Active regulation of the epidermal growth factor receptor by the membrane bilayer
- Department of Chemistry, Massachusetts Institute of Technology, United States
Figures
Figure 1 with 7 supplements
EGFR in different membrane environments.
(a) Full-length EGFR (gray) embedded in a nanodisc. The nanodisc is a lipid bilayer (beige) belted by an amphiphilic apolipoprotein (dark gray). EGFR consists of a 618-amino-acid extracellular region that binds EGF (orange), a 27-amino-acid transmembrane-spanning domain, and an intracellular region, which is a 37-amino-acid juxtamembrane domain, a 273-amino-acid kinase domain, and a 231-amino-acid disordered C-terminal tail. Green and maroon spheres indicate the donor and acceptor dyes, respectively (Figure 1—figure supplement 1). (b) Mean of zeta potential distributions for EGFR in nanodiscs containing increasing amounts of anionic lipids (0%, 15%, 30%, and 60% POPS). Error bars are from three technical replicates. (c) Ensemble fluorescence emission spectra ( nm) of EGFR-embedded Laurdan containing nanodiscs with increasing cholesterol.
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Figure 1—source data 1
Raw data underlying Figure 1b.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig1-data1-v1.csv
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Figure 1—source data 2
Raw data underlying Figure 1c.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig1-data2-v1.csv
Figure 1—figure supplement 1
Domains of EGFR.
EGFR consists of a 621-amino-acid extracellular region (ED), a 24-amino-acid transmembrane-spanning domain (TM), and an intracellular region, which is a 37-amino-acid juxtamembrane domain (JM), a 273-amino-acid kinase domain (KD), and a 231-amino-acid C-terminal tail (CTT). The JM is further divided into juxtamembrane-A (JM-A) and juxtamembrane-B (JM-B) domains. Residues 978–990 are defined as the N-terminal portion of the CTT (N-CTT), and residues 1070–1186 are defined as the C-terminal portion of the CTT (C-CTT). Residue numbering corresponds to EGFR excluding the signal sequence.
Figure 1—figure supplement 2
Production and characterization of full-length EGFR in nanodiscs.
(a) Cell-free reaction for the production of EGFR and nanodisc belt protein. Codon-optimized DNA of the receptor protein (EGFR-SNAP) and the belt protein (ApoA1Δ49) are incubated together with lipid vesicles with or without cholesterol and E. coli lysate at 25 °C. (b) Stain-free (left) and fluorescence (right) gel images of the His-tag purified sample show the presence of ApoA1Δ49 at 25 kDa and full-length EGFR at 160 kDa, which implies successful EGFR production and insertion into nanodiscs. The presence of the EGFR band alone in the fluorescence gel image indicates successful and specific labeling. (c) SDS-PAGE analysis of His-tag purified ApoA1Δ49 (1 µg) produced in cell-free reaction. The two lanes correspond to replicate samples under identical conditions. (d) Western blots were performed on labeled EGFR in nanodiscs. Anti-EGFR western blots (left) and anti-phosphotyrosine western blots (right) tested the presence of EGFR and its ability to undergo tyrosine phosphorylation, respectively, consistent with previous experiments on similar preparations (He et al., 2015; Quinn et al., 2019; Srinivasan et al., 2022). The two lanes in each blot correspond to replicate samples under identical conditions.
