Recent Applications of Fluorescence Recovery after Photobleaching (FRAP) to Membrane Bio-Macromolecules

Rayan, Gamal; Guet, Jean Erik; Taulier, Nicolas; Pincet, Frédéric; Urbach, W.

doi:10.3390/s100605927

Cited by 46 publications

(38 citation statements)

References 83 publications

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“…Other methods such as FRAP and FRAPP can also resolve multiple diffusion coefficients in a heterogenous population if the diffusion rates are sufficiently different (37). However, unlike the current approach, these methods do not directly measure the distribution of diffusion coefficients for individual particles (Fig.…”

Section: Resultsmentioning

confidence: 99%

Assembly of Membrane-Bound Protein Complexes: Detection and Analysis by Single Molecule Diffusion

2012

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Protein complexes assembled on membrane surfaces regulate a wide array of signaling pathways and cell processes. Thus a molecular understanding of the membrane surface diffusion and regulatory events leading to the assembly of active membrane complexes is crucial to signaling biology and medicine. Here we present a novel single molecule diffusion analysis designed to detect complex formation on supported lipid bilayers. The usefulness of the method is illustrated by detection of an engineered, heterodimeric complex in which two membrane-bound pleckstrin homology (PH) domains associate stably, but reversibly, upon Ca2+-triggered binding of calmodulin (CaM) to a target peptide from myosin light chain kinase (MLCKp). Specifically, when a monomeric, fluorescent PH-CaM domain fusion protein diffusing on a supported bilayer binds a dark MLCKp-PH domain fusion protein, the heterodimeric complex is observed to diffuse nearly 2-fold more slowly than the monomer because both of its twin PH domains can simultaneously bind to the viscous bilayer. In a mixed population of monomers and heterodimers, the single molecule diffusion analysis resolves and quantitates the rapidly diffusing monomer and slowly diffusing heterodimer subpopulations. The affinity of the CaM-MLCKp interaction is measured by titrating dark MLCKp-PH construct into the system, while monitoring the changing average diffusion coefficient of the fluorescent PH-CaM population, yielding a saturating binding curve. Strikingly, the apparent affinity of the CaM-MLCKp complex is ∼102-fold greater in the membrane system than in solution, apparently due both to faster complex association and slower complex dissociation on the membrane surface. More broadly, the present findings suggest that single molecule diffusion measurements on supported bilayers will provide an important tool for analyzing the 2D diffusion and assembly reactions governing the formation of diverse membrane-bound complexes, including key complexes from critical signaling pathways. The approach may also prove useful in pharmaceutical screening for compounds that inhibit membrane complex assembly or stability.

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Section: Resultsmentioning

confidence: 99%

Assembly of Membrane-Bound Protein Complexes: Detection and Analysis by Single Molecule Diffusion

2012

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“…However, the widespread application of these methods has largely neglected the fact that, unlike model planar or spherical surfaces, real biological interfaces are rather anisotropic or have a complex, asymmetric architecture. The effect of surface geometry on the measurement of dynamic parameters cannot be overemphasized [40–42]. As it turns out, diffusion of integral membrane proteins is significantly different from diffusion of luminal or cytosolic components, with t 1/2 varying by a factor of ~4, even when the membrane shapes are the same [37].…”

Section: Discussionmentioning

confidence: 99%

Dynamics and Structure-Function Relationships of the Lamin B Receptor (LBR)

Giannios¹,

Chatzantonaki²,

Georgatos³

2017

PLoS ONE

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The lamin B receptor (LBR) is a multi-spanning membrane protein of the inner nuclear membrane that is often employed as a “reporter” of nuclear envelope dynamics. We show here that the diffusional mobility of full-length LBR exhibits significant regional variation along the nuclear envelope, consistent with the existence of discrete LBR microdomains and the occurrence of multiple, asymmetrically-spaced anastomoses along the nuclear envelope-endoplasmic reticulum interface. Interestingly, a commonly used fusion protein that contains the amino-terminal region and the first transmembrane domain of LBR exhibits reduced mobility at the nuclear envelope, but behaves similarly to full-length LBR in the endoplasmic reticulum. On the other hand, carboxy-terminally truncated mutants that retain the first four transmembrane domains and a part or the whole of the amino-terminal region of LBR are generally hyper-mobile. These results suggest that LBR dynamics is structure and compartment specific. They also indicate that native LBR is probably “configured” by long-range interactions that involve the loops between adjacent transmembrane domains and parts of the amino-terminal region.

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“…The reader is referred to them, and to the original publications, for additional details that could not be included here because of space limitations. For the same reasons, this review cannot be exhaustive; for instance it does not discuss fluorescence correlation spectroscopy (FCS) [55,56], fluorescence recovery after photobleaching (FRAP) [57], and fluorescence approaches to study the electrostatic properties of membranes [44].…”

Section: Introductionmentioning

confidence: 99%

Fluorescence spectroscopy and molecular dynamics simulations in studies on the mechanism of membrane destabilization by antimicrobial peptides

Bocchinfuso

Bobone

Meisina

et al. 2011

Cell. Mol. Life Sci.

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Since their initial discovery, 30 years ago, antimicrobial peptides (AMPs) have been intensely investigated as a possible solution to the increasing problem of drug-resistant bacteria. The interaction of antimicrobial peptides with the cellular membrane of bacteria is the key step of their mechanism of action. Fluorescence spectroscopy can provide several structural details on peptide-membrane systems, such as partition free energy, aggregation state, peptide position and orientation in the bilayer, and the effects of the peptides on the membrane order. However, these "low-resolution" structural data are hardly sufficient to define the structural requirements for the pore formation process. Molecular dynamics simulations, on the other hand, provide atomic-level information on the structure and dynamics of the peptide-membrane system, but they need to be validated experimentally. In this review we summarize the information that can be obtained by both approaches, highlighting their versatility and complementarity, suggesting that their synergistic application could lead to a new level of insight into the mechanism of membrane destabilization by AMPs.

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Recent Applications of Fluorescence Recovery after Photobleaching (FRAP) to Membrane Bio-Macromolecules

Cited by 46 publications

References 83 publications

Assembly of Membrane-Bound Protein Complexes: Detection and Analysis by Single Molecule Diffusion

Assembly of Membrane-Bound Protein Complexes: Detection and Analysis by Single Molecule Diffusion

Dynamics and Structure-Function Relationships of the Lamin B Receptor (LBR)

Fluorescence spectroscopy and molecular dynamics simulations in studies on the mechanism of membrane destabilization by antimicrobial peptides

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