Diffusion as a Probe of the Heterogeneity of Antimicrobial Peptide−Membrane Interactions

Smith-Dupont, Kathryn B.; Guo, Lin; Gai, Feng

doi:10.1021/bi100426p

Cited by 14 publications

(43 citation statements)

References 68 publications

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“…In addition, we found in many cases that the diffusion times spread over a wide range of time and are too long to be ascribed to the diffusions of peptide oligomers. Thus, we tentatively attributed the observed heterogeneity in lipid diffusion to AMP-induced membrane domain formation (31). While this interpretation is consistent with several studies (16-27), our previous data in themselves do not elucidate the peptide's role.…”

Section: Discussionmentioning

confidence: 84%

“…As a control experiment, we have also measured the τ D -distribution of a mpX-GUV sample that contains a trace amount of fluorescently labeled mpX (1 nM) and lipid (i.e., 0.002% TR-DHPE) (31). As shown (Figure 4), simultaneous excitation of the florescent peptide and lipid results in a τ D -distribution that is drastically different from that resulting from only the diffusion of the peptide.…”

Section: Resultsmentioning

confidence: 99%

“…Recently we demonstrated that FCS is a sensitive method to probe AMP-membrane interactions (31). In particular, we showed that binding of an AMP can cause the diffusion times of the constituent lipids of two model membranes to significantly deviate from their intrinsic values (i.e., those measured in the absence of any peptides).…”

Section: Discussionmentioning

confidence: 99%

“…As shown (Figures 5 and 6), in both types of membranes mag2 exhibits a τ D -distribution that shows a high probability of occurrence within the time range of 100 – 600 μs. It is known that mpX binds to POPC membranes at least 10 times stronger than mag2 (42) and that in the entire concentration range studied mag2 is much less effective than mpX at altering the diffusivity of the lipids in POPC membranes (31). Thus, for POPC GUVs such fast diffusing species (i.e., those diffusing faster than the lipids) may correspond to peptides that are only weakly associated with the membrane surface.…”

Section: Discussionmentioning

confidence: 99%

“…Giant unilamellar vesicles were prepared according to an electroswelling method (35) and the details have been described previously (31). …”

Section: Methodsmentioning

confidence: 99%

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Diffusion as a Probe of Peptide-Induced Membrane Domain Formation

2011

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Recently we have shown that association with an antimicrobial peptide (AMP) can drastically alter the diffusion behavior of the constituent lipids in model membranes (Biochemistry 49,(4672)(4673)(4674)(4675)(4676)(4677)(4678). In particular, we found that the diffusion time of a tracer fluorescent lipid through a confocal volume measured via fluorescence correlation spectroscopy (FCS) is distributed over a wide range of timescales, indicating the formation of stable and/or transient membrane species that have different mobilities. A simple estimate, however, suggested that the slow diffusing species are too large to be attributed to AMP oligomers or pores that are tightly bound to a small number of lipids. Thus, we tentatively ascribed them to membrane domains and/or clusters that possess distinctively different diffusion properties. In order to further substantiate our previous conjecture, herein we study the diffusion behavior of the membrane-bound peptide molecules using the same AMPs and model membranes. Our results show, in contrast to our previous findings, that the diffusion times of the membrane-bound peptides exhibit a much narrower distribution that is more similar to that of the lipids in peptide-free membranes. Thus, taken together, these results indicate that while AMP molecules prompt domain formation in membranes, they are not tightly associated with the lipid domains thus formed. Instead, they are likely located at the boundary regions separating various domains and acting as mobile fences. KeywordsMembrane; Antimicrobial Peptide; Fluorescence Correlation Spectroscopy; Diffusion; Membrane Domain FormationThe mechanism of action of antimicrobial peptides (AMPs) has been the subject of extensive studies (1-7). Findings from these studies have prompted the formulation of several models of membrane disruption by AMPs. For example, the ability of AMPs to disrupt the structural integrity of the targeted cell membranes has been attributed to (a) barrel-stave or/and toroidal pore formation (8,9), (b) membrane-dissolution in a detergentlike manner (10,11), (c) formation of lipid-peptide domains (12-25), (d) segregation of anionic lipids and zwitterionic lipids (23-27), or (e) formation of non-lamellar phases (28-30). Recently, we have shown that the diffusivity of individual lipids in an AMP-bound membrane, probed via fluorescence correlation spectroscopy (FCS), provides a sensitive means to monitor how AMP binding affects the membrane's structure and dynamics (31), † Supported by the NIH (GM-065978) and the NSF (DMR05-20020).* To whom correspondence should be addressed; gai@sas.upenn.edu; Phone: 215-573-6256; Fax: 215-573-2112 even at very low peptide/lipid ratios. As shown (Figure 1), results obtained from two wellstudied AMPs, magainin 2 (mag2) and mastoparan X (mpX), showed that AMP-binding can drastically alter the lipid diffusion behavior, and that at relatively high peptide/lipid ratios the lipid diffusion times through a well defined confocal volume, acquired by repeating measurements, are...

