Dissecting the phase separation and oligomerization activities of the carboxysome positioning protein McdB
- Department of Molecular, Cellular, and Developmental Biology, University of Michigan-Ann Arbor, United States
- Department of Biological Chemistry, University of Michigan-Ann Arbor, United States
Figures
Figure 1 with 2 supplements
Defining a domain architecture of Se7942 McdB.
(A) Circular dichroism (CD) spectra of McdB at 20°C (black), 80°C (blue), and then returned to 20°C (magenta). Spectra show α-helical structure resilient to heat denaturation. (B) Sodium dodecyl …
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Figure 1—source data 1
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel corresponding to the trypsin digest gel in Figure 1B.
Bands A, B, and C as well as the full-length McdB are labeled.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig1-data1-v2.zip
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Figure 1—source data 2
Spreadsheet containing the raw data for circular dichroism (CD) curves shown in Figure 1A.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
Phase separation of Se7942 McdB across a range of buffer conditions during crystal screens.
Images taken during buffer screens for crystallography. McdB at 10 mg/ml in (50 mM KCl; 10 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), pH 10.2) was diluted into the buffers indicated below …
Figure 1—figure supplement 2
I-TASSER predictions for Se7942 McdB.
(A) McdB amino acid sequence and associated secondary structure predictions by I-TASSER. Each residue has a confidence score that ranges from 0 (least confident) to 9 (most confident). (B) Top 3 …
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Figure 1—figure supplement 2—source data 1
Spreadsheet with an editable version of the table in Figure 1—figure supplement 2C.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig1-figsupp2-data1-v2.zip
Figure 2 with 1 supplement
The α-helical domains of McdB form a trimer-of-dimers hexamer.
(A) Illustration of McdB truncations generated based on the predicted domain structure. (B) Circular dichroism (CD) spectra normalized by MW for the indicated McdB truncations. Spectra show …
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Figure 2—source data 1
Spreadsheet containing the raw data for circular dichroism (CD) curves shown in Figure 2B.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig2-data1-v2.xlsx
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Figure 2—source data 2
Spreadsheet containing the raw data for size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) curves shown in Figure 2C and an editable version of the table in Figure 2D.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
McdB truncations have unique secondary structures and display different oligomeric states.
(A) Circular dichroism (CD) spectra of full-length McdB and truncations. Curves from Figure 2B are overlayed onto a single graph. (B) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis …
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Figure 2—figure supplement 1—source data 1
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel corresponding to the McdB truncation mutants versus the His-SUMO tag as described in Figure 2—figure supplement 1B.
Full-length McdB, each truncation, and the His-SUMO tag are labeled.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig2-figsupp1-data1-v2.zip
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Figure 2—figure supplement 1—source data 2
Spreadsheet containing the raw data for circular dichroism (CD) curves shown in Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig2-figsupp1-data2-v2.xlsx
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Figure 2—figure supplement 1—source data 3
Spreadsheet containing the raw data for SEC curves shown in Figure 2—figure supplement 1C.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig2-figsupp1-data3-v2.xlsx
Figure 3
McdB from Se7942 forms liquid-like condensates via pH-dependent phase separation coupled to percolation (PSCP).
(A) Fluorescence recovery after photobleaching (FRAP) of McdB condensates at the indicated time points. Means and standard deviation (SD) from n = 8 condensates are shown. Representative …
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Figure 3—source data 1
Spreadsheet containing the raw data for fluorescence recovery after photobleaching (FRAP) curves shown in Figure 3A.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig3-data1-v2.xlsx
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Figure 3—source data 2
Spreadsheet containing the raw data for dynamic light scattering (DLS) curves shown in Figure 3D.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig3-data2-v2.xlsx
Figure 4 with 1 supplement
Truncations provide insight into the mechanisms of McdB condensate formation and stabilization.
(A) Representative DIC microscopy images of full-length and truncation mutants of McdB at 100 µM in 150 mM KCl and 20 mM HEPES, pH 7.2. (B) As in (A), but with increasing protein concentration as …
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Figure 4—source data 1
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel corresponding to the pelleting assay described in Figure 4C.
Full-length McdB and each truncation are labeled. Bands for the pellet and supernatant fractions are labeled.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig4-data1-v2.zip
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Figure 4—source data 2
Spreadsheet containing the raw data for gel quantification graphs shown in Figure 4C.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig4-data2-v2.xlsx
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Figure 4—source data 3
Spreadsheet containing the raw data for fluorescence recovery after photobleaching (FRAP) curves shown in Figure 4D.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig4-data3-v2.xlsx
Figure 4—figure supplement 1
McdB forms liquid-like condensates in both Ficoll and polyethylene glycol.
(A) Representative DIC microscopy images for WT McdB at 100 µM in 100 mM KCl, 20 mM HEPES, pH 7.2, and the addition of the indicated crowding agent. (B) Time course of images from (A) shows that …
Figure 5 with 3 supplements
McdB condensates can be solubilized by mutating basic residues in the N-terminal intrinsically disordered region (IDR) without affecting McdB structure.
(A) Representative DIC microscopy images of 50 µM McdB in 20 mM HEPES, pH 7.2 and increasing KCl concentration (top). Scale bar applies to all images. McdB condensates were pelleted (P) and run on a …
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Figure 5—source data 1
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel corresponding to the pelleting assay described in Figure 5A.
Full-length McdB is labeled. Bands for the pellet and supernatant fractions are labeled. The corresponding KCl concentration for each condition is labeled.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig5-data1-v2.zip
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Figure 5—source data 2
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel corresponding to the pelleting assay described in Figure 5B.
