E3 BIOSCIENCE
About
Assays
Protein ubiquitination is mediated by three distinct families of proteins: E1 ubiquitin activating enzymes (E1), E2 ubiquitin conjugating enzymes (E2), and E3 ubiquitin ligases (E3). Protein ubiquitination normally proceeds in two distinct steps. First, the E1 ubiquitin activating enzyme uses the energy of ATP hydrolysis to synthesize a covalent conjugate of ubiquitin with an E2 ubiquitin conjugating enzyme. The E2-ubiquitin conjugate is the activated ubiquitin intermediate, in which the C-terminus of ubiquitin is covalently linked to the active site cysteine of the E2 via a high-energy thioester bond. In the second step, an E3 ubiquitin ligase catalyzes transfer of ubiquitin from the E2-ubiquitin conjugate onto an amine group of a substrate protein.
Our technology uses Förster resonance energy transfer (FRET) for monitoring ubiquitination reactions. We manufacture FRET-active E2-Ub conjugates that enable sensitive and quantitative measurements of E3 ubiquitin ligase activity using a fluorescent ubiquitin discharge assay. E2-Ub conjugates are synthesized from E2s and ubiquitin labeled with two distinct fluorescent dyes that form a FRET pair. Depending on whether the second dye is a FRET acceptor or fluorescence quencher, the assays can be implemented in either the FRET or quenched fluorescence format.
FRET assays of E3 ubituitin ligase activity
FRET assays of E3 ligase activity are performed using E2-Ub conjugates labeled with a FRET pair of fluorophores. We normally use AF488 and AF594 fluorophores as FRET pairs in our E2-Ub reagents. The FRET signal is observed using a 480/20 nm excitation filter and a 630/20 emission filter. When purified FRET-active E2-Ub reagents are mixed with an active E3 ligase and an appropriate substrate, the progress of the ubiquitin transfer reaction can be monitored by recording the decay of the FRET signal over time.
The figure above shows one example of an experiment in which a FRET-active E2-Ub conjugate is dissociated upon incubation with a substrate protein and an E3 ubiquitin ligase. In this example 0.1 µM of the E2-Ub conjugate comprising AF488-labeled Ub (human ubiquitin) and AF594-labeled E2 (human UBE2N) was incubated with 0.1 µM of human UBE2V2(MMS2), 5 µM substrate protein (unlabeled human ubiquitin) and increasing concentrations (0 µM, 2.5 µM, 5.0 µM, 10 µM, 20 µM) of E3 ligase (human TRAF6 RING domain). (A) Measurement of fluorescence intensity using 480/20 nm excitation and 630/20 nm emission settings reveals a decrease in fluorescence over time as the E2-Ub conjugate is being consumed in the reaction. Reaction products can also be analyzed by SDS-PAGE. The SDS-PAGE gel imaged using AF488 fluorescence (C) and AF594 fluorescence (D) reveals that the E2-Ub conjugate is dissociated in the reaction, with the AF488-labeled ubiquitin incorporated into the di-ubiquitin product and the AF594-labeled UBE2N released in the free form.
The advantage of the FRET format is that the fluorescence signal of the acceptor fluorophore (measured with 580/20 nm excitation and 630/20 nm emission filters), can be used to remove all non-FRET sources of noise from the FRET signal (for example variability due to pipetting errors, bubbles in samples, etc.). The disadvantage is that the FRET format requires a fluorescence plate reader equipped with appropriate filters or monochromators. In contrast, quenched fluorescence assays (see below) can be performed on any plate reader capable of measuring FITC or GFP fluorescence.
Quenched fluorescence assays of E3 activity
E3 ubiquitin ligase activity assays can also be implemented in the quenched fluorescence format. To this end, we offer a selection of E2-Ub conjugates, in which the acceptor chromophore is a fluorescence quencher. For example, in a E2-Ub conjugate synthesized using AF488-labeled ubiquitin and an E2 labeled with SY9, the AF488 fluorescence is effectively quenched by the proximity of the SY9 quencher. When a quenched E2-Ub conjugate is incubated with an E3 ligase and an appropriate substrate, the progress of the ubiquitin transfer reaction can be followed as an increase of the AF488 fluorescence intensity over time.
FRET and quenched fluorescence assays of E1 activity
A similar approach can be used to assay activity of the E1 ubiquitin conjugating enzyme (UBA1) and related enzymes.
In an example above, an E1 activity assay is implemented in the FRET format (A). When AF488-labeled Ub and AF594-labeled E2 are incubated in the presence of E1 and ATP, the progress of E2-Ub synthesis can be followed by monitoring the FRET signal over time (B). In this example, 1 µM AF488-labeled Ub (human ubiquitin) and 1 µM AF594-labeled E2 (human UBE2N) were incubated in the presence of 5 mM ATP and increasing concentrations (0 µM, 0.06 µM, 0.13 µM, 0.25 µM) of E1 (human UBA1). SDS-PAGE analysis of the reaction products using AF488 fluorescence (C) and AF594 fluorescence (D) reveals that the E2-Ub conjugate product is formed from the two substrates of the reaction.
