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Measuring distances between TRPV1 and the plasma membrane using a noncanonical amino acid and transition metal ion FRET.

ABSTRACT: Despite recent advances, the structure and dynamics of membrane proteins in cell membranes remain elusive. We implemented transition metal ion fluorescence resonance energy transfer (tmFRET) to measure distances between sites on the N-terminal ankyrin repeat domains (ARDs) of the pain-transducing ion channel TRPV1 and the intracellular surface of the plasma membrane. To preserve the native context, we used unroofed cells, and to specifically label sites in TRPV1, we incorporated a fluorescent, noncanonical amino acid, L-ANAP. A metal chelating lipid was used to decorate the plasma membrane with high-density/high-affinity metal-binding sites. The fluorescence resonance energy transfer (FRET) efficiencies between L-ANAP in TRPV1 and Co(2+) bound to the plasma membrane were consistent with the arrangement of the ARDs in recent cryoelectron microscopy structures of TRPV1. No change in tmFRET was observed with the TRPV1 agonist capsaicin. These results demonstrate the power of tmFRET for measuring structure and rearrangements of membrane proteins relative to the cell membrane.

SUBMITTER: Zagotta WN

PROVIDER: S-EPMC4727949 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Measuring distances between TRPV1 and the plasma membrane using a noncanonical amino acid and transition metal ion FRET.

Zagotta William N WN Gordon Moshe T MT Senning Eric N EN Munari Mika A MA Gordon Sharona E SE

The Journal of general physiology 20160111 2

Despite recent advances, the structure and dynamics of membrane proteins in cell membranes remain elusive. We implemented transition metal ion fluorescence resonance energy transfer (tmFRET) to measure distances between sites on the N-terminal ankyrin repeat domains (ARDs) of the pain-transducing ion channel TRPV1 and the intracellular surface of the plasma membrane. To preserve the native context, we used unroofed cells, and to specifically label sites in TRPV1, we incorporated a fluorescent, n ...[more]

PMID: 26755770

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Project description:Förster resonance energy transfer (FRET) is an important mechanism for the estimation of intermolecular distances, e.g., in fluorescent labeled proteins. The interpretations of FRET experiments with standard Förster theory relies on the following approximations: (i) a point-dipole approximation (PDA) for the coupling between transition densities of the chromophores, (ii) a screening of this coupling by the inverse optical dielectric constant of the medium, and (iii) the assumption of fast isotropic sampling over the mutual orientations of the chromophores. These approximations become critical, in particular, at short intermolecular distances, where the PDA and the screening model become invalid and the variation of interchromophore distances, and not just orientations, has a critical influence on the excitation energy transfer. Here, we present a quantum chemical/electrostatic/molecular dynamics (MD) method that goes beyond all of the above approximations. The Poisson-TrEsp method for the ab initio/electrostatic calculation of excitonic couplings in a dielectric medium is combined with all-atom molecular dynamics (MD) simulations to calculate FRET efficiencies. The method is applied to analyze single-molecule experiments on a polyproline helix of variable length labeled with Alexa dyes. Our method provides a quantitative explanation of the overestimation of FRET efficiencies by the standard Förster theory for short interchromophore distances for this system. A detailed analysis of the different levels of approximation that connect the present Poisson-TrEsp/MD method with Förster theory reveals error compensation effects, between the PDA and the neglect of correlations in interchromophore distances and orientations on one hand and the neglect of static disorder in orientations and interchromophore distances on the other. Whereas the first two approximations are found to decrease the FRET efficiency, the latter two overcompensate this decrease and are responsible for the overestimation of the FRET efficiency by Förster theory.

Project description:Highlights • The main properties of coordinated magnetic nanoparticles (MNPs) including magnetic moment and saturated magnetisation).• The doping of transition metal ions into ferrite to precisely modulate its magnetic properties.• the potential mechanisms and recent advances in transition metal ion-doped MNPs (TMNPs) for bioimaging: magnetic resonance imaging and magnetic particle imaging and tumour therapy: e.g., magneto-mechanical killing, magnetothermal therapy, and drug delivery.• the current challenges and future trends of TMNPs in the biomedical field based on the latest advances by researchers. Magnetic nanoparticles (MNPs) have been extensively researched and implemented in biomedicine for more than half a century due to their non-invasive nature, ease of temporal and spatial manipulation, and considerable biocompatibility. However, the complex magnetic behaviour of MNPs is influenced by several parameters (e.g., particle size, shape, composition, core-shell structure, etc.), among which the amount of transition metal doping plays an important factor. For this reason, the doping of ferrite with transition metals has been used as an effective strategy to precisely tailor MNPs to achieve satisfactory performance in biomedical applications. In this review, we first introduced the main properties of coordinated MNPs (including magnetic moment and saturated magnetisation) and provide a comprehensive overview of the mechanistic studies related to the doping of transition metal ions into ferrite to precisely modulate its magnetic properties. We also highlighted the potential mechanisms and recent advances in transition metal ion-doped MNPs (TMNPs) for bioimaging (magnetic resonance imaging and magnetic particle imaging) and tumour therapy (e.g., magneto-mechanical killing, magnetothermal therapy, and drug delivery). Finally, we summarised the current challenges and future trends of TMNPs in the biomedical field based on the latest advances by researchers. Graphical abstract Image, graphical abstractTransition metal doped magnetic nanoparticles (TMNPs) for biomedical applications

Dataset Information

Measuring distances between TRPV1 and the plasma membrane using a noncanonical amino acid and transition metal ion FRET.

Publications

Measuring distances between TRPV1 and the plasma membrane using a noncanonical amino acid and transition metal ion FRET.

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