Structural Origins of Cholesterol Accelerated Lipid Flip-Flop Studied by Sum-Frequency Vibrational Spectroscopy

Allhusen, John S.; Kimball, Dylan R.; Conboy, John C.

doi:10.1021/acs.jpcb.6b01254

Cited by 22 publications

(30 citation statements)

References 93 publications

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“…As the lipid translocation rate (flipping) is very slow (τ 1/2 > 6 h in POPC membranes) , it is not surprising that no exchange of positively charged lipids between the two leaflets occurred during the time of our experiments. We did not find any data for the flipping rate of DC‐cholesterol (normal cholesterol moves freely between the two leaflets), but the presence of cholesterol derivatives has been reported to increase the flipping rate of phospholipids .…”

Section: Resultsmentioning

confidence: 57%

Delivery of membrane proteins into small and giant unilamellar vesicles by charge‐mediated fusion

et al. 2016

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One of the current challenges in synthetic biology is the production of stable membrane mimetic systems and the insertion of components in these systems. Here, we employ fusion of oppositely charged liposomes to deliver separately reconstituted membrane proteins into a common lipid bilayer. After a systematic evaluation of different lipid compositions by lipid mixing and size distribution analysis, suitable conditions were further investigated for proteoliposome fusion. With this technique, we functionally coreconstituted bo 3 oxidase and ATP synthase from Escherichia coli into unilamellar liposomes ranging from 100 nm to 50 lm in size. The presented method is a simple and versatile tool for oriented membrane protein reconstitution to produce biomimetic systems with increased complexity.

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Section: Resultsmentioning

confidence: 57%

Delivery of membrane proteins into small and giant unilamellar vesicles by charge‐mediated fusion

et al. 2016

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“…Although the term ''flip-flop'' is more commonly used to report on the translocation of lipids between leaflets, in our study, it is describing the similar movement of probe molecules as has been previously done by others (18)(19)(20)27,29,34,36,51). The expected rate of flip-flop is not well known because a large number of different experiments on different systems have produced a wide range of results (20,27,(62)(63)(64)(65)(66)(67)(68)(69)(70). It should be first noted that FM probes have been reported to be permanently bound to the outer leaflet of a bilayer membrane because of their double positive charge (71), incapable of translocating to the inner leaflet without pore or endocytosis mechanisms (72).…”

Section: Discussionmentioning

confidence: 79%

Second Harmonic Generation Spectroscopy of Membrane Probe Dynamics in Gram-Positive Bacteria

Miller

Brewer

Williams

et al. 2019

Biophysical Journal

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Bacterial membranes are complex mixtures with dispersity that is dynamic over scales of both space and time. To capture adsorption onto and transport within these mixtures, we conduct simultaneous second harmonic generation (SHG) and two-photon fluorescence measurements on two different gram-positive bacterial species as the cells uptake membrane-specific probe molecules. Our results show that SHG not only can monitor the movement of small molecules across membrane leaflets but also is sensitive to higher-level ordering of the molecules within the membrane. Further, we show that the membranes of Staphylococcus aureus remain more dynamic after longer times at room temperature in comparison to Enterococcus faecalis. Our findings provide insight into the variability of activities seen between structurally similar molecules in gram-positive bacteria while also demonstrating the power of SHG to examine these dynamics.

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“…Over a decade ago, we introduced a new method for directly measuring lipid bilayer asymmetry and lipid translocation using the nonlinear spectroscopic technique of sum-frequency vibrational spectroscopy (SFVS). ,,− SFVS is a coherent, nonlinear optical vibrational spectroscopy that is exquisitely sensitive to molecular symmetry, making it particularly useful in the study of membrane structure and dynamics. The theoretical foundations of SFVS are well-established, and many excellent reviews are available in the literature. − Only a brief discussion of the relevant aspects pertaining to lipid membrane asymmetry and translocation will be presented here.…”

Section: Introductionmentioning

confidence: 99%

The Ins and Outs of Lipid Flip-Flop

Allhusen

Conboy

2016

Acc. Chem. Res.

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Our current view of cellular membranes centers on the fluid-mosaic model, which envisions the cellular membrane as a "liquidlike" bilayer of lipids, cholesterol, and proteins that freely diffuse in two dimensions. In stark contrast, the exchange of materials between the leaflets of a bilayer was presumed to be prohibited by the large enthalpic barrier associated with translocating hydrophilic materials, such as a charged lipid headgroup, through the hydrophobic membrane core. This static picture with regard to lipid translocation (or "flip-flop" as it is affectionately known) has been a long-held belief in the study of membrane dynamics. The current accepted membrane model invokes specific protein flippase (inward moving), floppase (outward moving), and scramblase (bidirectional) enzymes that assist in the movement of lipids between the leaflets of cellular membranes. The low rate of protein-free lipid flip-flop has also been a cornerstone of our understanding of the bilateral organization of cellular membrane components, specifically the asymmetric distribution of lipid species found in the luminal and extracellular leaflets of the plasma membrane of eukaryotic cells. Much of the previous work contributing to our current understanding of lipid flip-flop has utilized fluorescent- or spin-labeled lipids. However, there is growing evidence that these lipid probes do not accurately convey the dynamics and thermodynamics of native (unlabeled) lipid motion. This Account summarizes our research efforts directed toward developing a deep physical and chemical understanding of protein-free lipid flip-flop in phospholipid membrane models using sum-frequency vibrational spectroscopy (SFVS). Our use of SFVS enables the direct measurement of native lipid flip-flop in model membranes. In particular, we have explored the kinetic rates and activation thermodynamics of lipid translocation as a means of deciphering the underlying chemical and physical directors governing this process. By means of transition state theory, the contributions from enthalpy and entropy on the activation energy barrier to lipid flip-flop have been explored in detail for a variety of lipid species and membrane compositions. Specifically, the effect of lipid structure and packing and the inclusion of cholesterol and transmembrane peptides on the rates and thermodynamics of lipid translocation have been investigated in detail. It is our hope that these studies will provide a new perspective on lipid translocation in biological membranes and the role of lipid flip-flop in generating and maintaining cell membrane lipid asymmetry.

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Structural Origins of Cholesterol Accelerated Lipid Flip-Flop Studied by Sum-Frequency Vibrational Spectroscopy

Cited by 22 publications

References 93 publications

Delivery of membrane proteins into small and giant unilamellar vesicles by charge‐mediated fusion

Delivery of membrane proteins into small and giant unilamellar vesicles by charge‐mediated fusion

Second Harmonic Generation Spectroscopy of Membrane Probe Dynamics in Gram-Positive Bacteria

The Ins and Outs of Lipid Flip-Flop

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