Using two-site binding models to analyze microscale thermophoresis data.

Area of Science:

• Biophysics
• Biochemistry
• Biotechnology

Background:

• Microscale thermophoresis (MST) is a powerful technique for quantifying bimolecular interactions.
• Existing MST analysis models primarily assume simple 1:1 binding, limiting applications for systems with multiple binding sites.
• The Hill equation is often used for multiple binding sites but has limitations for MST data analysis.

Purpose of the Study:

• To develop and present new analytical models for MST data analysis that accommodate 1:2 binding schemes.
• To provide models that account for distinct microscopic binding constants or cooperative binding effects.
• To assess the performance and applicability of these new models compared to existing methods.

Main Methods:

• Development of two new MST analytic models for 1:2 binding interactions.
• Model 1: Features two distinct microscopic dissociation constants (K_D(1) and K_D(2)).
• Model 2: Assumes molecular symmetry, resulting in a single macroscopic dissociation constant (K_D,M) and a cooperativity factor (α).

Main Results:

• The new models provide a more nuanced analysis of 1:2 binding interactions than the traditional 1:1 assumption.
• Performance assessment on simulated and real data demonstrates the utility and accuracy of the developed algorithms.
• The study discusses the general applicability of the Hill equation for MST data in light of these new models.

Conclusions:

• The introduced MST models significantly expand the analytical capabilities for studying complex biomolecular interactions.
• These models offer improved precision in determining dissociation constants for systems exhibiting 1:2 binding.
• Implementation within the PALMIST software facilitates broader adoption and application of these advanced analytical tools.