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Figure 1—figure supplement 2—source data 1
PDF file containing original SDS-PAGE gels for Figure 1—figure supplement 2b, indicating the relevant bands and experimental conditions.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig1-figsupp2-data1-v1.zip
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Figure 1—figure supplement 2—source data 2
Original files for SDS-PAGE gels for Figure 1—figure supplement 2b.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig1-figsupp2-data2-v1.zip
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Figure 1—figure supplement 2—source data 3
PDF file containing original SDS-PAGE gels for Figure 1—figure supplement 2c, indicating the relevant bands and experimental conditions.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig1-figsupp2-data3-v1.zip
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Figure 1—figure supplement 2—source data 4
Original files for SDS-PAGE gels for Figure 1—figure supplement 2c.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig1-figsupp2-data4-v1.zip
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Figure 1—figure supplement 2—source data 5
PDF file containing original SDS-PAGE gels for Figure 1—figure supplement 2d, indicating the relevant bands and experimental conditions.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig1-figsupp2-data5-v1.pdf
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Figure 1—figure supplement 2—source data 6
Original files for SDS-PAGE gels for Figure 1—figure supplement 2d.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig1-figsupp2-data6-v1.zip
Figure 1—figure supplement 3
Optimization of ApoA1 and EGFR co-expression in cell-free reactions.
Cell-free reactions were performed with different template ratios of ApoA1Δ49:EGFR (1:2 to 1:200) and sampled at various time points (2–19 hr). Labeled lysines were incorporated during synthesis, ensuring that only proteins expressed in the cell-free reaction were fluorescently labeled. EGFR (160 kDa) and ApoA1Δ49 (25 kDa) expressions were monitored by SDS-PAGE.
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Figure 1—figure supplement 3—source data 1
PDF file containing original SDS-PAGE gels for Figure 1—figure supplement 3, indicating the relevant bands and experimental conditions.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig1-figsupp3-data1-v1.zip
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Figure 1—figure supplement 3—source data 2
Original files for SDS-PAGE gels for Figure 1—figure supplement 3.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig1-figsupp3-data2-v1.zip
Figure 1—figure supplement 4
Characterization of EGFR-containing nanodiscs in different anionic membrane environments.
(a) Dynamic light scattering (DLS) of EGFR in 100% POPC nanodiscs in PBS buffer indicates ∼38 nm average size. (b) Jitter plot showing the size distribution of EGFR in 100% POPC nanodiscs from negative-stain transmission electron microscopy (TEM). The horizontal line represents the mean size (33.4±8.5 nm, N=134). A representative TEM image is shown below. (c) DLS of EGFR in 85% POPC, 15% POPS nanodiscs in PBS buffer indicates ∼40.7 nm average size. (d) Jitter plot showing the size distribution of EGFR in 85% POPC, 15% POPS nanodiscs from negative-stain TEM. The horizontal line represents the mean size (36.8±6.0 nm, N=116). A representative TEM image is shown below. (e) DLS of EGFR in 70% POPC, 30% POPS nanodiscs in PBS buffer indicates ∼54.8 nm average size. (f) Jitter plot showing the size distribution of EGFR in 70% POPC, 30% POPS nanodiscs from negative-stain TEM. The horizontal line represents the mean size (37.8±9.7 nm, N=223). A representative TEM image is shown below. (g) DLS of EGFR in 40% POPC, 60% POPS nanodiscs in PBS buffer indicates ∼46.2 nm average size. (h) Jitter plot showing the size distribution of EGFR in 40% POPC, 60% POPS nanodiscs from negative-stain TEM. The horizontal line represents the mean size (41.2±9.7 nm, N=133). A representative TEM image is shown below.
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Figure 1—figure supplement 4—source data 1
Raw data underlying Figure 1—figure supplement 4.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig1-figsupp4-data1-v1.csv
Figure 1—figure supplement 5
Characterization of EGFR-containing nanodiscs in membrane environments containing cholesterol.
