Enhanced FIDA-analysis of small-molecule-protein interactions
How to obtain fast, accurate, and reliable data to accelerate your biophysical insight?
The primary FIDA measurement is hydrodynamic radius (Rh). Based on direct measurements of size changes, it provides an array of information about stability, affinity, and kinetics. With the highly sensitive FIDA detectors, changes below 5% and, in many cases, down to 1% can be detected. Thereby, FIDA offers a unique ability to get biophysical insight based on direct structural measurement.
There are many examples of how the accuracy and sensitivity of FIDA Rh also enable the analysis of Small molecule-Protein interactions.
To increase data reliability, users withFIDA Neo detectors can also take advantage of ratiometric measurements to further enhance the sensitivity when analysing small-molecule interactions. We call this proprietary technology FIDA Lambda Dynamics, or just FIDA LD. It is based on fluorescent ratiometric measurements derived from standard FIDA experiments.
When doing FIDA analyses, the FIDA software also automatically collects the changes in the fluorescence intensity.It measures changes in fluorescence intensity that can occur when molecules (such as proteins or small ligands) bind to each other, or proteins change their conformational state due to environmental changes. We refer to that as FIDA BRIC. These measurements provide valuable information about binding/no-binding, solubility, conformational changes, sample loss, etc.
What is Lambda Dynamics?
At its core, Lambda Dynamics is all about capturing the minute changes in emission spectra that occur when molecules interact in a solution. These changes—lambda shifts—are not just noise; they carry valuable information about the interaction dynamics. To be more specific, Lambda Dynamics refers to the shifts in the wavelength (lambda) of the detected fluorescence signal, which occurs when binding events change the environment ofthe fluorophore or other optical markers. These shifts provide insights into the binding interactions and may indicate changes in the conformation, orientation or proximity of the molecules involved. Lambda Dynamics leverages principles also described as ‘lambda ratiometry’ (see Alexander Damchenko, 2010 & 2014). As noted by Damchenko, focusing on lambda dynamics is particularly valuable in studying intermolecular interactions because it provides a more reliable and detailed readout than single-wavelength measurements.
In practical terms, Lambda Dynamics takes the core idea of wavelength ratiometry and applies it to enhance the depth and reliability of biomolecular interaction studies. A unique background subtraction combined with the proprietary Fida technology allows for unprecedented Kd and in-solution kinetics accuracy.
FIDA’s Lambda Dynamics versus other methods
Comparison of dissociation constants obtained by different measurement methods. Each point of the binding curve in Fida LD is measured in triplicates.
Interaction constants (Kd´s) correspond to the interaction between the enzyme carbonic anhydrase and 3 small molecule inhibitors. The Kd´s obtained using Fida LD are measured using a Fida Neo instrument. The Fida LD methodology correlates very closely with other in-solution methodologies andSPR. Additional benefits of Fida LD include rapid assay development and setup,small sample volumes (nano-microliter), and accompanying quality control.
Tripling the insights on molecular interaction
Importantly, the primary FIDA measurement is hydrodynamic radius (Rh). With FIDA Rh it is possible to detect size changeless than 5%, and often less than 1%. Combined with FIDA BRIC and now FIDA LD, it provides a unique offering for accelerating your biophysical analysis. LambdaDynamics provides additional understanding of molecular interactions; insights that are not accessible through traditional fluorescence intensity measurements alone.
If you are not yet familiar with direct in-solution size change detection or Binding Related Intensity Change you can learn more here: link & link. In short, FIDA BRIC is designed to detect changes in signal intensity associated with binding events, meanwhile FIDA size measurement provides direct hydro dynamic radius size change down to less than 5%. Integrating Lambda Dynamics with FIDA BRIC and Fida Rh, researchers can achieve a new level of sensitivity and precision in detecting and characterising molecular interactions.
What extended capacities does it bring?
Lambda Dynamics provides several important benefits that are particularly relevant for researchers focused on molecular interaction studies:
- Detecting the (previously) undetectable: Lambda Dynamics is capable of detecting subtle lambda shifts that may be missed by other measurements or methods. This increased sensitivity is crucial for identifying complex biomolecular interactions that are often challenging to characterise.
- Efficiency: Lambda Dynamics are measured together with BRIC and hydrodynamic radius measurement: you get 3 assays in one
- In solution kinetics: Unlike traditional methods that may only offer Kd determination, Lambda Dynamics allows for quantifying kinetics of these interactions, offering a more nuanced and detailed understanding of the system at hand.
An advantage to researchers who seek enhanced precision, sensitivity and real-time capabilities.
Lambda Dynamics enhances the sensitivity, precision, and real-time capabilities of biomolecular interaction analysis. By capturing dynamic fluctuations in molecular interactions with unparalleled accuracy, Lambda Dynamics allows for a deeper and more nuanced understanding of these processes. Lambda Dynamics is relevant for researchers seeking new levels of understanding of complex modalities.
Demchenko, Alexander. (2010). The Concept of λ-Ratiometry in Fluorescence Sensing and Imaging. Journal of fluorescence. 20. 1099-128. 10.1007/s10895-010-0644-y.
Demchenko, Alexander. (2014). Practical aspects of wavelength ratiometry in the studies of intermolecular interactions. Journal of Molecular Structure. 1077. 10.1016/j.molstruc.2013.11.045.
Myszka, David. (2004). Analysis of small-molecule interactions using Biacore S51 technology. Analytical biochemistry. 329. 316-23. 10.1016/j.ab.2004.03.028.
Langer, A. et al. (2022). A New Spectral Shift-Based Method to Characterize Molecular Interactions. Assay and drug development technologies. 20. 10.1089/adt.2021.133.
