Method of oriented circular dichroism

Wu, Yinghao; Huang, Huey W.; Olah, Glenn A.

doi:10.1016/s0006-3495(90)82599-6

Cited by 251 publications

(326 citation statements)

References 19 publications

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“…This minority part did not contribute to the diffraction analysis but was included in the OCD measurement. In general, OCD is not sensitive to the peptide orientation within a few degrees (16). The OCD spectra shown below also indicated that the whole samples were aligned within a few degrees.…”

Section: Resultsmentioning

confidence: 71%

Interaction of Antimicrobial Peptides with Lipopolysaccharides

et al. 2003

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We study the interaction of antimicrobial peptides with lipopolysaccharide (LPS) bilayers to understand how antimicrobial peptides interact with the LPS monolayer on the outer membrane of Gramnegative bacteria. LPS in water spontaneously forms a multilamellar structure composed of symmetric bilayers. We performed X-ray lamellar diffraction and wide-angle in-plane scattering to study the physical characteristics of LPS multilayers. The multilayer alignment of LPS is comparable to phospholipids. Thus, it is suitable for the application of oriented circular dichroism (OCD) to study the state of peptides in LPS bilayers. At high hydration levels, the chain melting temperature in multilamella detected by X-ray diffraction is the same as that of LPS aqueous dispersions, as measured by calorimetry. LPS has a strong CD, but with a careful subtraction of the lipid background, the OCD of peptides in LPS is measurable. The method was tested successfully with melittin. It was then applied to two representative antimicrobial peptides, magainin and protegrin. At peptide concentrations comparable to the physiological conditions, both peptides penetrate transmembrane in LPS bilayers. The results imply that antimicrobial peptides readily penetrate the LPS monolayer of the outer membrane.

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Section: Resultsmentioning

confidence: 71%

Interaction of Antimicrobial Peptides with Lipopolysaccharides

et al. 2003

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“…However, if the helix axis has a transmembrane orientation, the incident light is parallel to the axis and several transitions are degenerate giving a characteristic CD spectrum with a positive amplitude around 200 nm and a shift of the negative amplitude to longer wavelengths (225-230 nm) (Fig. 1D) (for more information about OCD spectroscopy see (11) and references therein). CD spectroscopy on oriented supported bilayers has been successfully applied to distinguish membranesurface and transmembrane helix orientation (12).…”

Section: Resultsmentioning

confidence: 99%

pH (low) insertion peptide (pHLIP) inserts across a lipid bilayer as a helix and exits by a different path

Andreev

Karabadzhak

Weerakkody

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

145

197

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What are the molecular events that occur when a peptide inserts across a membrane or exits from it? Using the pH-triggered insertion of the pH low insertion peptide to enable kinetic analysis, we show that insertion occurs in several steps, with rapid (0.1 sec) interfacial helix formation, followed by a much slower (100 sec) insertion pathway to give a transmembrane helix. The reverse process of unfolding and peptide exit from the bilayer core, which can be induced by a rapid rise of the pH from acidic to basic, proceeds approximately 400 times faster than folding/insertion and through different intermediate states. In the exit pathway, the helix-coil transition is initiated while the polypeptide is still inside the membrane. The peptide starts to exit when about 30% of the helix is unfolded, and continues a rapid exit as it unfolds inside the membrane. These insights may guide understanding of membrane protein folding/unfolding and the design of medically useful peptides for imaging and drug delivery.folding kinetics | helix formation | lipid-peptide interaction | membranes | transmembrane helix

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“…Sigma alamethicin is a mixture of components, principally alamethicin I (85% by high-performance liquid chromatography) and alamethicin II (12%), which differ by one amino acid (36). The same material has been used in all previous studies (9,11,33,34,37,38). Two grades of melittin were used, the sequencing grade (product no.…”

Section: Methodsmentioning

confidence: 99%

Energetics of Pore Formation Induced by Membrane Active Peptides

2004

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Antimicrobial peptides are known to form pores in cell membranes. We study this process in model bilayers of various lipid compositions. We use two of the best-studied peptides, alamethicin and melittin, to represent peptides making two types of pores, that is, barrel-stave pores and toroidal pores. In both cases, the key control variable is the concentration of the bound peptides in the lipid bilayers (expressed in the peptide-lipid molar ratio, P/L). The method of oriented circular dichroism (OCD) was used to monitor the peptide orientation in bilayers as a function of P/L. The same samples were scanned by X-ray diffraction to measure the bilayer thickness. In all cases, the bilayer thickness decreases linearly with P/L and then levels off after P/L exceeds a lipid-dependent critical value, (P/L)*. OCD spectra showed that the helical peptides are oriented parallel to the bilayers as long as P/L < (P/L)*, but as P/L increases over (P/L)*, an increasing fraction of peptides changed orientation to become perpendicular to the bilayer. We analyzed the data by assuming an internal membrane tension associated with the membrane thinning. The free energy containing this tension term leads to a relation explaining the P/L-dependence observed in the OCD and X-ray diffraction measurements. We extracted the experimental parameters from this thermodynamic relation. We believe that they are the quantities that characterize the peptide-lipid interactions related to the mechanism of pore formation. We discuss the meaning of these parameters and compare their values for different lipids and for the two different types of pores. These experimental parameters are useful for further molecular analysis and are excellent targets for molecular dynamic simulation studies.Membrane active peptides, including antimicrobials and toxins, are known to induce transmembrane pores. The first peptide discovered to do so is alamethicin (1, 2). At first, alamethicin was thought to induce pores (which were detected by ion conduction) only by a transmembrane electric potential (see review in ref 3). However, numerous experiments (4-8) indicated that alamethicin could insert into bilayers in the absence of an external field (see review in ref 9). Although it was believed that alamethicin insertion would create pores, a direct correlation with ion conduction was difficult to establish. Later, with the combination of oriented circular dichroism (10, 11) and neutron diffraction (12, 13), we showed the direct correlation between alamethicin insertion (without voltage) and transmembrane pores. Two other extensively studied peptides, bee venom toxin melittin (14) and frog peptide magainin (15), also exhibited similar behaviors. Pores were evidently formed by both melittin (16-18) and magainin (19,20) because they caused leakage of fluorescent dyes from lipid vesicles. In the last 15 years, a great variety of antimicrobial peptides have been shown to induce transmembrane pores in bacterial cells as well as in lipid vesicles (see reviews in refs 2...

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Method of oriented circular dichroism

Cited by 251 publications

References 19 publications

Interaction of Antimicrobial Peptides with Lipopolysaccharides

Interaction of Antimicrobial Peptides with Lipopolysaccharides

pH (low) insertion peptide (pHLIP) inserts across a lipid bilayer as a helix and exits by a different path

Energetics of Pore Formation Induced by Membrane Active Peptides

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