Microscale Thermophoresis (MST) as a Tool to Study Binding Interactions of Oxygen-Sensitive Biohybrids

Microscale thermophoresis (MST) is a technique used to measure the strength of molecular interactions. MST is a thermophoretic-based technique that monitors the change in fluorescence associated with the movement of fluorescent-labeled molecules in response to a temperature gradient triggered by an IR LASER. MST has advantages over other approaches for examining molecular interactions, such as isothermal titration calorimetry, nuclear magnetic resonance, biolayer interferometry, and surface plasmon resonance, requiring a small sample size that does not need to be immobilized and a high-sensitivity fluorescence detection. In addition, since the approach involves the loading of samples into capillaries that can be easily sealed, it can be adapted to analyze oxygen-sensitive samples. In this Bio-protocol, we describe the troubleshooting and optimization we have done to enable the use of MST to examine protein–protein interactions, protein–ligand interactions, and protein–nanocrystal interactions. The salient elements in the developed procedures include 1) loading and sealing capabilities in an anaerobic chamber for analysis using a NanoTemper MST located on the benchtop in air, 2) identification of the optimal reducing agents compatible with data acquisition with effective protection against trace oxygen, and 3) the optimization of data acquisition and analysis procedures. The procedures lay the groundwork to define the determinants of molecular interactions in these technically demanding systems.. Authors: Jagilinki, Bhanu P. [Univ. of Oklahoma, Norman, OK (United States)]; Willis, Mark A. [Washington State Univ., Pullman, WA (United States)]; Mus, Florence [Univ. of Oklahoma, Norman, OK (United States)]; Sharma, Ritika [Univ. of Oklahoma, Norman, OK (United States)]; Pellows, Lauren M. [Univ. of Colorado, Boulder, CO (United States)]. DOE Contract: AC36-08GO28308. Subjects: 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 74 ATOMIC AND MOLECULAR PHYSICS; binding affinities; dissociation constant (Kd); electron transfer; enzyme-quantum dot biohybrids; microscale thermophoresis; nanocrystals; nitrogenase; quantum dots