Glycine insertion makes yellow fluorescent protein sensitive to hydrostatic pressure

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

Glycine insertion modulates the fluorescence properties of Aequorea victoria green fluorescent protein and its variants in their ambient environment

The green fluorescent protein (GFP) derived from Pacific Ocean jellyfish is an essential tool in biology. GFP-solvent interactions can modulate the fluorescent property of GFP. We previously reported that glycine insertion is an effective mutation in the yellow variant of GFP, yellow fluorescent protein (YFP). Glycine insertion into one of the β-strands comprising the barrel structure distorts its structure, allowing water molecules to invade near the chromophore, enhancing hydrostatic pressure or solution hydrophobicity sensitivity. However, the underlying mechanism of how glycine insertion imparts environmental sensitivity to YFP has not been elucidated yet. To unveil the relationship between fluorescence and β-strand distortion, we investigated the effects of glycine insertion on the dependence of the optical properties of GFP variants named enhanced-GFP (eGFP) and its yellow (eYFP) and cyan (eCFP) variants with respect to pH, temperature, pressure, and hydrophobicity. Our results showed that the quantum yield decreased depending on the number of inserted glycines in all variants, and the dependence on pH, temperature, pressure, and hydrophobicity was altered, indicating the invasion of water molecules into the β-barrel. Peak shifts in the emission spectrum were observed in glycine-inserted eGFP, suggesting a change of the electric state in the excited chromophore. A comparative investigation of the spectral shift among variants under different conditions demonstrated that glycine insertion rearranged the hydrogen bond network between His148 and the chromophore. The present results provide important insights for further understanding the fluorescence mechanism in GFPs and suggest that glycine insertion could be a potent approach for investigating the relationship between water molecules and the intra-protein chromophore

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

<p>(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.</p

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

<p>(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.</p

One amino acid insertion into YFP.

<p>(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.</p

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

<p>(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.</p

Fluorescence intensity change of YFP-3G in <i>E. coli</i> with increase of hydrostatic pressure.

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

Development of a Fully Automated Desktop Analyzer and Ultrahigh Sensitivity Digital Immunoassay for SARS-CoV-2 Nucleocapsid Antigen Detection

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has had a significant impact on public health and the global economy. Several diagnostic tools are available for the detection of infectious diseases, with reverse transcription-polymerase chain reaction (RT-PCR) testing specifically recommended for viral RNA detection. However, this diagnostic method is costly, complex, and time-consuming. Although it does not have sufficient sensitivity, antigen detection by an immunoassay is an inexpensive and simpler alternative to RT-PCR. Here, we developed an ultrahigh sensitivity digital immunoassay (d-IA) for detecting SARS-CoV-2 nucleocapsid (N) protein as antigens using a fully automated desktop analyzer based on a digital enzyme-linked immunosorbent assay. Methods: We developed a fully automated d-IA desktop analyzer and measured the viral N protein as an antigen in nasopharyngeal (NP) swabs from patients with coronavirus disease. We studied nasopharyngeal swabs of 159 and 88 patients who were RT-PCR-negative and RT-PCR-positive, respectively. Results: The limit of detection of SARS-CoV-2 d-IA was 0.0043 pg/mL of N protein. The cutoff value was 0.029 pg/mL, with a negative RT-PCR distribution. The sensitivity of RT-PCR-positive specimens was estimated to be 94.3% (83/88). The assay time was 28 min. Conclusions: Our d-IA system, which includes a novel fully automated desktop analyzer, enabled detection of the SARS-CoV-2 N-protein with a comparable sensitivity to RT-PCR within 30 min. Thus, d-IA shows potential for SARS-CoV-2 detection across multiple diagnostic centers including small clinics, hospitals, airport quarantines, and clinical laboratories

Targeting the Expression of Platelet-Derived Growth Factor Receptor by Reactive Stroma Inhibits Growth and Metastasis of Human Colon Carcinoma

The stromal cells within colon carcinoma express high levels of the platelet-derived growth factor receptor (PDGF-R), whereas colon cancer cells do not. Here, we examined whether blocking PDGF-R could inhibit colon cancer growth in vivo. KM12SM human colon cancer cells were injected subcutaneously (ectopic implantation) into the cecal wall (orthotopic implantation) or into the spleen (experimental liver metastasis) of nude mice. In the colon and liver, the tumors induced active stromal reaction, whereas in the subcutis, the stromal reaction was minimal. Groups of mice (n = 10) received saline (control), the tyrosine kinase inhibitor imatinib, irinotecan, or a combination of imatinib and irinotecan. Four weeks of treatment with imatinib and irinotecan significantly inhibited tumor growth (relative to control or single-agent therapy) in the cecum and liver but not in the subcutis. The combination therapy completely inhibited lymph node metastasis. Imatinib alone or in combination with irinotecan inhibited phosphorylation of PDGF-Rβ of tumor-associated stromal cells and pericytes. Combination therapy also significantly decreased stromal reaction, tumor cell proliferation, and pericyte coverage of tumor microvessels and increased apoptosis of tumor cells and tumor-associated stromal cells. These data demonstrate that blockade of PDGF-R signaling pathways in tumor-associated stromal cells and pericytes inhibits the progressive growth and metastasis of colon cancer cells