Fluorescence Principles

In addition to Spectral Shift, fluorescence intensity changes can also provide insights into protein-ligand interactions. Alterations in the local microenvironment of a fluorophore may not only shift the maximum emission wavelength but also affect the intensity or brightness of the emitted light. Fluorescence intensity is largely influenced by collisions between the fluorophore and the surrounding solvent, adjacent amino acid residues of a protein or a bound small molecule ligand (Figure 1). When a fluorophore is situated in a more shielded or less polar environment, it typically emits more light. Conversely, exposure to the solvent, where collisions are more frequent, often reduces fluorescence due to increased energy dissipation.

Figure 1: Depiction of how different microchemical environments can alter fluorescence intensity.

Structural Influence on Fluorescence

When a ligand binds to a protein, the protein may undergo structural or conformational changes. These changes can alter the fluorophore's exposure to the surrounding environment, leading to variations in fluorescence intensity. For example, if the binding event causes the fluorophore to be buried within a less accessible or more hydrophobic region (Figure 2), the emission of light might increase due to reduced exposure to quenching molecules. On the other hand, if the binding alters the structure in a way that brings the fluorophore closer to quenching polar water molecules, fluorescence may decrease.

In summary, the temperature response of the fluorophore can be affected by the following:

• Changes in the dynamics of the protein backbone
• Shielding/exposure to solvent due to conformational changes
• Increased or decreased contacts with flexible loops and amino acids

Figure 2: Protein ribbon representation displaying how ligand binding to a protein can alter the fluorophore's (highlighted in purple) exposure to the surrounding environment. 

Temperature-Related Intensity Change (TRIC)

Detecting these subtle changes at a constant temperature can be challenging. To address this, the second method in Monolith and Dianthus instruments–Temperature-Related Intensity Change or TRIC–introduces a small, controlled temperature increase, which is achieved by an extremely precise and rapid heating step using an infrared laser. This slight temperature increase can amplify the differences between bound and unbound state of the protein, making the fluorescence intensity changes easier to measure and can be mathematically expressed by the Stern Volmer Equation (Figure 3). TRIC has been identified as the primary factor contributing to the effects historically described and summarized as Microscale Thermophoresis (MST).

Figure 3: Stern Volmer Equation: The chemistry of the fluorophore is highlighted in green, changes of the environment in blue and temperature dependency in purple. 

The results from TRIC measurements are plotted in a similar way as in Spectral Shift to create a dose-response curve and to infer the binding affinity of a particular ligand (Figure 4). Importantly, TRIC can detect subtle binding interactions that might not induce significant spectral shifts.

Figure 4: Fluorescence traces at varying ligand concentrations (left). Fluorescence Intensity at 670nm is recorded and normalized before (Fcold) and after a temperature gradient is induced by an IR-laser. The TRIC signal (Fnorm = Fhot / Fcold) is plotted as a function of ligand concentration as a dose response curve on the right. The inflection point of the fitted curve equates to the K_d of the interaction.