Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

Margineanu, Anca; Chan, Jia Jia; Kelly, Douglas J.; Warren, Sean; Flatters, Delphine; Kumar, Sunil; Katan, Matilda; Dunsby, Christopher; French, Paul M. W.

doi:10.1038/srep28186

Cited by 96 publications

(70 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…When a fluorescent donor experiences FRET, its bulk fluorescence intensity and the lifetime of its excited state decrease. FRET can be used to monitor protein-protein interactions (PPIs) in biological systems such as living cells, tissues, and organisms [74][75][76]. A widely used method to measure FRET is fluorescence lifetime imaging microscopy (FLIM).…”

Section: Choosing the Optimal Fret Reporter Orientation For Estimatinmentioning

confidence: 99%

The Subcellular Localization and Oligomerization Preferences of NME1/NME2 upon Radiation-Induced DNA Damage

Radić

Šoštar

Weber

et al. 2020

IJMS

View full text Add to dashboard Cite

Nucleoside diphosphate kinases (NDPK/NME/Nm23) are enzymes composed of subunits NME1/NDPK A and NME2/NDPK B, responsible for the maintenance of the cellular (d)NTP pool and involved in other cellular processes, such as metastasis suppression and DNA damage repair. Although eukaryotic NDPKs are active only as hexamers, it is unclear whether other NME functions require the hexameric form, and how the isoenzyme composition varies in different cellular compartments. To examine the effect of DNA damage on intracellular localization of NME1 and NME2 and the composition of NME oligomers in the nucleus and the cytoplasm, we used live-cell imaging and the FRET/FLIM technique. We showed that exogenous NME1 and NME2 proteins co-localize in the cytoplasm of non-irradiated cells, and move simultaneously to the nucleus after gamma irradiation. The FRET/FLIM experiments imply that, after DNA damage, there is a slight shift in the homomer/heteromer balance between the nucleus and the cytoplasm. Collectively, our results indicate that, after irradiation, NME1 and NME2 engage in mutual functions in the nucleus, possibly performing specific functions in their homomeric states. Finally, we demonstrated that fluorophores fused to the N-termini of NME polypeptides produce the largest FRET effect and thus recommend this orientation for use in similar studies.

show abstract

Section: Choosing the Optimal Fret Reporter Orientation For Estimatinmentioning

confidence: 99%

The Subcellular Localization and Oligomerization Preferences of NME1/NME2 upon Radiation-Induced DNA Damage

Radić

Šoštar

Weber

et al. 2020

IJMS

View full text Add to dashboard Cite

show abstract

“…Another important application of FLI is in the imaging of large tissue at the macroscopic scale (MFLI). The applications hence range from high-throughput in vitro imaging 39 , ex vivo 40 or in vivo tissue imaging 41 for diagnostics, especially within the framework of optical guided surgery 42 , and preclinical studies 43 . Particularly, there is great interest in employing NIR MFLI as in this spectral window the background fluorescence is reduced, and deep tissue imaging can be performed with high sensitivity.…”

Section: Macroscopic Fluorescence Lifetime Imaging (Gated Iccd)mentioning

confidence: 99%

Ultra-fast fit-free analysis of complex fluorescence lifetime imaging via deep learning

Smith

Yao

Sinsuebphon

et al. 2019

Preprint

View full text Add to dashboard Cite

Fluorescence lifetime imaging (FLI) provides unique quantitative information in biomedical and molecular biology studies, but relies on complex data fitting techniques to derive the quantities of interest. Herein, we propose a novel fit-free approach in FLI image formation that is based on Deep Learning (DL) to quantify complex fluorescence decays simultaneously over a whole image and at ultra-fast speeds. Our deep neural network (DNN), named FLI-Net, is designed and model-based trained to provide all lifetime-based parameters that are typically employed in the field. We demonstrate the accuracy and generalizability of FLI-Net by performing quantitative microscopic and preclinical experimental lifetime-based studies across the visible and NIR spectra, as well as across the two main data acquisition technologies. Our results demonstrate that FLI-Net is well suited to quantify complex fluorescence lifetimes, accurately, in real time in cells and intact animals without any parameter settings. Hence, it paves the way to reproducible and quantitative lifetime studies at unprecedented speeds, for improved dissemination and impact of FLI in many important biomedical applications, especially in clinical settings.

show abstract

“…Currently most in-cell studies of proteins' structure employ nuclear magnetic resonance (NMR), or fluorescence spectroscopy (5,22), each method having its advantages and limitations. In-cell FRET (Förster energy transfer) is effective for monitoring protein-protein interactions, determining dissociation constants and identifying conformational changes of appropriately fluorophore-labeled target proteins (23)(24)(25). In addition, fluorescence correlation spectroscopy (FCS) can be used determine dissociation constants of protein oligomers in cells (26).…”

Section: Introductionmentioning

confidence: 99%

“…Nonetheless, many proteins display poor in-cell NMR signal qualities (line broadening) due to restricted motions and transient interactions with cellular components (3,32,33). In-cell studies using NMR and fluorescence techniques reported on protein folding and stability in cells (17,24,(34)(35)(36)(37)(38)(39)(40), structural changes of disordered proteins (41)(42)(43) and, to some extent, on protein association e and the stabilization of oligomeric protein forms (23,25,26). EPR (electron-paramagnetic resonance)-based double electron-electron resonance (DEER, also called PELDOR) spectroscopy has been suggested as an attractive alternative method to interrogate protein structures in cells (21,42,(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55).…”

Section: Introductionmentioning

confidence: 99%

In-cell destabilization of a homo-dimeric protein complex detected by DEER spectroscopy

Yang

Chen

Yang

et al. 2020

Preprint

View full text Add to dashboard Cite

The complexity of the cellular medium can affect proteins' properties and therefore in-cell characterization of proteins is essential. We explored the stability and conformation of BIR1, the first baculoviral IAP repeat domain of X-chromosome-linked inhibitor of apoptosis (XIAP), as a model for a homo-dimer protein in human HeLa cells.We employed double electron-electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd 3+ spin labels at three protein residues, C12 (flexible region), E22C and N28C (part of helical residues 26-31) in the N-terminal region. In contrast to predictions by excluded volume crowding theory, the dimer-monomer dissociation constant KD was markedly higher in cells than in solution and dilute cell lysate. As expected, this increase was recapitulated under conditions of high salt concentrations given that a conserved salt bridge at the dimer interface is critically required for association. Unexpectedly, however, also the addition of a crowding agent such as Ficoll destabilized the dimer, suggesting that Ficoll forms specific interactions with the monomeric protein. Changes in DEER distance distributions were observed for the E22C site, which displayed reduced conformational freedom in cells. Although overall DEER behaviors at E22C and N28C were compatible with a predicted compaction of disordered protein regions by excluded volume effects, we were unable to reproduce E22C properties in artificially crowded solutions. These results highlight the importance of in-cell DEER measurements to appreciate the complexities of cellular in vivo effects on protein structures and functions.

show abstract

Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

Cited by 96 publications

References 64 publications

The Subcellular Localization and Oligomerization Preferences of NME1/NME2 upon Radiation-Induced DNA Damage

The Subcellular Localization and Oligomerization Preferences of NME1/NME2 upon Radiation-Induced DNA Damage

Ultra-fast fit-free analysis of complex fluorescence lifetime imaging via deep learning

In-cell destabilization of a homo-dimeric protein complex detected by DEER spectroscopy

Contact Info

Product

Resources

About