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, 22, 256-268

Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling

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Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling

Shigekazu Tabata et al. iScience.

Abstract

Electron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM.

Keywords: Biochemistry Methods; Nanoparticles; Structural Biology.

Conflict of interest statement

The authors declare no competing interests.

Figures

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Figure 1
Figure 1
Specific Labeling of Protein Utilizing Complementary Recognition Pair of Peptide Tag and Reactive Zinc Complex (A) Schematic illustration of covalent labeling of tag-fused protein with zinc complex probe. (B) Binding-induced cysteine conjugation of α-helical peptide tag with zinc complex probe developed in the current study. (C) Sequences of the peptide tags. (D) Structures of the Zn(II)-DpaTyr probes.
Figure 2
Figure 2
Binding and Reactivity Profiles of Aspartate-Rich Peptide Tags (A) CD spectral change of peptide-a upon the addition of 1-2Zn(II). Conditions: [peptide-a] = 50 μM, [1-2Zn(II)] = 0, 25, 50, and 100 μM, 10 mM borate buffer, pH 8.0, 25°C. (B) Fluorescence titration profile of peptide-bem = 448 nm) upon the addition of 2-4Zn(II). Conditions: [peptide-b] = 0.05 μM, 50 mM HEPES buffer, 100 mM NaCl, pH 7.2, 25°C. Each plot represents the average value ±standard error of triplicate experiments. (C) Time-trace plot of the reaction of peptide-d with fluorogenic mCBI in the presence or absence of 2-4Zn(II). Conditions: [peptide-d] = 4 μM, [mCBI] = 120 μM, [2-4Zn(II)] = 14 μM, 50 mM HEPES, 100 mM NaCl, pH 7.2, 37°C. (D) Plot of pH-dependent reaction rate (Vo, ΔF) of peptide-d with mCBI in the presence or absence of 2-4Zn(II). Data were analyzed using Henderson-Hasselbach equation.
Figure 3
Figure 3
Fluorescence Labeling of helixD2-Tag-Fused Protein (A) Structures of the fluorescent Zn(II)-DpaTyr probes. (B) In-gel fluorescence (FL) and Coomassie Brilliant Blue (CBB) analysis of covalent labeling of MBP tagged with a helixD2-tag (upper panel) or CH6 tag (lower panel) by 10-4Zn(II). Labeling conditions: [tag-MBP] = 1 μM, [10-4Zn(II)] = 10 μM, 50 mM HEPES, 100 mM NaCl, 20 mM TCEP, pH 7.2, 37°C. (C) Time-trace plot of the fluorescence band intensity of the MBP labeled with 10-4Zn(II) (mean ± SD of triplicate experiment). (D and E) Fluorescence imaging of HEK293 cells expressing B2R fused with helixD2-tag (D) or CH6 tag (E) upon labeling with 10-4Zn(II). Labeling conditions: [10-4Zn(II)] = 4 μM, HEPES-buffered saline, pH 7.4, 37°C, 30 min. (F and G) (F) Fluorescence images of non-specific labeling on surface of HEK293 cells without expression of the tag-fused B2R. Labeling conditions: [10-4Zn(II) or 11-4Zn(II)] = 4 μM, HEPES-buffered saline, 20 μM TCEP, pH 7.4, 37°C, 30 min (G) Time-trace plot of the fluorescence intensity on the surface of HEK293 cells without expression of the tag-fused B2R (n = 10, mean ± SD).
Figure 4
Figure 4
EM Detection of helixD2-Tag-Fused B2R Protein on Freeze-Fracture Replicas (A) Schematic illustration of labeling states of a complementary replica pair. (B and C) EM images of the complementary replicas showing E-face (B) and P-face (C) from the same membrane. Scale bars, 1 μm. (Insets) Magnified images of the area framed in the main pictures. Arrowheads indicate 5-nm gold particles. Scale bars, 100 nm. (D) Comparison of the labeling density of 12-4Zn(II) on the E- and P-face images, respectively (****p < 0.001, Mann-Whitney U test; E-face, n = 11; P-face, n = 9, mean ± SE). (E) Comparison of the labeling density of anti-GFP antibody on the E- and P-face images, respectively (****p < 0.001, Mann-Whitney U test; E-face, n = 7; P-face, n = 15, mean ± SE).
Figure 5
Figure 5
EM Detection of helixD2-Tag-Fused B2R Protein on HEK Cells Using the Probe Conjugated with a 1.4-nm Gold Particle (A) Schematic illustration of labeling states of replicas. (B–D) Dark-field scanning transmission electron microscopy images of E-face replicas made from transfected (B and C) or non-transfected (D) HEK cells labeled with 13-4Zn(II) before aldehyde fixation. (C) and the inset on (D) are magnified images of the area framed in (B) and (D), respectively. White arrowheads indicate B2R clusters labeled with 13-4Zn(II). Scale bar: 200 nm in (B) and the main picture of (D) and 50 nm in (C) and the inset on (D). (E and F) Dark-field images of replicas made from transfected HEK cells labeled