Unique interactions between the chromophore and glutamate 16 lead to far-red emission in a red fluorescent protein

Abstract

mPlum is a far-red fluorescent protein with emission maximum at approximately 650 nm and was derived by directed evolution from DsRed. Two residues near the chromophore, Glu16 and Ile65, were previously revealed to be indispensable for the far-red emission. Ultrafast time-resolved fluorescence emission studies revealed a time dependent shift in the emission maximum, initially about 625 nm, to about 650 nm over a period of 500 ps. This observation was attributed to rapid reorganization of the residues solvating the chromophore within mPlum. Here, the crystal structure of mPlum is described and compared with those of two blue shifted mutants mPlum-E16Q and -I65L. The results suggest that both the identity and precise orientation of residue 16, which forms a unique hydrogen bond with the chromophore, are required for far-red emission. Both the far-red emission and the time dependent shift in emission maximum are proposed to result from the interaction between the chromophore and Glu16. Our findings suggest that significant red shifts might be achieved in other fluorescent proteins using the strategy that led to the discovery of mPlum.

Figure 1

Fluorescence spectra of mPlum and E16L variant. (A) Excitation (dashed lines) and emission (solid lines) spectra of mPlum at room (298 K, red) and low (77 K, blue) temperature. (B) Excitation (dashed) and emission (solid) spectra of mPlum-E16L variant at room (298 K, red) and low (77 K, blue) temperature.

Figure 2

Stereoview of portions of the final (2Fo-Fc) electron density maps. (A) mPlum, (B) E16Q, and (C) I65L variants. Maps are contoured at 1σ level. Atoms of the chromophore are shown in stick representation with nitrogen blue, sulfur yellow, and oxygen red. Note that I65 in panel B has two conformations (A and B).

Figure 3

Stereo images of chromophore environment comparison. The mPlum model (purple) is superposed onto (A) DsRed (orange), (B) mPlum E16Q (green), (C) mPlum I65L variant (orange), respectively. Note that in panel (B) Ile65 of E16Q variant shows two conformations. Hydrogen bonds are shown in dashed lines, labeled with approximate lengths in angstroms.