Background

Quantification of biomolecular interactions is essential as they are critical to drug design, pharmaceutical, and bioprocessing industries. There are several techniques used to determine the strength of molecular interactions including isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), and biolayer interferometry (BLI). Although these techniques have wide applications, there are several challenges associated with them in studying oxygen-sensitive samples. Microscale thermophoresis (MST), on the other hand, is a highly sensitive method where the intermolecular interactions are studied based on changes in fluorescence intensity of the target molecule as a function of the temperature-based difference in movement between bound and unbound states across a thermal gradient [1,2]. This Bio-protocol focuses on adapting the technique to analyze molecular interactions for highly oxygen-sensitive metalloenzymes and nanocrystalline quantum dots (QDs). We found that it was suboptimal to operate the NanoTemper MST within an anaerobic chamber, necessitating the development of a procedure to load and seal samples in an anaerobic chamber for analysis on the bench in the air. We achieved this by preparing the samples and sealing the MST capillaries inside the mBraun glove box, using capillary wax. Although this proved to be a successful strategy, there still was a need to protect extremely oxygen-sensitive samples like the MoFe protein and the Fe protein of nitrogenase from trace amounts of oxygen that might be introduced during sample preparation. Strong reducing agents like sodium dithionite (NaDT) are routinely used for this purpose; however, NaDT quenches the intensity of the fluorescence signal at even low concentrations. To overcome this, we have employed a novel, one electron-reduced viologen-based 1,1'-bis(3-sulfonatopropyl)-4,4'-bipyridinium radical anion [(SPr)₂V^•] that does not quench the fluorescence signal, instead of NaDT as a reducing agent in our experiments [3–5]. To check if the (SPr)₂V^• is a favorable and viable option, we have tested it for studying MoFe protein and Fe protein interactions. These proteins were selected because they are known to be extremely oxygen-sensitive; if our protocol with (SPr)₂V^• and sealing of the capillaries inside the mBraun works, this can be adapted as a reliable system to study interactions between any molecules that are oxygen-sensitive. After successfully validating our results, we have further developed MST protocols to study interactions of MoFe protein–cadmium sulfide (CdS) nanocrystal biohybrids. Specifically, we adapted the approach to examine CdS QDs of various sizes, which have been shown to interact with several metalloenzymes where the photoexcited conduction band electrons from the QDs could be transferred to the active site of the enzyme, triggering catalysis [6–9]. This emerging class of novel enzyme-QD biohybrids is of utmost significance as it could redefine bio-catalysis [10]. We have successfully demonstrated light-driven reduction of N₂ to NH₃ using novel MoFe-CdS nanocrystal biohybrids [11,12]. Although the reactivity of MoFe protein and CdS QD mixtures have been studied, there has been no characterization of the molecular interactions prior to our work [13]. We have now successfully developed the protocols for determining the dissociation constants (K_d) of these novel biohybrids involving MoFe protein and the CdS QDs. Here, we have articulated a detailed protocol for determining the K_d between the MoFe protein and 3.7 nm CdS QDs, which was reported in Pellows et al. [13].