with 13-4Zn(II) after aldehyde fixation. (F) A magnified image of the area framed in (E). Scale bar: 200 nm in (E) and 50 nm in (F). (G) Bright-field TEM image of E-face replica labeled with anti-FLAG antibody combined with 5-nm gold-particle-conjugated secondary antibody. Black arrowheads indicate B2R clusters labeled with 5-nm gold particles. Scale bar, 200 nm. (H) Comparison of the specific and background labeling density with 13-4Zn(II) and anti-FLAG antibody. “13-4Zn(II)” and “13-4Zn(II), fixed cells” indicate results from HEK cells labeled before and after aldehyde fixation, respectively (13-4Zn(II), ****p < 0.001, Mann-Whitney U test; specific labeling, n = 9 cells; background labeling, n = 13 cells; mean ± SE, anti-FLAG, ***p < 0.005, Mann-Whitney U test; specific labeling, n = 7 cells; background labeling, n = 7 cells; mean ± SE, 13-4Zn(II), fixed cells, ****p < 0.001, Mann-Whitney U test; specific labeling, n = 11 cells; background labeling, n = 15 cells; mean ± SE). (I) Distribution histograms of nearest neighbor distance (NND) of gold particles observed on replicas labeled with 13-Zn(II) (upper, n = 1748 particles) and anti-FLAG antibody (bottom, n = 998 particles). Fitted lines show Gaussian distribution fitting for the peaks. (J) Comparison of the number of particles per B2R cluster labeled with 13-4Zn(II) and anti-FLAG antibody. “Max 12 nm” and “Max 42 nm” mean the maximum NND of gold particles used for the definition of clusters labeled with 13-4Zn(II) and anti-FLAG antibody (13-4Zn(II) Max 12 nm; n = 10 cells, 13-4Zn(II) Max 42 nm, n = 10 cells; anti-FLAG Max 42 nm, n = 7 cells, mean ± SE, **p < 0.01, Mann-Whitney U test). (K) Comparison of the B2R cluster area evaluated by chemical labeling with 13-4Zn(II) and immunolabeling with anti-FLAG antibody (13-4Zn(II) Max 12 nm; n = 10 cells; 13-4Zn(II) Max 42 nm, n = 10 cells; anti-FLAG 42 nm, n = 7 cells; mean ± SE, n.s. p > 0.05, Mann-Whitney U test).
Figure 6
Figure 6
Comparison of Resolution between Chemical Labeling and Immunolabeling Methods (A and C) Schematic illustration of labeling states of the tagged-B2R on HEK cells labeled with 13-4Zn(II) (A) and anti-FLAG antibody combined with 1.4-nm gold-particle-conjugated secondary antibody (C). (B and D) EM images of ultrathin sections prepared from HEK cells labeled with 13-4Zn(II) (B) and anti-FLAG antibody (D), respectively. Gold particles were intensified by silver enhancement for 3–6 min. Distances between the particle center and the middle of the cell membrane were measured in sections tilted to obtain perpendicular views to the plasma membrane. Scale bars, 20 nm (left) and 10 nm (right). (E) Correlation of the intensified particle size and distance between particle centers and the middle of the cell membrane (orange dots, 13-4Zn(II); blue dots, anti-FLAG antibody). Significant positive correlation was detected for both (correlation coefficient = 0.24, p < 0.01, n = 147 for 13-4Zn(II); correlation coefficient = 0.26, p < 0.01, n = 228 for anti-FLAG antibody, Pearson correlation analysis). The distances for non-intensified 1.4-nm particles were deduced from linear fits extrapolated (broken orange and blue lines) to 1.4 nm (red dotted line). (F) Schematic illustration of positions of non-intensified 1.4-nm particles estimated by the two labeling methods in (E). The gradient circle indicates the deviation of the position.

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References

    1. Bulaj G., Kirtemme T., Goldenberg D.P. Ionization-reactivity relationship for cysteine thiol in polypeptides. Biochemistry. 1998;37:8965–8972. - PubMed
    1. Dean K.M., Palmer A.E. Advances in fluorescence labeling strategies for dynamic cellular imaging. Nat. Chem. Biol. 2014;10:512–523. - PMC - PubMed
    1. Faulk P.W., Taylor M.G. An immunocolloid method for the electron microscope. Immunochem. 1971;8:1081–1083. - PubMed
    1. Fijita A., Cheng J., Hirakawa M., Furukawa K., Kusunoki S., Fujimoto T. Ganglioside GM1 and GM3 in the living cell membrane from clusters susceptible to cholesterol depletion and chilling. Mol. Biol. Cell. 2007;18:2112–2122. - PMC - PubMed
    1. Flanagan M.E., Abramite J.A., Anderson D.P., Aulabaugh A., Dahal U.P., Gilbert A.M., Li C., Montgomery J., Oppenheimer S.R., Ryder T. Chemical and computational methods for the characterization of covalent reactive groups for the prospective design of irreversible inhibitors. J. Med. Chem. 2014;57:10072–10079. - PubMed

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