(a) Dynamic light scattering (DLS) of EGFR in 92.5% POPC, 7.5% cholesterol containing nanodiscs in PBS buffer indicates 32.8 nm average size. (b) Jitter plot showing the size distribution of EGFR in 92.5% POPC, 7.5% cholesterol nanodiscs from negative-stain transmission electron microscopy (TEM). The horizontal line represents the mean size (33.6±7.7 nm, N=154). A representative TEM image is shown below. (c) DLS of EGFR in 80% POPC, 20% cholesterol-containing nanodiscs in PBS buffer indicates 39.6 nm average size. (d) Jitter plot showing the size distribution of EGFR in 80% POPC, 20% cholesterol nanodiscs from TEM. The horizontal line represents the mean size (39.8±5.9 nm, N=188). A representative TEM image is shown below. (e) DLS of EGFR in EGFR in 62.5% POPC, 30% POPS, 7.5% cholesterol nanodiscs in PBS buffer indicates 33 nm average size. (f) Jitter plot showing the size distribution of EGFR in 62.5% POPC, 30% POPS, 7.5% cholesterol nanodiscs from TEM. The horizontal line represents the mean size (36.5±7.8 nm, N=159). A representative TEM image is shown below. (g) DLS of EGFR in 50% POPC, 30% POPC, 20% cholesterol nanodiscs in PBS buffer indicates 39.2 nm average size. (h) Jitter plot showing the size distribution of EGFR in 50% POPC, 30% POPC, 20% cholesterol nanodiscs from TEM. The horizontal line represents the mean size (39.2±6.3 nm, N=281). A representative TEM image is shown below.
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Figure 1—figure supplement 5—source data 1
Raw data underlying Figure 1—figure supplement 5.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig1-figsupp5-data1-v1.csv
Figure 1—figure supplement 6
Characterization of anionic content in EGFR-embedded nanodiscs with zeta potential (Her et al., 2016).
Zeta potential distributions for EGFR in nanodiscs containing increasing amounts of anionic lipids (0%, 15%, 30%, and 60% POPS). Maximum values from the distributions are shown in Figure 1b in the main text. Error bars are from three technical replicates.
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Figure 1—figure supplement 6—source data 1
Raw data underlying Figure 1—figure supplement 6.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig1-figsupp6-data1-v1.csv
Figure 1—figure supplement 7
Characterization of cholesterol (Ch) content in EGFR-embedded nanodiscs with Laurdan.
Ensemble fluorescence excitation spectra ( nm) of EGFR-embedded Laurdan containing nanodiscs with 0% cholesterol, 7.5% cholesterol, and 20% cholesterol. In the Laurdan excitation spectra, an increase in the excitation band centered around 390 nm is observed with the addition of increasing amounts of cholesterol to the EGFR-embedded nanodisc (Parasassi et al., 1994).
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Figure 1—figure supplement 7—source data 1
Raw data underlying Figure 1—figure supplement 7.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig1-figsupp7-data1-v1.csv
Figure 2 with 4 supplements
Membrane composition influences EGFR function through ATP binding.
(a) Full-length EGFR in nanodiscs with atto647N γ ATP (red sphere) and snap surface 488 or snap surface 594 (green sphere). (b) Extent of ATP binding in different anionic environments quantified using the intensity of atto 647 N (top) band normalized by the amount of EGFR produced as extracted from the intensity of ss488 band (center) as a function of negatively charged POPS lipids (bottom) in the absence (purple) and presence (orange) of EGF. Error bars from three independent biological replicates. (c) EGFR intracellular domain indicating ATP binding site-C-terminus distance and ATP-lipid contacts measured from molecular dynamics simulations. (d) Accessibility of ATP binding site quantified through the contact number between the ATP binding site and lipids in the absence (purple) and presence (orange) of EGF. Error bars from three equal partitions of the simulations. Probability distributions of the distance between residue 721, the closest residue to the ATP binding site (Honegger et al., 1987), and EGFR C-terminus for (e) neutral (0% POPS) and (f) 30% anionic lipids (30% POPS) without EGF (top); with 1 μM EGF (bottom). Dotted lines indicate the medians on all histograms with corresponding distances on upper x-axis (Table 1). (g) Schematic of multiparametric single-molecule confocal microscope. (h) Fluorescence intensity for a representative image ( nm) where green spots are immobilized EGFR nanodiscs. (i) Representative fluorescence time trace from single-molecule FRET experiments showing number of detected photons for each 100 ms interval as intensity traces (green for donor; red for acceptor) with the average for each period of constant intensity (black solid line) and the corresponding donor lifetime (black dashed line). smFRET donor lifetime distributions with atto647N γATP as acceptor and snap surface 594 as donor in (j) neutral (0% POPS) and (k) 30% anionic lipids (30% POPS) without EGF (top); with 1 μM EGF (bottom).
