首页|Substitution of the sole tryptophan of the cupredoxin, amicyanin, with 5-hydroxytryptophan alters fluorescence properties and energy transfer to the type 1 copper site
Substitution of the sole tryptophan of the cupredoxin, amicyanin, with 5-hydroxytryptophan alters fluorescence properties and energy transfer to the type 1 copper site
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NSTL
Elsevier
? 2022 Elsevier Inc.Amicyanin is a type 1 copper protein with a single tryptophan residue. Using genetic code expansion, the tryptophan was selectively replaced with the unnatural amino acid, 5-hydroxytryptophan (5-HTP). The 5-HTP substituted amicyanin exhibited absorbance at 300–320 nm, characteristic of 5-HTP and not seen in native amicyanin. The fluorescence emission maximum in 5-HTP substituted amicyanin is redshifted from 318 nm in native amicyanin to 331 nm and to 348 nm in the unfolded protein. The fluorescence quantum yield of 5-HTP substituted amicyanin mutant was much less than that of native amicyanin. Differences in intrinsic fluorescence are explained by differences in the excited states of tryptophan versus 5-HTP and the intraprotein environment. The substitution of tryptophan with 5-HTP did not affect the visible absorbance and redox potential of the copper, which is 10 ? away. In amicyanin and other cupredoxins, an unexplained quenching of the intrinsic fluorescence by the bound copper is observed. However, the fluorescence of 5-HTP substituted amicyanin is not quenched by the copper. It is shown that the mechanism of quenching in native amicyanin is F?rster, or fluorescence, resonance energy transfer (FRET). This does not occur in 5-HTP substituted amicyanin because the fluorescence quantum yield is significantly lower and the red-shift of fluorescence emission maximum decreases overlap with the near UV absorbance of copper. Characterization of the distinct fluorescence properties of 5-HTP relative to tryptophan in amicyanin provides a basis for spectroscopic interrogation of the protein microenvironment using 5-HTP, and long-distance interactions with transition metals.
5-hydroxytryptophanCopperCupredoxinFluorescence resonance energy transferGenetic code expansionTryptophan
Pastore A.J.、Davidson V.L.、Ficaretta E.、Chatterjee A.
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Burnett School of Biomedical Sciences College of Medicine University of Central Florida
NSTL
Elsevier
