Figure 1.
Fluctuation of integrated peptides.
(a) A topology diagram of sfGFP. β-strands, α-helices, and the chromophore are represented by gray arrows, black rectangles, and a white star, respectively. The DEVD peptide was integrated into the A (D23/G24), B (G51/K52), C (D102/D103), D (G134–L137), E (Q157/K158), F (D173/G174), G (G189/D190), and H (E213/K214) sites. (b) Root mean square fluctuation (RMSF) of the integrated peptides. The RMSF values represent the atomic fluctuations of each residue throughout 4.5–9.0 ns trajectories. Average fluctuation distances are also indicated. (c) Superimposed structures at every 0.5 ns throughout the 4.5–9.0 ns trajectory for mutant proteins in MD simulations. The integrated peptides are highlighted in magenta.
Figure 2.
Analysis of sfGFP mutants with peptide integration around site D.
(a) Peptide sequences of the sfGFP and mutants. The integration region of each mutant is highlighted in black and the integrated peptide sequences are shown in white letters. (b) Root mean square fluctuation (RMSF) values of peptides sequences of m1–m6 are shown. Average fluctuation distances are also indicated. (c) Fluorescence spectra obtained following excitation at 480 nm (m1–m5). The fluorescence intensity of sfGFP at 510 nm was used as a reference. Relative intensities of mutant proteins are also indicated. (d) Proteolytic resistance of mutants m1–m5. Cleaved fragments were analyzed after 24-h treatment with caspase-3 using western blotting. Protease resistance (PR%) was calculated from the band intensity of cleaved fragments (Cleaved) compared with that of remaining full-length proteins (Full). (e) Linear correlation between the average fluctuation of the integrated peptides and protease resistance. (f) Comparison of sfGFP mutant tolerance based on the position of integration. The relative fluorescent intensity (RFU) at 510 nm compared with that of sfGFP, the protease resistance (%) evaluated using western blotting, and structural fluctuation (Å) of the various peptides calculated by MD simulation are shown.
Figure 3.
Analysis of gFPSs containing polypeptides of various lengths at K131–L137.
(a) Peptide sequences of gFPSs. The amino acid sequence of sfGFP is shown at the top. The integrated peptide sequences of m7–m12 are shown in white letters. (b) Root mean square fluctuation (RMSF) values of K131–L137 in sfGFP and m7–m12 are shown. Average fluctuation distances are also indicated. (c) Fluorescence spectra following excitation at 480 nm for sfGFP and m7–m11. The fluorescence intensity of sfGFP at 510 nm was used as a reference. Relative intensities of gFPSs are also indicated. (d) Proteolytic resistance of sfGFP and m7–m11. Cleaved fragments were analyzed after 24-h treatment with caspase-3 by western blotting. Protease resistance (PR%) was calculated from the band intensity of cleaved fragments (Cleaved) compared with that of remaining full-length proteins (Full). (e) Comparison of gFPS tolerance with regard to the peptide length. The relative fluorescent intensity (RFU) at 510 nm compared with that of sfGFP, the protease resistance (%) evaluated using western blotting, and structural fluctuation (Å) of the various peptides calculated by MD simulation are shown.
Figure 4.
Analysis of the gFPSs containing HER2-BPs.
(a) Superimposed structures of sfGFP, mH1, mH2, mH3, mH4, and mH5 at every 0.5 ns throughout the 4.5–9.0 ns trajectory of the MD simulations. The K131–L137 integration sites are highlighted in magenta. (b) Representative surface structures for sfGFP, mH1, mH2, mH3, mH4, and mH5. Amino acids (N135–L137) in the sfGFP and integrated HER2-BPs of mH1–mH5 are also shown using the ball and stick model. These peptides are highlighted in blue, cyan, green, yellow, orange, red, and magenta for the 1st–7th amino acids, respectively.
Figure 5.
Binding assays for the gFPSs containing HER2-BPs.
(a) Fluorescence (FL) and bright field (BF) micrographs of HER2-positive N87 cells treated with sfGFP, mH1, mH2, mH3, mH4, and mH5 for 16 h. Exposure time = 1/200 s. Bar = 50 µm. (b) FL and BF micrographs of HER2-positive N87 cells treated with sfGFP, mH1C, mH2C, mH3C, mH4C, and mH5C for 16 h. Exposure time = 1/200 s or 1/2 s. Bar = 50 µm.
Figure 6.
Molecular display of gFPS on the cell surface of yeast.
(a) Bright field (BF) and fluorescence (FL) micrographs of yeast cells harboring empty control (pULD1) or gFPS-displaying plasmids (pULD1-gFPS). Bar = 10 µm (b) BF, green fluorescence (FL green), and red fluorescence (FL red) micrographs of gFPS- or gFPS-HER2-BP 1-displaying yeast cells treated with R-HER2-ECD for 3 h. Bar = 20 µm.
Figure 7.
A one-step peptide screening system using gFPS.
DNA libraries encoding gFPS with random peptides are introduced into the host (e.g., phage, bacteria, yeast, mammalian cells), resulting in the display of structurally constrained peptides on the host cell surface (A). The host cells that express peptides that bind to the target are easily selected based on gFPS fluorescence (B). The plasmid in the selected host is amplified and the DNA sequence of the corresponding peptide is determined (C). The identified peptides can be tested in vivo using structurally constrained forms such as circularized peptides (D) or following integration into in vivo scaffolds such as immunoglobulin G (IgG) molecules (E). These peptides retain unique structures that specifically bind to the target molecule with high affinity in vivo.