Emitting State of 5‑Hydroxyindole, 5‑Hydroxytryptophan, and 5‑Hydroxytryptophan Incorporated in Proteins

5-Hydroxy-L-tryptophan (5HW) has been biosynthetically incorporated in many proteins to facilitate their characterization using fluorescence spectroscopy. An attractive feature of this tryptophan analogue is its absorbance at 310−320 nm, allowing its specific excitation in a Trp background. The red-shift in absorbance upon introduction of a hydroxyl group at the 5-position of Trp or indole was found to be due to a lowering of the 1Lb transition energy. It was therefore believed that 5HW only features 1Lb emission. Recently, calculations for 5-hydroxyindole (5HI) in water revealed 1La is the emitting state, and the same was predicted for 5HW incorporated in proteins. To clarify which state emits in 5HI and 5HW, we present here excitation anisotropy spectra of these probes and of four proteins labeled with 5HW at a surface exposed position. Our data clearly show 1Lb is the emitting state of 5HI, 5HW, and 5HW in three of the proteins investigated. For one protein mixed emission was observed, and the decay kinetics were found strongly dependent on the emission wavelength. This work provides the first experimental evidence that 1La can be the emitting state for this Trp analogue incorporated in a protein.