Journal: Molecular cell
Article Title: Sequence grammar underlying the unfolding and phase separation of globular proteins.
doi: 10.1016/j.molcel.2022.06.024
Figure Lengend Snippet: Figure 5. Molecular chaperones suppress phase separation (A) In the absence of chaperones, the dilute phase consists of folded and unfolded barnase. There exists a phase equilibrium when the total concentration of barnase, ctot, is greater than csat. In the presence of chaperones, barnase in the dilute phase consists of three dominant states: folded, unfolded free, and unfolded bound to chaperones. At the same total concentration of barnase as in the absence of chaperones, barnase cannot phase separate because ctot is less than the saturation concentration needed in the presence of chaperones, cchaperone sat , given that chaperone binding reduces the concentration of free unfolded barnase. (B) Basic model for chaperone function. Hsp40 binds the unfolded molecule and forms a ternary complex with Hsp70 in the ATP-bound state. ATP hydrolysis leads to the release of Hsp40 and the formation of a high-affinity complex between Hsp70 and the unfolded substrates. (C) csat for the L14A barnase-optoDroplet variant construct transiently transfected in Neuro2a cells in the absence or presence of different dosages of the Hsp70 inhibitor VER-155008. Dashed line corresponds to the csat of L14A in the absence of the inhibitor. Error bars denote the standard deviation from 50 bootstrapped trials. (D) csat for the L14A barnase-optoDroplet variant construct in the absence or presence of overexpressed chaperones. Bars with arrows indicate that a csat value could not be extracted for these systems and must be at least above the value of the bar. Error bars denote the standard deviation from 50 bootstrapped trials. (E) Confocal images of Neuro2a cells transfected with V5-tagged DNAJB1 (Hsp40) or HSPA1A (Hsp70). Cells were stained by immunofluorescence for the V5-tag (Cy5), and the nucleus was stained with Hoechst 33342. Graphs show the quantitation of immunofluorescence. Data shown as paired samples from individual cells. Paired t test results shown; * p < 0.05. See also Figure S4 and Table S4.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-V5 antibody Abcam Cat#Ab27671; RRID:AB_471093 Anti-HSPA1A antibody Abcam Cat#Ab5439; RRID:AB_304888 Goat anti-mouse Cyanine5 Life technologies Cat#A10524; RRID:AB_2534033 Chemicals, peptides, and recombinant proteins VER-155008 Sigma-Aldrich Cat#SML0271 Opti-MEM Thermo Fisher Scientific Cat#31985062 Dulbecco’s Modified Eagle Medium Thermo Fisher Scientific Cat#21013024 Fetal bovine serum Thermo Fisher Scientific Cat#10099141 L-glutamine Thermo Fisher Scientific Cat#25030081 Lipofectamine 3000 Thermo Fisher Scientific Cat#L3000015 Hoechst 33342 Thermo Fisher Scientific Cat#H3570 Hank’s Balanced Salt Solution Thermo Fisher Scientific Cat#14025092 Digitonin Sigma-Aldrich Cat#D141 Complete EDTA-free protease inhibitor cocktail Sigma-Aldrich Cat#11836170001 Pierce Trypsin protease, MS grade Thermo Fisher Scientific Cat#90057 TMT10plex Isobaric label reagent set plus TMT11-131C label reagent Thermo Fisher Scientific Cat#A34808 Critical commercial assays BCA protein assay Thermo Fisher Scientific Cat#23227 Deposited data Raw proteomics data ProteomeXchange Consortium PRIDE: PXD033716 Raw and analyzed data This paper; Zenodo Zenodo data: https://doi.org/ 10.5281/zenodo.6603909 Raw image data used to assemble figures This paper; Mendeley Mendeley data: https://doi.org/ 10.17632/3rrp4zy7p3.1 Experimental models: Cell lines Mouse: Neuro2a cells American Type Culture Collection Cat#CCL-131 Human: HEK293T cells Laboratory of Greg Mosely, Monash University, Australia N/A Recombinant DNA DDX4 IDR–mCherry–Cry2 Shin et al., 2017 N/A Barnase WT– mCherry–Cry2 This paper N/A Barnase I55G– mCherry–Cry2 This paper N/A Barnase V45T– mCherry–Cry2 This paper N/A Barnase I25A– mCherry–Cry2 This paper N/A Barnase I88A– mCherry–Cry2 This paper N/A Barnase L14A– mCherry–Cry2 This paper N/A Barnase I51A– mCherry–Cry2 This paper N/A Barnase L89G– mCherry–Cry2 This paper N/A Barnase I96G– mCherry–Cry2 This paper N/A Barnase I55G, L89G– mCherry–Cry2 This paper N/A Barnase I88G– mCherry–Cry2 This paper N/A (Continued on next page) Molecular Cell 82, 3193–3208.e1–e8, September 1, 2022 e1
Techniques: Concentration Assay, Binding Assay, Variant Assay, Construct, Transfection, Standard Deviation, Staining, Quantitation Assay
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