Non-fluorescent transient states of tyrosine as a basis for label-free protein conformation and interaction studies

• Published in: Scientific reports Vol. 14; no. 1; p. 6464
• Main Authors: Bagheri, Niusha, Chen, Hongjian, Rabasovic, Mihailo, Widengren, Jerker
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 18.03.2024; Nature Publishing Group UK; Nature Portfolio
• Subjects: Amino acids; Calmodulin; Calmodulin - metabolism; Coloring Agents; Fluorescence; Protein Conformation; Protein structure; Proteins; Spectrometry, Fluorescence - methods; Tryptophan; Tryptophan - chemistry; Tyrosine; Tyrosine - chemistry
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-024-57054-6

The amino acids tryptophan, tyrosine, and phenylalanine have been extensively used for different label-free protein studies, based on the intensity, lifetime, wavelength and/or polarization of their emitted fluorescence. Similar to most fluorescent organic molecules, these amino acids can undergo transitions into dark meta-stable states, such as triplet and photo-radical states. On the one hand, these transitions limit the fluorescence signal, but they are also highly environment-sensitive and can offer an additional set of parameters, reflecting interactions, folding states, and immediate environments around the proteins. In this work, by analyzing the average intensity of tyrosine emission under different excitation modulations with the transient state monitoring (TRAST) technique, we explored the photo physics of tyrosine as a basis for such environment-sensitive readouts. From how the dark state transitions of tyrosine varied with excitation intensity and solvent conditions we first established a photophysical model for tyrosine. Next, we studied Calmodulin (containing two tyrosines), and how its conformation is changed upon calcium binding. From these TRAST experiments, performed with 280 nm time-modulated excitation, we show that tyrosine dark state transitions clearly change with the calmodulin conformation, and may thus represent a useful source of information for (label-free) analyses of protein conformations and interactions.