Fluorescence Technologies for Monitoring Interactions Between Biological Molecules In Vitro - Progress in Molecular Biology and Translational Science - page 112

Interaction Fluorescent probe

Intrinsic
Extrinsic

Protein / protein
Protein / nucleic acids
Protein / membrane

Time Fluorescent effect

Steady state
Kinetics
Lifetime

Solvatochromism
Anisotropy
FRET

Figure 4.2 Fluorescence technology to probe biological molecules.

inorganic and organic compounds, temperature, pH, and the concentration
of the fluorescent molecules. The effects of these parameters vary widely
from one fluorophore to another. In particular, the absorption and emission
spectra, as well as the quantum yield, can be affected by the polarity of the
environment. This phenomenon, more generally known as “solvat-
ochromism,” can be defined as the ability of a fluorescent molecule to un-
dergo changes in position and/or intensity of absorption or emission bands
according to solvent polarity variation. 5 The solvatochromic effect, hyp-
sochromic (blue) or bathochromic (red) shifts, refers to a strong dependence
of the absorption and emission spectra on solvent polarity ( Fig. 4.3 ). Since
the polarities of the ground and excited states of a fluorophore are different, a
change in solvent polarity will induce different stabilization of the ground
and excited states and thus a modification of the energy between these elec-
tronic states. 5 Consequently, variations in the position, intensity, and shape
of the absorption and emission spectra can be used as a direct indicator of
solvent changes and local environment modification.
In proteins, although the aromatic residues Trp, Phe, and Tyr can poten-
tially be used as fluorescent probes, Trp is by far the most used intrinsic probe.
Tryptophan is an important intrinsic solvatochromic probe that can be useful
as a natural sensor of the conformational state of a protein or for assessment of
the nature of its environment. 6-9 The indole group of the side chain
confers maximum absorbance near 280 nm and maximum emission
intensity located between 310 and 350 nm on the tryptophan residue
depending on the environment. The use of denaturants, surfactants, or