Glycine insertion makes yellow fluorescent protein sensitive to hydrostatic pressure

Abstract

Fluorescent protein-based indicators for intracellular environment conditions such as pH and ion concentrations are commonly used to study the status and dynamics of living cells. Despite being an important factor in many biological processes, the development of an indicator for the physicochemical state of water, such as pressure, viscosity and temperature, however, has been neglected. We here found a novel mutation that dramatically enhances the pressure dependency of the yellow fluorescent protein (YFP) by inserting several glycines into it. The crystal structure of the mutant showed that the tyrosine near the chromophore flipped toward the outside of the β-can structure, resulting in the entry of a few water molecules near the chromophore. In response to changes in hydrostatic pressure, a spectrum shift and an intensity change of the fluorescence were observed. By measuring the fluorescence of the YFP mutant, we succeeded in measuring the intracellular pressure change in living cell. This study shows a new strategy of design to engineer fluorescent protein indicators to sense hydrostatic pressure.

Figure 1. One amino acid insertion into YFP.

(A) Structure around the chromophore of YFP (yellow; PDB ID: 1yfp). Red, oxygen. Blue, nitrogen. (B) Schematic drawing of the one amino acid insertion method. (C) Absorbance (left) and emission (right) spectra of the YFP mutants. Orange, wild-type YFP; red, glutamate insertion; blue, histidine insertion; green, methionine insertion; magenta, tyrosine insertion. Emission spectra were obtained at 488 nm excitation. The intensities are normalized as to the peak intensity of wild-type YFP. Concentrations are constant among all data. (D) Spectral shifts of the peak of absorbance (green) and emission (yellow) spectra of the YFP mutants. Lower alphabets stand for the inserted amino acid. Amino acids are arranged according to the van der Waals radius. (E) Fluorescence intensity (red), chloride sensitivity (magenta), and pH sensitivity (orange) of the YFP mutants. The values are normalized as to those of wild-type YFP. Lower alphabets stand for the inserted amino acid. The chloride sensitivity is defined as the ratio of the fluorescence intensities at 0 mM and 200 mM KCl. The pH sensitivity is defined as the ratio of the fluorescence intensities at pH 8.0 and 7.0.

Figure 2. Effect of insertion of 'G' rich fragments into YFP.

(A) Absorbance (blue) and emission (red) spectra of YFP-1G (upper), YFP-3G (middle), and YFP-6G (lower). Green, absorbance of YFP; yellow, emission of YFP. Emission spectra were obtained at 488 nm excitation. The intensities are normalized as to each peak intensity. (B) Spectral shifts of absorbance (green) and emission (yellow), fluorescence intensities (red), and pH dependency (orange) of YFP-nG that is defined as the ratio of the fluorescence intensities at pH 8.0 and 7.0. The intensities are normalized as to wild-type YFP. (C) Fluorescence photographs of YFP and YFP-3G solutions on blue-light (488 nm excitation) transilluminator. The concentrations of proteins were 0.33 mg/ml. We set both samples on side by side, and took the photograph simultaneously.

Figure 3. Crystal structure of YFP inserted 'G' and ‘GGG’.

(A) Overlay structure diagram showing front (left) and side (right) views of YFP (yellow; PDB ID: 1yfp), YFP-1G (cyan), and YFP-3G (green). The missing residues of YFP-3G are indicated by dotted line. (B) Close-up view of the YFP-3G chromophore shown with the 2Fo-Fc density map. (C) Superposition of the structures of YFP (yellow), YFP-1G (cyan), and YFP-3G (green) around the chromophore. Residues interact with the chromophore are shown in stick model with the main-chain backbone trace of β7, β10 and β11. Oxygen and nitrogen atoms are colored in red and blue, respectively. The right panel is viewed from the bottom of the left panel. (D) Water molecules near the chromophore ring. The stick models of YFP-1G (left) and YFP-3G (middle) and YFP (right) are colored in cyan, green and yellow, respectively. YFP-1G and YFP-3G are shown with the 2Fo-Fc density map. Water molecules are represented by red ball. The arrows indicate water molecules filling the space where Tyr145 of YFP was located. Oxygen and nitrogen atoms are colored in red and blue, respectively.

Figure 4. Hydrostatic pressure dependency of the YFP, YFP-1G and YFP-3G.

(A, B, C) Fluorescence spectra of YFP (A), YFP-1G (B), and YFP-3G (C) between 0.1 and 50 MPa (red to blue). The traces represent the averages of six individual trials. All spectra are normalized with the spectrum at 0.1 MPa. (D) Peak shifts of the fluorescence spectra of YFP (red), YFP-1G (blue), and YFP-3G (green) between 0.1 and 50 MPa. (E) Pressure dependence of the peak fluorescence intensities of YFP (red), YFP-1G (blue), and YFP-3G (green) between 0.1 and 50 MPa. The values are normalized with the value at 0.1 MPa. All emission spectra were obtained at 488 nm excitation. Error bars, standard deviation.

Figure 5. Fluorescence intensity change of YFP-3G in E. coli with increase of hydrostatic pressure.

(A) Fluorescence image of E. coli cells expressing YFP-3G at 0.1, 30 and 50 MPa. Insertions are the enlarged images of the single E. coli cell. (B) Time curse of fluorescent intensity of single E. coli expressing YFP-3G with the change of hydrostatic pressure. Values are the applied pressure. (C) Pressure dependence of the fluorescence intensities of E. coli expressing YFP (red) and YFP-3G (green) at 0.1-50 MPa (N = 74-151). Error bars, standard error.