A Fluorescent Protein Scaffold for Presenting Structurally Constrained Peptides Provides an Effective Screening System to Identify High Affinity Target-Binding Peptides

<div><p>Peptides that have high affinity for target molecules on the surface of cancer cells are crucial for the development of targeted cancer therapies. However, unstructured peptides often fail to bind their target molecules with high affinity. To efficiently identify high-affinity target-binding peptides, we have constructed a fluorescent protein scaffold, designated gFPS, in which structurally constrained peptides are integrated at residues K131–L137 of superfolder green fluorescent protein. Molecular dynamics simulation supported the suitability of this site for presentation of exogenous peptides with a constrained structure. gFPS can present 4 to 12 exogenous amino acids without a loss of fluorescence. When gFPSs presenting human epidermal growth factor receptor type 2 (HER2)-targeting peptides were added to the culture medium of HER2-expressing cells, we could easily identify the peptides with high HER2-affinity and -specificity based on gFPS fluorescence. In addition, gFPS could be expressed on the yeast cell surface and applied for a high-throughput screening. These results demonstrate that gFPS has the potential to serve as a powerful tool to improve screening of structurally constrained peptides that have a high target affinity, and suggest that it could expedite the one-step identification of clinically applicable cancer cell-binding peptides.</p></div

Analysis of gFPSs containing polypeptides of various lengths at K131–L137.

<p>(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.</p

Molecular display of gFPS on the cell surface of yeast.

<p>(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.</p

Analysis of sfGFP mutants with peptide integration around site D.

<p>(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.</p

Fluctuation of integrated peptides.

<p>(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.</p

Analysis of the gFPSs containing HER2-BPs.

<p>(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.</p

Binding assays for the gFPSs containing HER2-BPs.

<p>(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.</p