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.

The aromatic amino acids tryptophan, tyrosine, and phenylalanine have been extensively used for different label-free protein studies. These investigations extract information on protein conformations and interactions from the emitted fluorescence's intensity, wavelength, and/or polarization. Like most fluorescent organic compounds, these amino acids also undergo transitions into dark meta-stable states, including triplet and photo-radical states. These transitions are notably sensitive to the surrounding environment, offering an additional set of parameters that reflect the protein's interactions, folding states, and immediate surroundings.

Transient State (TRAST) monitoring has been developed to quantify fluorophore transition dynamics by recording the average fluorescence intensity in response to a modulated excitation. In this work, we performed TRAST experiments to investigate tyrosine autofluorescence and used it to detect conformational changes in calmodulin, a calcium-binding protein containing two tyrosine residues. A photophysical model for tyrosine was established, and it was revealed how tyrosine's dark state transitions changed with excitation intensity, solvent pH, and redox conditions. The TRAST experiments demonstrated that tyrosine's dark state transitions could serve as valuable information sources for label-free analyses of protein conformations and interactions.

De aromatiska aminosyrorna tryptofan, tyrosin och fenylalanin har använts i stor utsträckning för olika inmärkningsfria proteinstudier. Dessa undersökningar extraherar information om proteinkonformationer och interaktioner från den emitterade fluorescens intensiteten, dess våglängd och/eller polarisering. Liksom de flesta fluorescerande organiska föreningar genomgår dessa aminosyror också övergångar till mörka metastabila tillstånd, inklusive triplett- och fotoradikaltillstånd. Dessa övergångar är särskilt känsliga för den omgivande miljön, och erbjuder en extra uppsättning parametrar som återspeglar proteinets interaktioner, vikningstillstånd och omedelbara omgivningar.

Transient State (TRAST) monitorering har utvecklats för att kvantifiera fluoroforövergångsdynamik genom att registrera den genomsnittliga fluorescensintensiteten som svar på en modulerad excitation. I detta arbete utförde vi TRAST-experiment för att undersöka tyrosinautofluorescens och använde den för att detektera konformationsförändringar i calmodulin, ett kalciumbindande protein som innehåller två tyrosiner. En fotofysikalisk modell för tyrosin etablerades, och hur tyrosins mörka tillståndsövergångar förändrades med excitationsintensitet, lösningsmedels pH och redoxförhållanden kunde faststållas. TRAST- experimenten visade att tyrosins mörka tillståndsövergångar kan fungera som värdefulla informationskällor för inmärkningsfria analyser av proteinkonformationer och interaktioner.