Photophysics and optical switching in green fluorescent protein mutants

Abstract

We demonstrate by using low-temperature high-resolution spectroscopy that red-shifted mutants of green fluorescent protein are photo-interconverted among three conformations and are, therefore, not photostable "one-color" systems as previously believed. From our experiments we have further derived the energy-level schemes governing the interconversion among the three forms. These results have significant implications for the molecular and cell biological applications of the green fluorescent protein family; for example, in fluorescence resonant energy transfer experiments, a change in "color" on irradiation may not necessarily be due to energy transfer but can also arise from a photo-induced conversion between conformers of the excited species.

Figure 1

Absorption spectra of wild-type GFP and the red-shifted mutants S65T, RS-GFP, and EYFP at T = 1.6 K. The maxima of the absorption bands and the 0–0 transitions of the three conformers of wt-GFP are indicated.

Figure 2

(a) Excitation (blue curve) and emission (red curve) spectra of RS-GFP at 1.6 K before (—) and after (---) burning into I_0–0 at 499 ± 1 nm (right hole). Before burning, only the I-form is present. After burning, the vibronic bands of the B-form between 450 and 475 nm increase (blue, ---); thus, I→B. By burning into B_0–0 at 476 ± 1 nm, a hole is produced (Left), and the original spectrum recovers; thus, B→I. After burning into the I_0–0, the A-form is also photoinduced, which is reflected in the fluorescence spectrum. In the region from 400 to 490 nm there is no signal before burning (red, —), but there is fluorescence after burning (red, ---). (b) Excitation spectra of A with λ_det = 450 nm. Optical switching between A and I occurs. (1) The A-form has been produced by burning into I_0–0 at 499 ± 1 nm. (2) After burning a hole in the 0–0 transition of A at 434 ± 1 nm, the intensity of the whole A-spectrum decreases whereas that of the I-form increases (not shown). (3) After subsequent burning into I, the population of the A-form increases again.

Figure 3

Energy-level diagrams of the red-shifted mutants RS-GFP (Top), S65T (Middle), and EYFP (Bottom). The forms A, I, and B photointerconvert as indicated by the diagonal arrows. The relative populations of the three forms differ for the various mutants. In RS-GFP only the I-form is populated at 1.6 K before burning; by burning into I, the A- and B-forms are photoinduced. In S65T, all forms are populated at 1.6 K, with the I-form having the lowest ground-state level. In EYFP, the most populated form is B; I and A are significantly less populated at 1.6 K. Their populations can be enhanced by burning into B.