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Figure 2—source data 1
PDF file containing original SDS-PAGE gels for Figure 2b, indicating the relevant bands and experimental conditions.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig2-data1-v1.zip
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Figure 2—source data 2
Original files for SDS-PAGE gels for Figure 2b.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig2-data2-v1.zip
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Figure 2—source data 3
Raw data underlying Figure 2b.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig2-data3-v1.csv
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Figure 2—source data 4
Raw data underlying smFRET distribution in Figure 2j.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig2-data4-v1.csv
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Figure 2—source data 5
Raw data underlying smFRET distribution in Figure 2k.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig2-data5-v1.csv
Figure 2—figure supplement 1
Characterization of ss594-labeled EGFR nanodiscs in different anionic lipid environments.
(a) Ensemble fluorescence excitation spectra ( nm) of ss594-labeled EGFR nanodiscs with 0% POPS, 15% POPS, 30% POPS, and 60% POPS lipids. (b) Ensemble fluorescence emission spectra ( nm) of ss594-labeled EGFR nanodiscs in 0% POPS, 15% POPS, 30% POPS, and 60% POPS lipids. (c) Ensemble time-correlated single photon counting measurements for ss594-labeled EGFR nanodiscs in 0% POPS, 15% POPS, 30% POPS, and 60% POPS lipids. The instrument response function (IRF) is shown in gray.
Figure 2—figure supplement 2
Characterization of cy5-labeled EGFR nanodiscs in different anionic lipid environments.
(a) Ensemble fluorescence excitation spectra ( nm) of cy5-labeled nanodiscs in 0% POPS, 15% POPS, 30% POPS, and 60% POPS lipids. (b) Ensemble fluorescence emission spectra ( nm) of cy5-labeled nanodiscs in 0% POPS, 15% POPS, 30% POPS, and 60% POPS lipids. (c) Ensemble time-correlated single photon counting measurements were performed for cy5-labeled nanodiscs in 0% POPS, 15% POPS, 30% POPS, and 60% POPS. The instrument response function (IRF) is shown in gray.
Figure 2—figure supplement 3
Single ss594-labeled EGFR-embedded nanodiscs.
(a) Confocal fluorescence image of immobilized constructs of EGFR in nanodiscs labeled with ss594 ( nm). (b) Representative intensity time trace from a single construct. The number of detected photons for each 100 ms interval was calculated and used to generate a fluorescence intensity trace (green) with the average intensity for the emissive period overlaid (black). (c) Histogram of the arrival times of detected photons generates the donor lifetime decay profile. Representative decay profiles of EGFR (green) with fit curve (black). The instrument response function (IRF) is shown in gray.
Figure 2—figure supplement 4
Jitter plots of the donor lifetime distributions from smFRET experiments to measure the distance between the ATP binding site and the C-terminus of EGFR.
Distributions of the donor lifetime from the histograms in Figure 2j and k of main text are represented as jitter plots along with the donor-only samples. The horizontal line indicates the median lifetime. One-way ANOVA was performed to obtain the p-values (Supplementary file 1A). The median value of the donor-only distribution in each lipid environment was used as the reference value for calculations of the donor-acceptor distances for all smFRET measurements in that environment.
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Figure 2—figure supplement 4—source data 1
Raw data underlying Figure 2—figure supplement 4.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig2-figsupp4-data1-v1.csv
Figure 3 with 6 supplements
EGFR intracellular conformations depend on membrane composition of the nanodisc.
