Although fluorescence tools for biological study abound, some processes, such as the interactions between proteins and metabolites, are challenging to investigate with the tools that currently exist. However, a research group led by professor Jeremy Mills at Arizona State University (ASU) is working to change that by using a fluorescent noncanonical amino acid to generate new proteins. The purpose of the ASU study is to develop new protein-based fluorescence tools that will be broadly applicable to the study of biological systems. A key aspect of the study is the generation of atomic-level pictures of many of the new proteins. The pictures “provide a great deal of information about how the binding of biotin changes the fluorescence properties of the proteins,” Mills said. The researchers designed protein constructs and experiments and performed spectroscopic and structural characterizations of five streptavidin protein mutants that contained the fluorescent noncanonical amino acid (fNCAA), which is named l-(7-hydroxycoumarin-4-yl) ethylglycine (7-HCAA). The characterizations were performed at sites close to the binding site of the streptavidin’s substrate. Assistant professor Jeremy Mills from ASU’s School of Molecular Sciences and the Biodesign Institute’s Center for Molecular Design and Biomimetics. Courtesy of ASU. The team found that many of the mutant proteins exhibited altered fluorescence spectra in response to biotin binding. Some proteins demonstrated an increase in fluorescence intensity, while others experienced a decrease. Changes in the red-shifted and blue-shifted emission maxima were also observed. To collect diffraction data, the researchers purified and crystallized the proteins. They obtained structural data for three of the five mutant proteins. The crystal structures helped the team understand how the interactions between the 7-HCAA amino acid and the functional groups could be responsible for the changes in the 7-HCAA spectra. The team’s atomic-level characterization of the new proteins could provide a starting point for the development of new protein-based fluorescent sensors of biological activity. “This information lays a foundation for the development of new fluorescent proteins that will help further the legacy that fluorescent proteins have already forged in the study of biological systems,” Mills said. New protein-based fluorescent tools are needed to study biological systems in ways that might be very difficult to do using existing fluorescent tools. The study and data collected by the ASU team is a step toward understanding how to harness the power of proteins to help guide future efforts to rationally design new fluorescent tools. “The protein studies of Jeremy Mills are typified by outstanding scholarship and a relentless commitment to making critical advances that will benefit science and society at large,” said Tijana Rajh, director of the ASU School of Molecular Sciences. The research was published in Biochemistry (www.doi.org/10.1021/acs.biochem.1c00291).