What is Spectral Shift?
Fluorescence Principles
NanoTemper’s Spectral Shift technology harnesses fluorescence to detect small changes in emitted light during a biomolecular interaction, in solution, and under isothermal conditions.
In the most basic description, fluorescence is an event where a molecule in its resting state (S0) absorbs a photon of light (hνA) and enters an excited state (S0-->S1). Then, to return to the resting state, the molecule rapidly emits a photon (hνF) of lesser energy after vibrational relaxation; the amount of relaxation and subsequent energy loss strongly depends on the local environment, such as temperature, solvent (buffer), and steric hinderance (Figure 1).
Figure 1: Jablonski diagram describing the absorption and emission of a photon.
The amount of energy loss is varied over multiple relaxation pathways that specifies the bandwidth, and the shape of the emission spectrum generated. The emission spectrum is therefore defined by its consistent local environment.
Spectral Shift Technology
The Spectral Shift technology takes advantage of the fact that when the local chemical environment around the dye changes—such as after a ligand is bound—the emission wavelength can shift (Figure 2.A). By simultaneously measuring fluorescence at two different wavelengths, 650 nm and 670 nm, and plotting the ratio between these two intensities against ligand concentration, the binding affinity (Kd) can be derived (Figure 2.B).
Figure 2.A: Depiction of how Spectral Shift can be detected by a dual wavelength measurement (left) and an illustration of how the fluorescence detection is technically performed in the instrument (right). 2.B: Dose response plot of the recorded ratio of 670nm/650nm fluorescence against ligand concentration. The inflection point of the fitted curve equates to the Kd of the interaction.
The chemical environment changes that occur upon ligand binding are made up by a combination of different factors including:
- ligand proximity
- conformational changes in protein
- hydrophobicity and charge distribution on surface
Figure 3 displays unbound and bound state of a protein. Upon ligand binding the surface of the protein changes, which includes a redistribution of hydrophobic/hydrophilic areas and water molecules.
Figure 3: Surface hydrophobicity map of protein in unbound form (left) and after ligand binding (right). Orange and turquoise shades denote the most hydrophilic and hydrophobic areas, respectively.
Mathematically, the Spectral Shift can be described by the Lippert-Mataga equation (Figure 4), where the magnitude of the Spectral Shift relates to differences in the dipole moments between the excited (µe) and ground state (µg) of the dye, as well as the effective volume that the dye can probe (a3), and changes in the dye environment (△f).
Figure 4: Lippert-Mataga equation with added color-coded sections for spectral shift (yellow), dye chemistry (green) and dye environment (blue).
References