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Protein interactions

Biomolecular interactions

Inside cells specific interactions between biomolecules are involved in almost any physiological process. Sensing extracellular signals is a matter of receptor to adapter interactions and an intricate network of structural protein interactions maintains the shape of the cell. Finding interactions between proteins involved in common cellular functions is a way to get a broader view of how they work co-operatively in a cell. One way to observe biomolecular interactions is by doing Forster Resonance Energy Transfer (FRET) measurements. In this article some examples of different interactions are given, with the link to the paper in question.

Protein-Protein Interactions

Signal transduction pathways inside cells involve the coupling of ligand-receptor interactions to many intracellular events. These events include phosphorylation by tyrosine kinases and/or serine/threonine kinases. Protein phosphorylation change enzyme activities and protein conformations. The eventual outcome is an alteration in cellular activity and changes in the program of genes expressed within the responding cells. Phosphorylation dynamics can be imaged by FRET, by labelling two proteins-, domains-, or phospho-epitopes that come in close proximity during a phosphorylation event.

Epidermal-Growth Factor Receptor (EGFR) phosphorylation with the eYFP-(acceptor)-labelled phosphotyrosine-binding domain and eCFP (donor)-tagged EGFR.Beta-secretase (BACE) phosphorylation with BACE-GFP (donor) transfected cells fixed and stained with phosphoserine-Cy3 (acceptor).

When the enzyme is labelled by one fluorophore of a FRET pair, and the substrate by the other, FRET is expected when the enzyme cleaves the substrate.

Presenilin 1 (PS1) is a critical component of the gamma-secretase complex. This complex is involved in the cleavage of several substrates, including the amyloid precursor protein (APP). By FLIM-FRET is shown that the low-density receptor-related protein (LRP) is a PS1 interactor and can compete with APP for gamma-secretase enzymatic activity.

Endosome fusion can also be imaged by FRET:

Fusion between primary endocytic vesicles and/ or sorting endosomes.

If you would like to know more about antibody fluorophores, like lifetime information of the Alexa dyes, please contact us.

Conformational Changes

When the N-terminus is tagged with the donor fluorophore and the C-terminus with the acceptor fluorophore (or vice versa), the conformational change of the macromolecule can be visualised by the occurrence of FRET. In the 'open' conformation no FRET will occur, while the 'closed' conformation will cause FRET. Different dyes bind to different regions in DNA and so FRET occurrence can give information on the condensation of DNA:

An example is the staining of nuclei with Hoechst, that binds to AT-rich regions and with 7-AAD (7-aminoactinomycin D) that binds to GC-rich regions. These stained nuclei give a non-homogenous FRET signal in total nuclei, hence an increased FRET efficiency is shown when the cell progresses from G1 to G2/M (condensed DNA formation) phase.

Oligomerization kinetics is used to reveal the composition of macromolecules, and can be observed by FRET:

Conformation of leptin receptors expressed in the cell membrane.

Lipid-protein interactions

Interactions between lipids and proteins can be visualised by FRET by incorporation of fluorescent lipids in the membrane and fluorescence-tagged peripheral membrane proteins.

Forster Resonance Energy Transfer

Forster Resonance Energy Transfer (FRET) is the non-radiative transfer of energy from a molecule in the excited state (donor) to a molecule in the ground state (acceptor). A fluorescent donor molecule can return to the ground state by losing its energy through emission of a photon (fluorescence), or by transferring its energy to a nearby (1 - 9nm) acceptor molecule (FRET). Compared to a molecule that exhibits no FRET, the donor has more options to lose its energy. Therefore, it returns faster to the ground state, which decreases its lifetime.

FRET is a useful tool to quantify molecular dynamics like interactions of two fluorophores by microscopy. The proteins under investigation are labelled with donor fluorophores or acceptor fluorophores. Interaction between the two fluorophores is accompanied by direct energy transfer from donor to acceptor (FRET). When FRET occurs, it means that the two proteins of interest are in such close proximity that they can interact with each other.

During FRET, a quantum of energy is transferred from a donor fluorophore to an acceptor fluorophore in a nonradiative process. So, in case of no FRET, the donor fluorophore is excited and emits photons. The acceptor fluorophore does not emit photons, because it is not excited. In case of FRET, the donor fluorophore is excited, but in stead of emitting all its energy as photons, it transfers some of its energy to the acceptor fluorophore that becomes excited and emits light.

Summarising, in case of no FRET only the donor fluorophore emits photons, and in case of FRET both donor and acceptor emit photons.

FRET only occurs if...

