For Spectral Shift analysis, the ratio of 670nm/650nm is plotted against the logarithmic ligand concentration. Similarly to TRIC, the ‘direction’ of the binding curve has no influence on the numerical value of the detected K_d.

In contrast to TRIC, some qualitative information about conformational changes around the dye can be deduced from the curve’s direction. Shifts towards shorter wavelengths (blue-shift) occur when the environment around the dye becomes more polar. Shifts towards longer wavelengths (red-shift) indicate that the environment around the dye became more hydrophobic.
 

Shifts in the emission spectrum of the dye are typically caused by changes to how the water molecules are arranged around the dye or by alterations in the surface hydrophobicity of the protein (Figure 1). While the emission of most fluorophores shifts towards longer wavelengths in less polar environments, NanoTemper has optimized their proprietary dyes in the opposite way—a physical effect known as “negative solvatochromism”. Negative solvatochromism occurs whenever the ground state of a dye is more stable in polar environments than its excited state.

Figure 1: Idealized fluorescence emission spectrum (purple) of a NanoTemper RED fluorophore and how this emission spectrum can spectrally shift to higher (red) or lower (blue) wavelengths due to chemical environment changes.

Hence, a Red-Shift correlates with an increase in local hydrophobicity of the dye and displacement of hydration water shell around the dye – typically caused by the following.

• Binding of a hydrophobic small molecule

• Protein-protein interaction

• Attachment of a dye to a protein surface

Figure 2. Red-shifting spectral shift dose response curve of p38 against SB203580 (left) and a respective visual molecular depiction (right).  

A Blue-Shift, on the other hand, comes from a decrease in local hydrophobicity of the dye and restoration of the hydration shell – typically caused by the following.

• Binding of a hydrophilic small molecule 
• Dissociation of a dye from a protein surface

Figure 3. Blue-shifting spectral shift dose response curve of maltose-binding protein (MBP) against maltose (left) and a respective visual molecular depiction (right).