Full-length McdB is labeled. Bands for the pellet and supernatant fractions are labeled. The corresponding pH for each condition is labeled.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig5-data2-v2.zip
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Figure 5—source data 3
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel corresponding to the pelleting assay described in Figure 5E.
Full-length McdB and each glutamine-substitution mutant are labeled. Bands for the pellet and supernatant fractions are labeled.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig5-data3-v2.zip
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Figure 5—source data 4
Spreadsheet containing the raw data for gel quantification graphs shown in Figure 5A, B.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig5-data4-v2.xlsx
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Figure 5—source data 5
Spreadsheet containing the raw data for size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) curves shown in Figure 5D and an editable version of the associated table.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig5-data5-v2.xlsx
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Figure 5—source data 6
Spreadsheet containing the raw data for gel quantification graphs shown in Figure 5E.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig5-data6-v2.xlsx
Figure 5—figure supplement 1
Multidimensional phase diagrams for Se7942 McdB.
Turbidity-based phase diagrams for McdB across varying protein concentration, KCl concentration, and pH. Data points represent the mean and error bars represent standard deviation (SD) from at least …
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Figure 5—figure supplement 1—source data 1
Spreadsheet containing the raw data for turbidity assay graphs shown in Figure 5—figure supplement 1S.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig5-figsupp1-data1-v2.zip
Figure 5—figure supplement 2
Alanine scanning of basic residues in the N- and C-termini of McdB.
(A) Table showing the sequence of WT McdB compared to the terminal A-substitution mutants. Acidic and basic residues are colored red and blue, respectively. A-substitutions are bolded. (B) …
Figure 5—figure supplement 3
Mutations to the C-terminal domain (CTD) destabilize the trimer-of-dimers hexamer.
(A) Table showing the net charge and amino acid sequence of wild-type McdB compared to the Q-substitution mutants in the CTD. Acidic and basic residues are colored red and blue, respectively. …
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Figure 5—figure supplement 3—source data 1
Spreadsheet containing the raw data for size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) curves shown in Figure 5—figure supplement 3S and an editable version of the associated table.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig5-figsupp3-data1-v2.zip
Figure 6 with 1 supplement
Net charge of the intrinsically disordered region (IDR) can be used to tune the solubility of McdB condensates.
(A) Table showing the net charge and N-terminal IDR sequence of wild-type McdB compared to the glutamine (Q)-substitution mutants. Acidic and basic residues in the IDR are colored red and blue, …
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Figure 6—source data 1
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel corresponding to the pelleting assay described in Figure 6C.
Full-length McdB and each glutamine-substitution mutant are labeled. Bands for the pellet and supernatant fractions are labeled.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig6-data1-v2.zip
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Figure 6—source data 2
Spreadsheet containing the raw data for gel quantification graphs shown in Figure 6C.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig6-data2-v2.xlsx
Figure 6—figure supplement 1
Circular dichroism (CD) spectra of wild-type McdB and N-terminal glutamine-substitution mutants.
(A) Table showing the net charge and N-terminal intrinsically disordered region (IDR) sequence of wild-type McdB compared to the glutamine-substitution mutants. Acidic and basic residues in the IDR …
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Figure 6—figure supplement 1—source data 1
Spreadsheet containing the raw data for circular dichroism (CD) curves shown in Figure 6—figure supplement 1S.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig6-figsupp1-data1-v2.zip
Figure 7
Net charge of the intrinsically disordered region (IDR) affects McdB solubility in E. coli.
(A) Representative fluorescence microscopy images monitoring the expression of the indicated constructs over time. Scale bar applies to all images. (B) Quantification of the proportion of cells …
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Figure 7—source data 1
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel corresponding to the protein expression levels described in Figure 7C.
Bands representing mCherry alone, mCh-McdB[wt], and mCh-McdB[−3] are boxed and labeled. The time after induction is indicated.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig7-data1-v2.zip
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Figure 7—source data 2
Spreadsheet containing the raw data for foci count quantification graphs shown in Figure 7B.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig7-data2-v2.xlsx
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Figure 7—source data 3
Spreadsheet containing the raw data for gel quantification graphs shown in Figure 7C.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig7-data3-v2.xlsx
Figure 8 with 1 supplement
McdB[−3], which results in a high degree of condensate solubilization in vitro and in E. coli, alters the soluble fraction of McdB and carboxysome Rubisco levels in vivo.
(A) Representative fluorescence microscopy images of the indicated strains. Scale bar = 5 µm and applies to all images. Pearson’s correlation coefficients (PCCs) are shown for mNG-McdB and RbcS-mTQ …
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Figure 8—source data 1
Spreadsheet containing the raw data for graphs shown in Figure 8B, C.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig8-data1-v2.zip
Figure 8—figure supplement 1
Deletion of McdA causes no additional loss of carboxysome positioning in McdB[−3] strain.
(A) Quantification of RbcS-mTQ foci per micron from n > 500 cells. Medians and interquartile ranges are displayed. ****p < 0.001 based on Kruskal–Wallis analysis of variance (ANOVA). (B) …
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Figure 8—figure supplement 1—source data 1
Spreadsheet containing the raw data for graphs shown in Figure 8—figure supplement 1A, B.
- https://cdn.elifesciences.org/articles/81362/elife-81362-fig8-figsupp1-data1-v2.zip
Figure 9
Proposed model of Se7942 McdB domain structure and self-association.
The central coiled-coil (CC) domain is necessary and sufficient for dimerization and driving condensate formation. The α-helical C-terminal domain (CTD) trimerize McdB dimers and increases the …
