholipid membranes will be presented in greater detail.

Anti-Bacterial Agents↗

PubMed1996

Conformation of the acylation site of palmitoylgramicidin in lipid bilayers of dimyristoylphosphatidylcholine.

Gramicidin A(gA) can be palmitoylated by means of an ester linkage to the OH group of the terminal ethanolamine that sits at the membrane-water interface in the functional gA channel. We have investigated palmitoyl-gA as a model transmembrane acylprotein. Ethanolamine-d(4) (NH(2)CD(2)CD(2)OH) was incorporated into gA by total synthesis, and a portion of the labeled gA was palmitoylated. Solid-state (2)H-NMR spectra of acyl- and nonacyl-gA in hydrated dimyristoylphosphatidylcholine (DMPC) bilayers were compared. The spectra for both oriented and nonoriented samples at 4 and at 40 degrees C indicate that the ethanolamine of gA is highly mobile prior to acylation, but essentially immobile after palmitoylation. The (2)H quadrupolar splittings allow the conformation of the ethanolamine group in acyl-gA to be determined. By combining our data with the previously determined quadrupolar splittings for deuterium labels on the palmitoyl chain [Vogt, T.C.B., Killian, J.A., & de Kruijff, B. (1994) Biochemistry 33, 2063-2070], we also propose a model for the acyl chain. The ethanolamine group rotates over Leu(10) and toward the outside of the gA channel's cylinder upon acylation, so that the attached acyl chain passes between the side chains of Trp(9) and Leu(10). To accommodate the acyl chain, the six-membered portion of the indole ring of Trp(9) is displaced by about 0.9 angstroms, by means of 1-2 degree rotations in chi(1) and chi(2).

Gramicidin↗

PubMed1995

Palmitoylation-induced conformational changes of specific side chains in the gramicidin transmembrane channel.

To gain insight into the structural consequences of acylation for membrane proteins, we have covalently attached palmitic acid to the ethanolamine end of gramicidin A (gA), which functions as a well-characterized cation-selective membrane channel. Next, we investigated by NMR methods the effect of acylation on the side chains of Trp9, Leu10, and Trp11, which are expected to be close to the acyl chain, and of Val7, which is expected to be far from the acyl chain. Two-dimensional NMR spectroscopy in a sodium dodecyl sulfate (SDS) environment suggests that one of the beta-hydrogens of Leu10 of gA is severely shielded by a nearby aromatic ring. This shielding disappears upon acylation. Deuterium NMR spectra for labeled samples in hydrated dimyristoylphosphatidylcholine (DMPC) bilayers show that, for the major gA conformation, the (deuterated) side chains of Trp9 and Leu10 are markedly influenced by acylation, whereas the side chains of Val7 and Trp11 are essentially unaffected. The NMR results in both environments suggest that the indole ring of Trp9 is situated near the side chain of Leu10 and moves away upon acylation. We propose that acylation provides a subtle mechanism to modulate protein and lipid interactions and to regulate the stability and function of proteins within membranes.

Acylation↗

PubMed1994

The influence of acylation on the lipid structure modulating properties of the transmembrane polypeptide gramicidin.

In order to get insight into the effect of acylation of a transmembrane polypeptide on the interaction of the polypeptide with the membrane lipids we used 31P-NMR to investigate the influence of acylated gramicidins on the polymorphic phase behavior of hydrated dispersions of 1-palmitoyllysophosphatidylcholine (lyso-PC), 1,2-dioleoylphosphatidylcholine (DOPC) and 1,2-dielaidoylphosphatidylethanolamine (DEPE). Palmitoylgramicidin induces a micelle to extended bilayer organization in lyso-PC with a slightly lower efficiency than the parent gramicidin molecule. In DOPC and DEPE acylgramicidins induce the formation of HII phase at the expense of a bilayer organization with a similar high efficiency as gramicidin. The ability of acylgramicidin to induce lipid mixing between vesicles prepared of DOPC was decreased relative to gramicidin. The results are discussed in the light of the proposed models for gramicidin-induced HII phase formation and emphasize that gramicidin itself has a very strong lipid structure modulating activity.

