A New Spectral Shift-Based Method to Characterize Molecular Interactions.

There are many fluorescence-based applications that can be used to characterize molecular interactions. However, available methods often depend on site-specific labeling techniques or binding-induced changes in conformation or size of the probed target molecule. To overcome these limitations, we applied a ratiometric dual-emission approach that quantifies ligand-induced spectral shifts with sub-nanometer sensitivity. The use of environment-sensitive near-infrared dyes with the method we describe enables affinity measurements and thermodynamic characterization without the explicit need for site-specific labeling or ligand-induced conformational changes. We demonstrate that in-solution spectral shift measurements enable precise characterization of molecular interactions for a variety of biomolecules, including proteins, antibodies, and nucleic acids. Thereby, the described method is not limited to a subset of molecules since even the most challenging samples of research and drug discovery projects like membrane proteins and intrinsically disordered proteins can be analyzed.

MST and TRIC Technology to Reliably Study PROTAC Binary and Ternary Binding in Drug Development.

PROTACs have shown promise as a new class of therapy, with a unique mechanism of action orthogonal to traditional small molecules that are used to regulate protein activity. Their novel MOA utilizing the body's natural protein degradation machinery degrades a protein of interest rather than inhibiting its function. This strategy has several advantages over conventional small-molecule inhibitors, e.g., higher sensitivity, less off-target effects, and greater target space. However, unlocking the potential of PROTACs necessitates drug discovery techniques that can support the complexity of the novel MOA. In this chapter, we describe the application of MicroScale Thermophoresis (MST) and Temperature-Related Intensity Change (TRIC) to characterize both the binary and ternary binding of PROTACs with target proteins and ubiquitin ligases along with an efficient determination of the cooperativity of the ternary complex formation. The assay development and experimental procedure to characterize the well-described BET PROTAC MZ1 show how MST and TRIC can be applied as a fast and highly sensitive method for PROTAC discovery.

Development of a Microscale Thermophoresis-Based Method for Screening and Characterizing Inhibitors of the Methyl-Lysine Reader Protein MRG15.

MRG15 is a transcription factor containing the methyl-lysine reader chromodomain. Despite its involvement in different physiological and pathological states, to date the role of this protein has not been fully elucidated due to the lack of a specific and potent chemical probe.In this work, we report the development of a microscale thermophoresis (MST)-based assay for the study of MRG15-ligand binding interactions. After the development, the assay was validated using a small focused library and UNC1215 as the reference compound, to yield the identification of 10 MRG15 ligands with affinities ranging from 37.8 nM to 59.1 µM.Hence, our method is robust, convenient, and fast and could be applied to other methylation reader domain-containing proteins for the identification of new chemical probes.

Quantifying the Interaction of Phosphite with ABC Transporters: MicroScale Thermophoresis and a Novel His-Tag Labeling Approach.

The combination of MicroScale Thermophoresis (MST) and near-native site-specific His-tag labeling enables simple, robust, and reliable determination of the binding affinity between proteins and ligands. To demonstrate its applicability for periplasmic proteins, we provide a detailed protocol for determination of the binding affinity of phosphite to three ABC transporter periplasmic-binding proteins from environmental microorganisms. ABC transporters are central to many important biomedical phenomena, including resistance of cancers and pathogenic microbes to drugs. The protocol described here can be used to quantify protein-ligand and protein-protein interactions for other soluble, membrane-associated and integral membrane proteins.

The structural basis of nanobody unfolding reversibility and thermoresistance.

Nanobodies represent the variable binding domain of camelid heavy-chain antibodies and are employed in a rapidly growing range of applications in biotechnology and biomedicine. Their success is based on unique properties including their reported ability to reversibly refold after heat-induced denaturation. This view, however, is contrasted by studies which involve irreversibly aggregating nanobodies, asking for a quantitative analysis that clearly defines nanobody thermoresistance and reveals the determinants of unfolding reversibility and aggregation propensity. By characterizing nearly 70 nanobodies, we show that irreversible aggregation does occur upon heat denaturation for the large majority of binders, potentially affecting application-relevant parameters like stability and immunogenicity. However, by deriving aggregation propensities from apparent melting temperatures, we show that an optional disulfide bond suppresses nanobody aggregation. This effect is further enhanced by increasing the length of a complementarity determining loop which, although expected to destabilize, contributes to nanobody stability. The effect of such variations depends on environmental conditions, however. Nanobodies with two disulfide bonds, for example, are prone to lose their functionality in the cytosol. Our study suggests strategies to engineer nanobodies that exhibit optimal performance parameters and gives insights into general mechanisms which evolved to prevent protein aggregation.

Near-native, site-specific and purification-free protein labeling for quantitative protein interaction analysis by MicroScale Thermophoresis.

MicroScale Thermophoresis (MST) is a frequently used method for the quantitative characterization of intermolecular interactions with several advantages over other technologies. One of these is its capability to determine equilibrium constants in solution including complex biological matrices such as cell lysates. MST requires one binding partner to be fluorescent, which is typically achieved by labeling target proteins with a suitable fluorophore. Here, we present a near-native, site-specific in situ labeling strategy for MST experiments that enables reliable measurements in cell lysates and that has distinct advantages over routine covalent labeling techniques. To this end, we exploited the high-affinity interaction of tris-NTA with oligohistidine-tags, which are popular for purification, immobilization or detection of recombinant proteins. We used various DYE-tris-NTA conjugates to successfully label His-tagged proteins that were either purified or a component of cell lysate. The RED-tris-NTA was identified as the optimal dye conjugate with a high affinity towards oligohistidine-tags, a high fluorescence signal and an optimal signal-to-noise ratio in MST binding experiments. Owing to its emission in the red region of the spectrum, it also enables reliable measurements in complex biological matrices such as cell lysates allowing a more physiologically realistic assessment and eliminating the need for protein purification.