How to Measure and Analyze Tryptophan Fluorescence in Membranes Properly, and Why Bother?

Ladokhin, Alexey S.; Jayasinghe, Sajith; White, Stephen H.

doi:10.1006/abio.2000.4773

Cited by 424 publications

(505 citation statements)

References 28 publications

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“…In these cases, titration with bacterial cells did not cause significant variations in the fluorescent signal (Supplementary Figure 3), demonstrating the absence of any relevant scattering-related artifacts. 17,21 This conclusion was confirmed also by following peptide/bacteria association through variations in the dansyl fluorescence time-decay, which is not affected by scattering (Supplementary Figure 4). We calculated the fraction of peptide bound to bacterial membranes from the a Determined as the fraction of colony forming units (CFU) in a sample incubated for 2 h with different peptide concentrations, as compared with the CFU of the control at time zero, i.e., (4.5 ± 0.5) × 10 8 CFU/mL.…”

supporting

confidence: 53%

How Many Antimicrobial Peptide Molecules Kill a Bacterium? The Case of PMAP-23

Luca²,

et al. 2014

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Antimicrobial peptides (AMPs) kill bacteria mainly through the perturbation of their membranes and are promising compounds to fight drug resistance. Models of the mechanism of AMPs-induced membrane perturbation were developed based on experiments in liposomes, but their relevance for bacterial killing is debated. We determined the association of an analogue of the AMP PMAP-23 to Escherichia coli cells, under the same experimental conditions used to measure bactericidal activity. Killing took place only when bound peptides completely saturated bacterial membranes (10 6 −10 7 bound peptides per cell), indicating that the "carpet" model for the perturbation of artificial bilayers is representative of what happens in real bacteria. This finding supports the view that, at least for this peptide, a microbicidal mechanism is possible in vivo only at micromolar total peptide concentrations. We also showed that, notwithstanding their simplicity, liposomes represent a reliable model to characterize AMPs partition in bacterial membranes.

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confidence: 53%

How Many Antimicrobial Peptide Molecules Kill a Bacterium? The Case of PMAP-23

Luca²,

et al. 2014

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“…When introduced to anionic membrane models, all peptides displayed significant blue shifts in Trp k max values, ranging from 14 to 25 nm. These results provide evidence of peptide insertion into the bilayer 41 and effective removal of the peptides from the bulk aqueous environment even at concentrations below their MIC values. In contrast, the same series of peptides displays variable behavior in the presence of zwitterionic vesicles, which generally correlates with their R t values.…”

Section: Fluorescencementioning

confidence: 70%

Membrane interactions of designed cationic antimicrobial peptides: The two thresholds

2008

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ABSTRACT:Novel cationic antimicrobial peptides (CAPs) designed in our lab-typified by sequences such as KKKKKKAAX-AAXAAXAA-NH 2 , where X 5 Phe/Trp-display high antibacterial activity but exhibit little or no hemolytic activity towards human red blood cells even at high doses.To clarify the mechanism of their selectivity for bacterial versus mammalian membranes and to increase our understanding of the relationships between primary sequence and bioactivity, a library of derivatives was prepared by increasing segmental hydrophobicity, in which systematic substitutions of Ala for two, three, or four Leu residues were made. Conformationally constrained dimeric and cyclic derivatives were also synthesized. The peptides were examined for activity against pathogenic bacteria (Pseudomonas aeruginosa),

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“…Peptide partitioning into lipid bilayers was determined spectroscopically by fluorescence methods described by White et al(35;36). Partitioning of peptides into lipid bilayers was Fluorescence was recorded using excitation wavelength of 270 nm and scanning for emission between 290 and 500 nm with 8 nm bandwidths.…”

Section: Peptide Partitioningmentioning

confidence: 99%

“…Scans were subtracted for background and lipid scattering effects. and compensated for scattering loss by scaling to free tryptophan samples at the same lipid concentration (36). From these data, the area under the curve was taken as a measure of the intensity (I) of tryptophan fluorescence.…”

Section: Peptide Partitioningmentioning

confidence: 99%

β-Sheet Pore-Forming Peptides Selected from a Rational Combinatorial Library: Mechanism of Pore Formation in Lipid Vesicles and Activity in Biological Membranes

et al. 2007

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In a previous report we described the selection of potent, β-sheet pore-forming peptides from a combinatorial library designed to mimic membrane-spanning β-hairpins (Rausch JM, Marks JR and Wimley WC, (2005) PNAS, 102:10511-5). Here, we characterize their mechanism of action and compare the structure-function relationships in lipid vesicles to their activity in biological membranes. The pore-forming peptides bind to membrane interfaces and self-assemble into β-sheets that cause a transient burst of graded leakage across the bilayers. Despite the continued presence of the structured peptides in the bilayer, at most peptide concentrations leakage is incomplete and ceases quickly after peptide addition with a deactivation half-time of several minutes. Molecules up to 3,000 Da escape from the transient pores, but much larger molecules do not. Fluorescence spectroscopy and quenching showed that the peptides reside mainly on the bilayer surface and are partially exposed to water, rather than in a membrane-spanning state. The "carpet" or "sinking raft" model of peptide pore formation offers a viable explanation for our observations and suggests that the selected pore formers function with a mechanism that is similar to the natural pore-forming antimicrobial peptides. We therefore also characterized the antimicrobial and cytotoxic activity of these peptides. All peptides studied, including non pore-formers, had sterilizing antimicrobial activity against at least some microbes, and most have low activity against mammalian cell membranes. Thus, the structurefunction relationships that were apparent in the vesicle systems are similar to, but do not correlate completely with the activity of the same peptides in biological membranes. However, of the peptides tested, only the pore-formers selected in the high throughput screen have potent, broad-spectrum sterilizing activity against Gram-positive and Gram-negative bacteria as well as against fungi, while having only small lytic effects on human cells.A wide variety of pore-forming proteins and peptides take advantage of the discontinuous hydrophobicity of membrane-spanning β-sheets, which makes them well suited for undergoing transitions between hydrophilic and hydrophobic environments. For example, the β-barrel protein toxins including perfringolysin-O (1), α-hemolysin (2), and the anthrax protective antigen(3) pre-assemble a protein scaffold on membrane surfaces and then undergo a spontaneous transition in which loop sequences change conformation and are inserted into the membrane to form β-barrel pores. Similarly, there are many pore-forming, antimicrobial peptides (AMPs) 1 that are soluble in water and which self assemble into β-sheet-rich pores in membranes(4-6). To explore the structural determinants of folding and self-assembly of β- † Supported by the National Institutes of Health grant GM060000 *Corresponding author: Phone: 504-988-7076; Fax:504-988-2739, Email: wwimley@tulane.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available ...

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How to Measure and Analyze Tryptophan Fluorescence in Membranes Properly, and Why Bother?

Cited by 424 publications

References 28 publications

How Many Antimicrobial Peptide Molecules Kill a Bacterium? The Case of PMAP-23

How Many Antimicrobial Peptide Molecules Kill a Bacterium? The Case of PMAP-23

Membrane interactions of designed cationic antimicrobial peptides: The two thresholds

β-Sheet Pore-Forming Peptides Selected from a Rational Combinatorial Library: Mechanism of Pore Formation in Lipid Vesicles and Activity in Biological Membranes

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