Acylation↗

PubMed1994

Structure and dynamics of the acyl chain of a transmembrane polypeptide.

We have used acylated analogs of gramicidin as a model to study the interaction between a covalently coupled fatty acid and the hydrophobic part of a membrane-spanning protein in a bilayer environment. The acyl chain was covalently coupled to the C-terminal ethanolamine group of gramicidin which is located near the membrane interface, mimicking a situation found in acylated proteins. Either perdeuterated palmitic acid or palmitic acid deuterated at only C2, C3, C5-6, C7-8, C9, or C13 was coupled to gramicidin and examined by 2H-NMR in oriented bilayers of dimyristoylphosphatidylcholine. In this way, quadrupolar splittings of deuterons at specific carbons were assigned. The quadrupolar splittings and T1 values were compared to those of free palmitic acid in oriented bilayers, with and without gramicidin. The results indicate that the covalently coupled fatty acid is highly immobilized near the carboxyl terminus because double quadrupolar splittings and very low T1 values (4 ms) were found for the -CD2- deuterons at carbon atoms C2 and C3. Control experiments with free fatty acid showed single quadrupolar splittings and higher T1 values for this segment of the fatty acid. Molecular modeling of the carboxy-terminal segment of the covalently coupled acyl chain suggested that it has a defined structure with a bend near its attachment site. In contrast, the methyl end (C10-C16) of the covalently coupled fatty acid had quadrupolar splittings and T1 values very similar to those found for free fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Gramicidin↗

PubMed1992

Influence of acylation on the channel characteristics of gramicidin A.

The influence of acylation on the conductance, average duration, and channel-forming potency of channels formed by gramicidin A analogues was investigated using single-channel and multichannel techniques. Lauroyl-, myristoyl-, palmitoyl-, stearoyl-, and oleoylgramicidin A were prepared by covalent coupling of that fatty acid to the C-terminal ethanolamine group. Acylation of gramicidin A does not affect the single-channel conductance or the minichannel frequency in diphytanoylphosphatidylcholine/n-decane black lipid membranes. However, the average duration of all acylgramicidin channels was increased approximately 5-fold as compared to unmodified gramicidin A, which has a duration of 0.9 s at 200-mV applied potential. Somewhat surprisingly the rate of channel formation of the acylgramicidins is decreased relative to gramicidin A: lauroyl- and stearoylgramicidin are approximately 200 times less effective in channel formation as compared to gramicidin A. We conclude that channels formed by the acylgramicidins and by gramicidin A are structurally and conformationally equivalent.

Acylation↗

PubMed1991

Synthesis of acylated gramicidins and the influence of acylation on the interfacial properties and conformational behavior of gramicidin A.

Five gramicidin A analogs were synthesized in which various acyl chains, differing in length and unsaturation, were covalently coupled to the C-terminal ethanolamine group. The analogs were characterized by various spectroscopic techniques and their molecular properties were investigated using monolayer techniques and circular dichroism. It is demonstrated that neither the interfacial properties nor the conformational behavior of gramicidin A at the air/water interface are seriously affected upon acylation. It is proposed that at the limiting area the gramicidin molecule is oriented with its C-terminus towards the subphase with the covalently coupled acylchain located parallel to the helical axis in between the protruding tryptophans. Circular dichroism experiments, in which gramicidin-containing vesicles were prepared from different organic solvents, indicate that the presence of a covalently coupled fatty acylchain tends to stabilize the beta 6.3 helical conformation. It is demonstrated that, like for gramicidin A, also for the acylgramicidins the single-stranded beta 6.3 helical conformation, or channel conformation, is the preferred conformation upon incorporation in bilayers.

Acylation↗