Structural Origins of Altered Spectroscopic Properties upon Ligand Binding in Proteins Containing a Fluorescent Noncanonical Amino Acid

Patrick R. Gleason, Bethany Kolbaba-Kartchner, J. Nathan Henderson, Erik P. Stahl, Chad R. Simmons, Jeremy H. Mills

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2577-2585
Number of pages9
JournalBiochemistry
Volume60
Issue number34
DOIs
StatePublished - Aug 31 2021

ASJC Scopus subject areas

  • Biochemistry

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