  1. The donor fluorescence emission spectrum overlaps with the acceptor absorbance.

  2. The donor and acceptor fluorophores are in close proximity (i.e. 1 - 9nm, which is at the scale of protein size).

  3. The transition dipole moments of the donor and acceptor fluorophores are not perpendicular.

FRET pairs

To let FRET occur, the emission spectrum of the donor fluorophore has to overlap the excitation spectrum (absorbance) of the acceptor fluorophore. Some examples are BFP-YFP, CFP-YFP, GFP-DsRed, GFP-Cy3, GFP-mOrange, YFP-RFP, and Cy3-Cy5.

Browser based calculator to find the critical distance and FRET efficiency with known spectral overlap.

Related Posts

Ion imaging

For ion imaging, several fluorescent indicators (sensor, construct, tracer, etc) are available that have a change in quantum yield upon ion binding. This means that they emit photons with different energy, thus have different emission wavelength. Their fluorescence lifetime could also change. Therefore, there are two methods in which ion imaging can be done by use of indicators: the ratiometric method and the FLIM method.

Another method ion imaging is by the use of Forster Resonance Energy Transfer based (FRET-based) indicators that change their conformation upon ion binding. Upon the conformational change of a FRET-based indicator, its FRET efficiency changes, which is used as indicator of ion concentration. Examples of these indicators are cameleons. Cameleons are genetically-encoded fluorescent indicators for Ca2+ based on green fluorescent protein variants and calmodulin (CaM).

Reference: Miyawaki A, Griesbeck O, Heim R, Tsien RY. "Dynamic and quantitative Ca2+ measurements using improved cameleons". Proc Natl Acad Sci USA (PNAS) 96(5):2135-40 (1999)

Demonstration of the Lambert Instruments Toggel camera for single-image FLIM (siFLIM) detection of histamine-induced alterations in Ca2+ concentration. Tiny oscillations in Ca2+ levels (~2.5 s periods) are observed after addition of histamine. Such small and rapid transients would go completely unnoticed when recorded by conventional FLIM.  Video courtesy of the Netherlands Cancer Institute.

Demonstration of the Lambert Instruments Toggel camera for single-image FLIM (siFLIM) detection of histamine-induced alterations in Ca2+ concentration. Tiny oscillations in Ca2+ levels (~2.5 s periods) are observed after addition of histamine. Such small and rapid transients would go completely unnoticed when recorded by conventional FLIM.

Video courtesy of the Netherlands Cancer Institute.

Calcium Imaging

Calcium (Ca2+) is important for signal transduction pathways.

Proton (pH) Imaging

The intracellular proton (H+) concentration (pH), as well as intracellular calcium, is important in the regulation of cellular functions including growth, differentiation, motility, exocytosis and endocytosis. To study this in more detail, measurements of the intracellular pH of resting cells can be done and the pH fluctuations inside cells after environmental perturbations can be followed.

Reference: Hai-Jui Lin, Petr Herman, and Joseph R. Lakowicz. "Fluorescence Lifetime-Resolved pH Imaging of Living Cells". Cytometry Part A 52A:77–89 (2003).

Zinc Imaging

Zinc (Zn2+) is involved in enzyme catalysis, protein structure, protein-protein interactions, and protein-oligonucleotide interactions. Zinc interacts with extracellular binding sites, which are likely to include binding sites involved in the subsequent translocation of this ion to the cell interior. Inside the cell, Zinc binds to cytosolic and organelle binding sites or is taken up by intracellular organelles.

Sodium Imaging

Sodium (Na+) is important in the signal transduction in the central nerve system.

Magnesium Imaging

Many enzymes (like kinases) require the presence of magnesium Mg2+ ions for their catalytic action, especially enzymes utilising ATP.

Chloride Imaging

Chloride (Cl-) plays a role in the central nervous system.


Potassium (K+) plays a role in cell growth and cell viability.

Indicators for Ion Imaging by FLIM

  • BCECF (pH)

  • Bis-BTC (heavy metals)

  • Calcium-crimson (Calcium, orange excitation)

  • Calcium-green (Calcium, blue excitation)

  • Carboxyfluorescein (pH)

  • Carboxy-SNAFL-1 (pH)

  • Carboxy-SNAFL-2 (cytosol pH)

  • DM-NERF dextrans (lysosoml pH)

  • Fluo-3 (Calcium)

  • Fura-2 (Calcium)

  • LysoSensor DND-160 (lysosomal pH)

  • LysoSensor probe (pH)

  • Magnesium-green (Magnesium)

  • Mag-quin-1 (Magnesium)

  • Mag-quin-2 (Magnesium)

  • MQAE (Chloride)

  • Newport Green DCF (Zinc)

  • OG-514 carboxylic acid dextrans (lysosoml pH)

  • PBFI (Potassium)

  • Quin-2 (Calcium, blue excitation)

  • SPQ (Chloride)