posted on 2021-08-20, 17:00authored byPatrick
R. Gleason, Bethany Kolbaba-Kartchner, J. Nathan Henderson, Erik P. Stahl, Chad R. Simmons, Jeremy H. Mills
Fluorescent
noncanonical amino acids (fNCAAs) could serve as starting
points for the rational design of protein-based fluorescent sensors
of biological activity. However, efforts toward this goal are likely
hampered by a lack of atomic-level characterization of fNCAAs within
proteins. Here, we describe the spectroscopic and structural characterization
of five streptavidin mutants that contain the fNCAA l-(7-hydroxycoumarin-4-yl)ethylglycine
(7-HCAA) at sites proximal to the binding site of its substrate, biotin.
Many of the mutants exhibited altered fluorescence spectra in response
to biotin binding, which included both increases and decreases in
fluorescence intensity as well as red- or blue-shifted emission maxima.
Structural data were also obtained for three of the five mutants.
The crystal structures shed light on interactions between 7-HCAA and
functional groups, contributed either by the protein or by the substrate,
that may be responsible for the observed changes in the 7-HCAA spectra.
These data could be used in future studies aimed at the rational design
of fluorescent, protein-based sensors of small molecule binding or
dissociation.