The structure of a far-red fluorescent protein, AQ143, shows evidence in support of reported red-shifting chromophore interactions

Abstract

Engineering fluorescent proteins (FPs) to emit light at longer wavelengths is a significant focus in the development of the next generation of fluorescent biomarkers, as far-red light penetrates tissue with minimal absorption, allowing better imaging inside of biological hosts. Structure-guided design and directed evolution have led to the discovery of red FPs with significant bathochromic shifts to their emission. Here, we present the crystal structure of one of the most bathochromically shifted FPs reported to date, AQ143, a nine-point mutant of aeCP597, a chromoprotein from Actinia equina. The 2.19 Å resolution structure reveals several important chromophore interactions that contribute to the protein's far-red emission and shows dual occupancy of the green and red chromophores.

Keywords: chromoprotein; near-infrared, bathochromic shift; red fluorescent protein.

Figure 1

Alignment of the chromophores and C-terminal cysteine from each of the eight monomers in the asymmetric unit. The modeled cis phenolate is shown in turquoise. The N-acylamine and N-acylimine are present in the green and red chromophores, respectively.

Figure 2

Chromophore contacts in AQ143. Residues that directly interact with the chromophore or help to co-ordinate structural waters (red spheres) are shown along with the immediate hydrogen-bonding network. A representative chromophore was chosen (chain E) to illustrate the contacts. Hydrogen bonds (dotted lines) are shown for interactions with the chromophore. The modeled cis conformation is shown in turquoise, along with two putative hydrogen bonds to its hydroxyl group. Two hydrogen bonds to the acylimine oxygen from Glu41 and a co-ordinated water can be seen in the right of the figure.