Fluorescence Technologies for Monitoring Interactions Between Biological Molecules In Vitro - Progress in Molecular Biology and Translational Science - page 118

2. MONITORING PROTEIN/SUBSTRATE INTERACTIONS
Protein/partner interactions, such as enzyme/substrate, protein/
nucleic acid, and protein/nucleotide, play a crucial role in the regulation
of biological pathways at the extracellular, cellular, viral, or drug delivery
level. Development of fluorescence technologies using both natural and
synthetic probes has provided new and appropriate tools to decipher the
nature, strength, and impact on the life cycle of a wide range of molecular
interactions. Protein/ligand complexes can be probed by intrinsic trypto-
phan fluorescence at both steady-state and pre-steady-state levels. However,
most proteins contain more than one tryptophan residue, which constitutes
a limitation for the analysis of the specific interaction of one domain of the
protein. Therefore, in most cases, extrinsic fluorescence constitutes an
attractive alternative as a large panel of fluorescent dyes with well-suited
spectral properties and more appropriate quantum yields for sensitive detec-
tion and single-molecule assays.
2.1. Protein/nucleotide interactions
Nowadays, fluorescent- or caged-nucleotide analogs are widely used as
probes for enzyme activities and as sensors for screening of inhibitors and/or
activators. 20-22 Nucleotide chemistry has focused on the design of specific
fluorescently labeled nucleotides to investigate either enzymatic parameters
or interactions with partners (activator, inhibitor, etc.) of nucleotide
binding proteins, such as nucleotide kinases, protein kinases, GTPases,
ATPases, and so on. 23-26 Nucleotides can be modified on their base
(benzo-, etheno-), on the phosphate (XTP- g -naphthalene, g -[(6-amino)
hexyl]-, g -(sulfo-1-naphthyl)amide, methylanthranylate (Mant)), and on
the sugar (trinitrophenyl-, Mant-) moieties, depending on the nature/
structure of the nucleotide binding site of the enzyme ( Fig. 4.6 ). New
chemistry has also been proposed using the amino hexyl linker, allowing
labeling with a large panel of available dyes with the amino group
( Fig. 4.6 ). The binding of nucleotide analogs to proteins leads to large
modifications of their extrinsic fluorescence, which can be used as a sensor
for the determination of enzymatic parameters as well as for screening of
inhibitors or natural substrates. However, it is important to keep in mind
that the presence of the dye can modify the binding properties of the
nucleotide, and displacement experiments of labeled nucleotides using
unlabeled nucleotides are essential to validate the approach. For example,