Using High Throughput Differential Scanning Fluorimetry to Obtain Binding Parameters

Interaction analysis by the SUPR-DSF offers high-speed and label-free measurements across the analyte binding concentration range. The measurements are free in solution and are not influenced by surface effects, mass transport or refractive index buffer issues. The measurements confirm binding is specific by detecting induced stability changes in the protein-ligand complex. This is depicted from the shift in the emitted fluorescence as it melts, measured as a function of ligand concentration, from which the KD can be calculated. This method uses commercially available, low cost, low volume 384-well microplates, and is capable of screening thousands of samples for stability in a day to confirm binding.

Binding induced stability changes are readily apparent even for very weak binding partners (e.g., fragment libraries), confirming the binding is specific and of structural/functional importance. The SUPR-DSF can be integrated into existing robotic solutions, making very large-scale screens easy and rapid to complete. The below investigation applies the SUPR-DSF platform to the study of binding effects of a ligand with carbonic anhydrase
Results
The fraction unfolded melting curves for Carbonic Anhydrase I (hCA-I) from Human Erythrocytes with Trifluoromethanesulfonimide (TFMSA) are shown in Figure 1. The curves represent the average of the triplicate values. The largest standard error (0.07oC) is smaller than the symbol size. The control (No Ligand) had a Tm of 58.7°C, which agreed with reported values.[1] Examining Figure 1, you can clearly see TFMSA stabilizes hCA-I by shifting the melting curves to the right at higher concentrations. The saturating dose of 2 mM shifted the Tm by 10.0°C to 68.7°C. The results for all the samples, including Tonset, Tm, and van 't Hoff enthalpies (ΔHm) are shown in Table 1.
Figure 2 illustrates the melting temperatures of hCA-I as a function of TFMSA concentration. Fitting the measured melting temperatures to standard equations[2] with a 95% confidence interval (CI)[3] found multiple binding sites. An initial binding site dissociation constant of 2.1 µM (CI: 0.82 µM – 3.85 µM) which is comparable with literature values[4] and a second binding site dissociation constant at 174.2 µM (CI: 82.8 µM – 424.5 µM).

Conclusion
The use of the SUPR-DSF to study binding interactions has demonstrated that this technique offers a viable and unique solution. The SUPR-DSF offers an intrinsic fluorescence-based high-throughput methodology that is free in solution, with all measurements carried out directly in the 384-well plate-based format. The SUPR-DSF rapidly provided highly precise data with excellent sensitivity in the study of Carbonic Anhydrase with TFMSA. The values obtained agree with previously published literature values for KD and prove that the SUPR-DSF can be used as either a pre-screening or confirmational tool for ligand binding studies.