(a) Full-length EGFR in nanodiscs with partially anionic lipids (red). The negatively charged residues on the C-terminal tail are indicated in red, and the positively charged residues on the kinase domain are indicated in blue. smFRET donor fluorescence lifetime distributions in (b) 100% POPC, 0% POPS; 85% POPC, 15% POPS; 70% POPC, 30% POPS; 40% POPC, 60% POPS without EGF (top); with 1 μM EGF (bottom). (c) Full-length EGFR in nanodiscs with cholesterol (teal). (d) smFRET donor fluorescence lifetime distributions in 92.5% POPC, 7.5% cholesterol; 80% POPC, 20% cholesterol without EGF (top); with 1 μM EGF (bottom). (e) Full-length EGFR in nanodiscs with cholesterol and anionic lipids. (f) smFRET donor fluorescence lifetime distributions in 62.5% POPC, 30% POPS, 7.5% cholesterol; 50% POPC, 30% POPS, 20% cholesterol without EGF (top); with 1 μM EGF (bottom). Dotted lines indicate the maxima from a global fit of all lifetime distributions to a double Gaussian distribution model using maximum likelihood estimation. The maxima correspond to a compact and an open conformation with a distance of 8 nm and 12 nm, respectively, between the EGFR C-terminal tail and the membrane bilayer. (g) The amplitude of the open conformation (in %) in all the eight different membrane compositions in the absence (purple) and presence of EGF (orange). (h) The change in amplitude induced by EGF (black). The amplitude change upon EGF addition is high (22–55%) in 0–30% POPS but reduces drastically (0–6%) upon introduction of cholesterol in the lipid bilayer. The error bars in (g) and (h) are from the global fit.
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Figure 3—source data 1
Raw data underlying smFRET distribution in Figure 3b.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig3-data1-v1.csv
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Figure 3—source data 2
Raw data underlying smFRET distribution in Figure 3d.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig3-data2-v1.csv
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Figure 3—source data 3
Raw data underlying smFRET distribution in Figure 3f.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig3-data3-v1.csv
Figure 3—figure supplement 1
Single Cy5-labeled EGFR-embedded nanodiscs.
(a) Confocal fluorescence image of immobilized constructs of EGFR in nanodiscs containing a labeled Cy5 lipid ( nm). (b) Representative intensity time trace from a single construct. The number of detected photons for each 100ms interval was calculated and used to generate a fluorescence intensity trace (red) with the average intensity for the emissive period overlaid (black).
Figure 3—figure supplement 2
Jitter plots of the donor lifetime distributions from smFRET experiments to measure the distance between the membrane and the C-terminus of EGFR.
Distributions of the donor lifetime from the histograms in Figure 3b of main text are represented as jitter plots along with the donor-only samples. The horizontal line indicates the median lifetime. One-way ANOVA was performed to obtain the p-values (Supplementary file 1C). NS, not significant. The median value of the donor-only distribution in each lipid environment was used as the reference value for calculations of the donor-acceptor distances for all smFRET measurements in that environment.
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Figure 3—figure supplement 2—source data 1
Raw data underlying Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig3-figsupp2-data1-v1.csv
Figure 3—figure supplement 3
EGFR intracellular domain conformation is correlated with ATP binding.
(a) Correlation analysis between distance of EGFR C-terminus from the bilayer and extent of ATP binding in the (top) absence of EGF (Pearson correlation R=0.75) and (bottom) presence of EGF (Pearson correlation R=0.88).
Figure 3—figure supplement 4
Jitter plots of the donor lifetime distributions from smFRET experiments to measure the distance between the membrane and the C-terminus of EGFR.
Distributions of the donor lifetime from the histograms in Figure 3d of main text are represented as jitter plots along with the donor-only samples. The horizontal line indicates the median lifetime. One-way ANOVA was performed to obtain the p-values (Supplementary file 1G). NS, not significant. The median value of the donor-only distribution in each lipid environment was used as the reference value for calculations of the donor-acceptor distances for all smFRET measurements in that environment.
