Optical unfolding - Dianthus α
Optical Unfolding is a measurement mode available with Dianthus α that allows you to assess protein thermal stability using extrinsic fluorescence. Regular Dianthus instruments with Spectral Shift optics or NT.23 instruments can be physically upgraded to enable this functionality. This guide explains the principles behind Optical Unfolding and goes over recommended applications.
Measurement Principle
Protein thermal stability is a fundamental biophysical property that reflects the folded state of a protein. When proteins are heated, they undergo a transition from their native folded state to an unfolded state. This transition can be monitored using fluorescence-based methods.
Optical Unfolding uses extrinsic fluorescent dyes that exhibit different fluorescence properties depending on the protein's folding state. As the protein unfolds, hydrophobic regions become exposed, altering the dye's local environment and changing its fluorescence emission spectrum.
The Optical Unfolding measurement involves:
- Sample heating: An infrared laser heats the sample from ambient temperature to approximately 80°C over 60 seconds
- Fluorescence monitoring: The Spectral Shift ratio (fluorescence at two emission wavelengths) is recorded continuously during heating
- Melting curve generation: The ratio change over time produces a sigmoidal curve representing the unfolding transition
The inflection point (IP) is the time at which 50% of the protein population has unfolded. This parameter is analogous to the melting temperature (Tm) used in other thermal stability methods, but is reported as a time rather than a temperature due to the rapid heating approach.
What Assay Conditions Do I Have to Consider for Optical Unfolding?
Concentration: The concentration range is similar to Spectral Shift assays, with a typical range between 5 nM and 20 nM. Fluorescence will decrease upon temperature increase, therefore sub-nanomolar concentrations should be tested first.
Labelling: For affinity-based labelling, the protein-dye ratio should be adjusted closer to 5:1 to ensure that upon temperature increase the dye stays bound to the target protein.
Volume: Volume should be consistent throughout all wells. The recommended volume is 12 µL (compatible with Spectral Shift assays). It is very important to seal the plate prior to measurement.
How Long Does the Unfolding Measurement Take and What Is the Throughput?
Per data point / per well: Laser time can be selected between 30 seconds and 60 seconds.
Per plate: A full 384-well plate measured at 60 seconds per well will take approximately 8 hours.
The recommended workflow is to select hits and compounds of interest rather than measuring the whole plate to reduce measurement time.
Does the Heating Time Correlate with the Melting Temperature on Prometheus?
Yes, there is a strong correlation between inflection time on Dianthus and unfolding temperature on Prometheus, but there is no direct conversion of inflection time to unfolding temperature.
The inflection time strongly depends on the sample volume. A larger volume is heated more slowly than a small volume, which is why it is very important to keep a consistent volume and to seal the plate prior to measurement.
Inflection times do not represent the melting temperature, as the unfolding lags behind the actual sample temperature. A 12 µL sample is heated to approximately 80°C in 60 seconds.
Applications for Optical Unfolding
Protein Quality Assessment
Before starting binding experiments, verify that your protein is properly folded and stable. A well-behaved protein should show:
- A clear sigmoidal unfolding transition
- Reproducible inflection point across replicates
No evidence of aggregation (irregular curve shapes)
Binding Validation
Ligand binding often stabilises proteins, shifting the unfolding transition to higher temperatures (later inflection times). By comparing the unfolding behaviour of protein alone versus protein with ligand, you can:
- Confirm that binding has occurred
- Obtain an independent measure of binding (orthogonal to affinity measurements)
- Distinguish true binders from non-binders or artefacts
Identifying Problematic Compounds
Aggregators
Compounds that induce protein aggregation typically cause destabilisation or irregular unfolding curves. The melting curve may show:
- Decreased inflection point (earlier unfolding)
- Loss of sigmoidal shape
- Increased baseline noise
Fluorescent Compounds
Auto-fluorescent compounds can interfere with the measurement by contributing to the fluorescence signal. This may result in:
- Abnormal baseline fluorescence
- Irregular curve shapes
- Concentration-dependent artefacts
Characterising Multi-Component Systems
Optical Unfolding is particularly useful for studying complex systems such as Ternary complexes.
For molecules like PROTACs that bring together two proteins, a possible experimental setup can be:
- E3 ligase alone: Baseline stability
- Binary complex (E3 ligase + PROTAC): Stabilisation from PROTAC binding
- Ternary complex (E3 ligase + PROTAC + target protein): Additional stabilisation from protein-protein interactions
The ternary complex typically shows greater stabilisation than the binary complex due to the additional protein-protein interface, providing evidence of ternary complex formation.