Azatryptophans endow proteins with intrinsic blue fluorescence

Abstract

Our long-term goal is the in vivo expression of intrinsically colored proteins without the need for further posttranslational modification or chemical functionalization by externally added reagents. Biocompatible (Aza)Indoles (Inds)/(Aza)Tryptophans (Trp) as optical probes represent almost ideal isosteric substitutes for natural Trp in cellular proteins. To overcome the limits of the traditionally used (7-Aza)Ind/(7-Aza)Trp, we substituted the single Trp residue in human annexin A5 (anxA5) by (4-Aza)Trp and (5-Aza)Trp in Trp-auxotrophic Escherichia coli cells. Both cells and proteins with these fluorophores possess intrinsic blue fluorescence detectable on routine UV irradiations. We identified (4-Aza)Ind as a superior optical probe due to its pronounced Stokes shift of approximately 130 nm, its significantly higher quantum yield (QY) in aqueous buffers and its enhanced quenching resistance. Intracellular metabolic transformation of (4-Aza)Ind into (4-Aza)Trp coupled with high yield incorporation into proteins is the most straightforward method for the conversion of naturally colorless proteins and cells into their blue counterparts from amino acid precursors.

Fig. 1.

AnxA5, (Aza)Inds, and bioincorporation experiments. (A) Chemical structures of Ind, (4-Aza)Ind, and (5-Aza)Ind. (B) Ribbon plot of human anxA5 (side view) with Trp 187 buried in the hydrophobic pocket at the convex side of the molecule. (C) Expression profiles of anxA5 in cell lysates of E. coli ATCC 49980 grown in minimal medium with Ind and both (Aza)Inds. Note that comparably high expression levels of anxA5 were achieved with all Ind isosteres. (1) Noninduced cell lysate, (2) Trp-anxA5 (wild type-anxA5), (3) (4-Aza)Trp-anxA5, and (4) (5-Aza)Trp-anxA5.

Fig. 2.

Normalized fluorescence emission spectra of free Inds and (Aza)Inds in buffered aqueous solutions and of the parent protein and its Aza-variants on excitation at 280 nm. Comparison of the fluorescence spectra of free Ind with free (4-Aza)Ind (A) and (5-Aza)Ind (C); note the dramatic red-shift in the (4-Aza)Ind emission spectrum (70 nm). The emission maximum of free (5-Aza)Ind is less substantially red-shifted (54 nm). The fluorescence emission maximum of (4-Aza)Trp-anxA5 (B) is 107 nm red-shifted and that of (5-Aza)Trp-anxA5 (D) is 96 nm red-shifted compared with Trp-anxA5. Note also the spectral shoulder at 365 nm of (4-Aza)Trp-anxA5 (see the text for more details).

Fig. 3.

Secondary structure of (Aza)Trp-anxA5 and the parent Trp-protein. Far-UV CD spectra from 200–260 nm were recorded at 4°C.

Fig. 4.

Fluorescence profiles of (4-Aza)Ind and (7-Aza)Ind and related proteins in aqueous buffered solution. (A) Fluorescence spectra of (4-Aza)Ind (solid line) and (7-Aza)Ind (dashed line) on excitation at the respective absorbance maximum. Note the strong red-shift (33 nm) in the profile of (4-Aza)Ind (λ_max,em = 418 nm) when compared with (7-Aza)Ind (λ_max,em = 385 nm) along with markedly higher fluorescence intensity of (4-Aza)Ind. (B) Emission profiles (normalized to protein fluorescence) of (4-Aza)Trp-anxA5 (solid line) and (7-Aza)Trp-anxA5 (dashed line) on excitation at 280 nm. Note that the emission maximum λ_max,em of (4-Aza)Trp-anxA5 (λ_max,em = 423 nm) is 65 nm more red-shifted than of (7-Aza)Ind (λ_max,em = 358 nm). The spectral shoulder of (4-Aza)Trp-anxA5 in the range between 350 and 370 nm is discussed in the text.