In another example, an E1 activity assay is implemented in the quenched fluorescence format (A). When AF488-labeled Ub and SY9-labeled E2 are incubated in the presence of E1 and ATP, the progress of E2-Ub synthesis can be followed by monitoring the AF488 fluorescence intensity over time (B). In this example, 1 µM AF488-labeled Ub (human ubiquitin) and 1 µM SY9-labeled E2 (human UBE2N) were incubated in the presence of 5 mM ATP and increasing concentrations (0 µM, 0.06 µM, 0.13 µM, 0.25 µM) of E1 (human UBA1). Measurement of fluorescence intensity using 480/20 nm excitation and 520/20 nm emission settings reveals a decrease in the intensity of the AF488 fluorescence signal vs time as the quenched FRET-active E2-Ub conjugate is being formed in the reaction. (C) SDS-PAGE analysis by AF488 fluorescence reveals that the E2-Ub conjugate product is formed from the two substrates of the reaction.
Reagents
Cat.#: 488UB-594D3-1-50
Purified FRET-active E2-Ub thioester conjugate comprising recombinant human UBE2D3 labeled with AF594 and recombinant human Ub labeled with AF488. 50 µL of 1 µM frozen conjugate (>10 reactions). Shipped on dry ice.
$ contact for price
Cat.#: 488UB-594N-1-50
Purified FRET-active E2-Ub thioester conjugate comprising recombinant human UBE2N labeled with AF594 and recombinant human Ub labeled with AF488. 50 µL of 1 µM frozen conjugate (>10 reactions). Shipped on dry ice.
$ contact for price
Cat.#: 488UB-594W-1-50
Purified FRET-active E2-Ub thioester conjugate comprising recombinant human UBE2W labeled with AF594 and recombinant human Ub labeled with AF488. 50 µL of 1 µM frozen conjugate (>10 reactions). Shipped on dry ice.
$ contact for price
Cat.#: 488UB-SY9D3-1-100
Purified quenched E2-Ub thioester conjugate comprising recombinant human UBE2D3 labeled with SY9 quencher and recombinant human Ub labeled with AF488. 100 µL of 1 µM frozen conjugate (>20 reactions). Shipped on dry ice.
$ contact for price
Cat.#: 488UB-SY9N-1-100
Purified quenched E2-Ub thioester conjugate comprising recombinant human UBE2N labeled with SY9 quencher and recombinant human Ub labeled with AF488. 100 µL of 1 µM frozen conjugate (>20 reactions). Shipped on dry ice.
$ contact for price
Cat.#: 488UB-SY9W-1-100
Purified quenched E2-Ub thioester conjugate comprising recombinant human UBE2W labeled with SY9 quencher and recombinant human Ub labeled with AF488. 100 µL of 1 µM frozen conjugate (>20 reactions). Shipped on dry ice.
$ contact for price
Cat.#: 594UB-BH3N-1-100
Purified quenched E2-Ub thioester conjugate comprising recombinant human UBE2N labeled with BH3 quencher and recombinant human Ub labeled with AF594. 100 µL of 1 µM frozen conjugate (>20 reactions). Shipped on dry ice.
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Cat.#: CY55UB-BH3D3-1-100
Purified quenched E2-Ub thioester conjugate comprising recombinant human UBE2D3 labeled with BH3 quencher and recombinant human Ub labeled with Cy5.5. 100 µL of 1 µM frozen conjugate (>20 reactions). Shipped on dry ice.
$ contact for price
Custom Reagents
Cat.#: ???-???-?-??
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contact@e3bioscience.com
Contact
We would love to help you achieve your research goals using our technology! Please email us with any questions. We offer steep discounts for high-throughput screening campaigns and will be happy to synthesize custom reagents for you. We can also advise on how to configure your equipment for assays and reagents described here.
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REFERENCES
Abstract
TRIM5α is an E3 ubiquitin ligase of the TRIM family that binds to the capsids of primate immunodeficiency viruses and blocks viral replication after cell entry. Here we investigate how synthesis of K63-linked polyubiquitin is upregulated by transient proximity of three RING domains in honeycomb-like assemblies formed by TRIM5α on the surface of the retroviral capsid. Proximity of three RINGs creates an asymmetric arrangement, in which two RINGs form a catalytic dimer that activates E2-ubiquitin conjugates and the disordered N-terminus of the third RING acts as the substrate for N-terminal autoubiquitylation. RING dimerization is required for activation of the E2s that contribute to the antiviral function of TRIM5α, UBE2W and heterodimeric UBE2N/V2, whereas the proximity of the third RING enhances the rate of each of the two distinct steps in the autoubiquitylation process: the initial N-terminal monoubiquitylation (priming) of TRIM5α by UBE2W and the subsequent extension of the K63-linked polyubiquitin chain by UBE2N/V2. The mechanism we describe explains how recognition of infection-associated epitope patterns by TRIM proteins initiates polyubiquitin-mediated downstream events in innate immunity.