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Figure 3—figure supplement 4—source data 1
Raw data underlying Figure 3—figure supplement 4.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig3-figsupp4-data1-v1.csv
Figure 3—figure supplement 5
Jitter plots of the donor lifetime distributions from smFRET experiments to measure the distance between the membrane and the C-terminus of EGFR.
Distributions of the donor lifetime from the histograms in Figure 3f of main text are represented as jitter plots along with the donor-only samples. The horizontal line indicates the median lifetime. One-way ANOVA was performed to obtain the p-values (Supplementary file 1J). NS, not significant. The median value of the donor-only distribution in each lipid environment was used as the reference value for calculations of the donor-acceptor distances for all smFRET measurements in that environment.
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Figure 3—figure supplement 5—source data 1
Raw data underlying Figure 3—figure supplement 5.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig3-figsupp5-data1-v1.csv
Figure 3—figure supplement 6
smFRET donor lifetime distributions in DMPC and POPC nanodiscs.
smFRET donor fluorescence lifetime distributions in (a) 100% DMPC and (b) 100% POPC without EGF (top); with 1 μM EGF (bottom). Distributions of the donor lifetime from the histograms on the left in 100% DMPC (c) and 100% POPC (d) are represented as jitter plots along with the donor-only samples. The horizontal line indicates the median lifetime. One-way ANOVA was performed to obtain the p-values (Supplementary file 1C). The median value of the donor-only distribution in each lipid environment was used as the reference value for calculations of the donor-acceptor distances for all smFRET measurements in that environment.
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Figure 3—figure supplement 6—source data 1
Raw data underlying Figure 3—figure supplement 6.
- https://cdn.elifesciences.org/articles/108789/elife-108789-fig3-figsupp6-data1-v1.csv
Figure 4
Membrane composition influences EGFR conformation and function.
(Top) In healthy cells, EGF binding promotes transmembrane conformational coupling between the extracellular and intracellular domains of EGFR, enabling kinase activation through established signaling mechanisms. (Middle) Our results demonstrate that in membranes with high anionic lipid content, electrostatic repulsion between the negatively charged lipids and the kinase domain overrides EGF-induced transmembrane conformational coupling. (Bottom) Our results demonstrate that in cholesterol-rich membranes, increased membrane rigidity overrides EGF-induced transmembrane conformational coupling. These findings establish membrane composition as a dominant regulator of EGFR signaling, independent of EGF binding.
Tables
Table 1
Median distances between the ATP binding site (residue 721) and the C-terminal end of EGFR from smFRET experiments and simulations.
The distance values were extracted as the medians of the distributions in Figure 2e, f, j and k in the main text. The numbers in parentheses indicate the 95% confidence interval for experiments and the minimal and maximal median value from three equal partitions of data for simulations. Ro = 7.5 nm for snap surface 594 and atto 647 N.
| Sample conditions | Experiment distance (nm) | Simulation distance (nm) |
|---|---|---|
| 0% anionic lipids | ||
| EGFR, -EGF | 8.1 [8.0, 8.2] | 7.0 [6.0, 8.0] |
| EGFR,+EGF | 8.6 [8.5, 8.7] | 5.8 [5.2, 7.2] |
| 30% anionic lipids | ||
| EGFR, -EGF | 9.1 [8.9, 9.2] | 5.7 [5.7, 5.7] |
| EGFR,+EGF | 11.6 [11.1, 12.6] | 7.5 [6.2, 9.2] |
Table 2
Median distances between the membrane and C-terminal end of the protein from smFRET experiments.