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Section: Discussionmentioning

confidence: 84%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…Giant unilamellar vesicles were prepared according to an electroswelling method (35) and the details have been described previously (31). …”

Section: Methodsmentioning

confidence: 99%

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Diffusion as a Probe of Peptide-Induced Membrane Domain Formation

2011

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Characterization of sodium dodecylsulphate and dodecylphosphocholine mixed micelles through NMR and dynamic light scattering

Manzo

Carboni

Rinaldi

et al. 2013

Magnetic Reson in Chemistry

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The complexity of biological membranes leads to the use of extremely simplified models in biophysical investigations of membrane-bound proteins and peptides. Liposomes are probably the most widely used membrane models due, especially, to their versatility in terms of electric charge and size. However, liquid-state NMR suffers the lack of such a model, because even the smallest liposomes slowly tumble in solution, resulting in a dramatic signals broadening. Micelles are typically used as good substitutes, with sodium dodecylsulphate (SDS) and dodecylphosphocholine (DPC) being the most widely employed surfactants. However, they are always used separately to mimic prokaryotic and eukaryotic membranes, respectively, and accurate investigations as a function of surface charge cannot be performed. In this work, the critical micelle concentration (CMC) of binary mixtures with different SDS/DPC ratios has been determined by following the chemical shift variation of selected (1)H and (31)P NMR signals as a function of total surfactant concentration. The regular solution theory and the Motomura's formalism have been applied to characterize the micellization both in water and in phosphate buffer saline, and results were compared with those obtained directly from the experimental NMR chemical shift. The ζ-potential and size distribution of the mixed micelles have been estimated with dynamic light scattering measurements. Results showed that SDS and DPC are synergic and can be used together to prepare mixed micelles with different negative/zwitterionic surfactants molar ratio.

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Spectroscopic studies of protein folding: Linear and nonlinear methods

2011

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Although protein folding is a simple outcome of the underlying thermodynamics, arriving at a quantitative and predictive understanding of how proteins fold nevertheless poses huge challenges. Therefore, both advanced experimental and computational methods are continuously being developed and refined to probe and reveal the atomistic details of protein folding dynamics and mechanisms. Herein, we provide a concise review of recent developments in spectroscopic studies of protein folding, with a focus on new triggering and probing methods. In particular, we describe several laser-based techniques for triggering protein folding/unfolding on the picosecond and/or nanosecond timescales and various linear and nonlinear spectroscopic techniques for interrogating protein conformations, conformational transitions, and dynamics.

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Diffusion as a Probe of the Heterogeneity of Antimicrobial Peptide−Membrane Interactions

Cited by 14 publications

References 68 publications

Diffusion as a Probe of Peptide-Induced Membrane Domain Formation

Diffusion as a Probe of Peptide-Induced Membrane Domain Formation

Characterization of sodium dodecylsulphate and dodecylphosphocholine mixed micelles through NMR and dynamic light scattering

Spectroscopic studies of protein folding: Linear and nonlinear methods

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