The distance values were extracted from the distributions shown in Figure 3b in the main text. The numbers in parentheses indicate the 95% confidence interval for experiments. Asterisk (*) indicates distance was beyond the FRET range for the snap surface 594 and Cy5 dye pair (Ro = 8.4 nm; Sisamakis et al., 2010).
| Sample conditions | Distance (nm) |
|---|---|
| 0% POPS, 100% POPC; -EGF | 12.1 [11.6, 12.7] |
| 0% POPS, 100% POPC; +EGF | 8.8 [8.2, 9.6] |
| 15% POPS, 85% POPC; -EGF | 9.2 [8.8, 9.6] |
| 15% POPS, 85% POPC; +EGF | 11.9 [11.4, 12.4] |
| 30% POPS, 70% POPC; -EGF | 9.2 [9.1, 9.3] |
| 30% POPS, 70% POPC; +EGF | *13.9 [12.5, 14.4] |
| 60% POPS, 40% POPC; -EGF | 11.4 [11.1, 11.7] |
| 60% POPS, 40% POPC; +EGF | 11.2 [10.9, 11.6] |
Table 3
Median distances between the membrane and C-terminal end of the protein from smFRET experiments.
The distance values were extracted from the distributions shown in Figure 3d and f in the main text. The numbers in parentheses indicate the 95% confidence interval for experiments. Asterisk (*) indicates distance was beyond the FRET range for the snap surface 594 and Cy5 dye pair (Ro = 8.4 nm; Sisamakis et al., 2010).
| Sample conditions | Experiment distance (nm) |
|---|---|
| 92.5% POPC, 7.5% Cholesterol; -EGF | 12.1 [11.6, 12.6] |
| 92.5% POPC, 7.5% Cholesterol; +EGF | 12.2 [11.5, 13.2] |
| 80% POPC, 20% Cholesterol; -EGF | 11.1 [10.7, 11.5] |
| 80% POPC, 20% Cholesterol; +EGF | 10.9 [10.6, 11.2] |
| 62.5% POPC, 30% POPS, 7.5% Cholesterol, -EGF | 10.4 [10.2, 10.5] |
| 62.5% POPC, 30% POPS, 7.5% Cholesterol, +EGF | 10.3 [10.2, 10.4] |
| 50% POPC, 30% POPS, 20% Cholesterol, -EGF | *12.9 [11.5, 12.3] |
| 50% POPC, 30% POPS, 20% Cholesterol, +EGF | *13.0 [11.5, 12.1] |
| 100% DMPC; -EGF | 11.4 [11.1, 11.6] |
| 100% DMPC; +EGF | 8.1 [8.1, 8.2] |
Author response table 1
| Two-Gaussian | Three-Gaussian | ||
|---|---|---|---|
| µ1 | 1.33 ns | µ1 | 1.38 ns |
| σ1 | 0.34 ns | σ1 | 0.36 ns |
| µ2 | 2.71 ns | µ2 | 2.64 ns |
| σ2 | 0.67 ns | σ2 | 0.45 ns |
| µ3 | 3.43 ns | ||
| σ3 | 0.64 ns |
Author response table 2
| Sample | FWHM of DLS distribution (nm) |
|---|---|
| 100 % POPC | 10 |
| 85 %POPC, 15 % POPS | 19.1 |
| 70 %POPC, 30 % POPS | 8.2 |
| 40 %POPC, 60 % POPS | 18.6 |
| 92.5 %POPC, 7.5 % Cholesterol | 8.7 |
| 80 %POPC, 20 % Cholesterol | 21.6 |
| 62.5 %POPC, 30% POPS, 7.5 % Cholesterol | 23.3 |
| 50 %POPC, 30% POPS, 20 % Cholesterol | 12.5 |
Additional files
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Supplementary file 1
Statistical analyses, sample sizes, model selection, and supporting data for single-molecule FRET measurements across membrane compositions and experimental conditions.
- https://cdn.elifesciences.org/articles/108789/elife-108789-supp1-v1.pdf
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MDAR checklist
- https://cdn.elifesciences.org/articles/108789/elife-108789-mdarchecklist1-v1.pdf
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Active regulation of the epidermal growth factor receptor by the membrane bilayer
eLife 14:RP108789.
https://doi.org/10.7554/eLife.108789.3
