Materials and reagents

Biological materials

1. Proteins:

a. Recombinant full-length murine prion protein (rPrP, residues 23–231) without any tag. PrP has an intrinsic ability to coordinate divalent transition metals in the octapeptide region (also termed octarepeats, OR) that we take advantage of to purify using Ni²⁺-affinity chromatography (explained in Section C) [11]. Mouse PrP has five ORs that can bind up to 4 copper ions, and two additional His in the N-terminal domain can also bind Cu²⁺, making it six sites for Cu²⁺

Note: Recombinantly expressed PrP (rPrP) at 70–100 μM is stored in low protein binding microcentrifuge tubes in ultrapure H₂O at -20 °C for up to 6 months. We found that rPrP protein stability and solubility as well as secondary and tertiary structures are maintained using these conditions. In addition, rPrP does not phase-separate in H₂O; checking this is essential, as LLPS-forming proteins cannot be stored in a condition where phase separation is favored.

b. rPrP labeled with Alexa Fluor 647 (Alexa Fluor^® 647 carboxylic acid, succinimidyl ester) by formation of a stable dye-protein conjugate, as the reactive dye contains a succinimidyl ester moiety that reacts with primary amines of proteins such as the N-terminus and the side-chain of lysine residues (Thermo Fisher, catalog number: A20173) (see [12] for the labeling procedure and consult the manufacturer’s manual)

2. Plasmids:

a. pET-41, in which mouse PrP cDNA (corresponding to amino acid residues 23–230, mature protein) was cloned into NdeI and HindIII sites (original HisTag in the plasmid was removed) for expression in Escherichia coli (refer to Section A)

b. pEYFP-N1 for mammalian expression of PrP^C-EYFP-GPI (kindly gifted by Marilene H. Lopes, USP, Brazil) (refer to Section J)

c. pCR3-EYFP-GPI (DAF) for mammalian expression of YFP-GPI (kindly gifted by Daniel Legler, Universität Konstanz, Germany) (refer to Section J)

3. Human embryonic kidney cell line (HEK293 cells) (ATCC, CRL-1573) (refer to Sections J, K, L, and N)

4. Escherichia coli BL21 (DE3) (Thermo Scientific, catalog number: EC0114) (refer to Section A)

Reagents

1. Ultrapure water, treated by reverse osmosis, filtered, and deionized (resistivity of 18.2 MΩ·cm and pH 6.9 at 25 °C)

2. Copper (II) chloride dihydrate (CuCl₂·2H₂O) (Sigma-Aldrich, catalog number: 307483)

3. 30% (w/v) hydrogen peroxide (H₂O₂) (Millipore, catalog number: 88597)

4. 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) (C₈H₁₈N₂O₄S) (Sigma-Aldrich, catalog number: V900477)

5. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P3911)

6. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 484016)

7. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S1679)

8. Calcium chloride dihydrate (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: V900269)

9. Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Sigma-Aldrich, catalog number: M2670)

10. Polyethylene glycol (PEG) 4000 (Sigma-Aldrich, catalog number: 81240)

11. Alexa Fluor^® 647 Protein Labeling kit (Thermo Fisher, catalog number: A20173)

12. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 30721)

13. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881)

14. Phosphate-buffered saline (PBS) (Thermo Fisher, catalog number: 10010001)

15. Recombinant PrP expression and purification (Sections B and C)

a. 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) (C₄H₁₁NO₃) (Sigma-Aldrich, catalog number: 252859)

b. Ethylenediaminetetraacetic acid (EDTA) (C₁₀H₁₄N₂Na₂O₈·2H₂O) (Sigma-Aldrich, catalog number: E4884)

c. Disodium hydrogen phosphate (Na₂HPO₄) (Sigma-Aldrich, catalog number: S9763)

d. α-D-Glucopyranosyl β-D-fructofuranoside (sucrose) (C₁₂H₂₂O₁₁) (Sigma-Aldrich, catalog number: S8501)

e. Lysozyme from chicken egg white (Sigma-Aldrich, catalog number: L6876)

f. CelLytic B cell lysis reagent (Sigma-Aldrich, catalog number: B7435)

g. Sodium dihydrogen phosphate (NaH₂PO₄) (Sigma-Aldrich, catalog number: S0751)

h. UltraPure guanidine hydrochloride (CH₆ClN₃) (Invitrogen, catalog number: 15502016)

i. 1,3-Diaza-2,4-cyclopentadiene (imidazole) (C₃H₄N₂) (Sigma-Aldrich, catalog number: I202)

j. Agar (Becton Dickinson Bioscience, catalog number: 214010)

k. Luria-Bertani (LB) broth (Becton Dickinson Bioscience, catalog number: 244620)

l. Super optimal broth (SOC) (Sigma-Aldrich, catalog number: S1797)

m. Phenylmethanesulfonyl fluoride (PMSF) (C₇H₇FO₂S) (Sigma-Aldrich, catalog number: P7626)

n. HisTrap HP, 5 mL (Cytiva, catalog number: 17524802)

o. SnakeSkin dialysis tubing 10 K MWCO (Thermo Scientific, catalog number: 68100)

p. Triton X-100 (Sigma-Aldrich, catalog number: X100)

q. Colloidal Coomassie G-250 stain for protein polyacrylamide gels (Bio-Rad, catalog number: 1610803)

16. Media for cell culture (Sections J, L, K, and N)

a. Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, catalog number: D5546)

b. Fetal bovine serum (FBS) (Gibco, catalog number: A5670801)

c. Penicillin-Streptomycin 100× (Sigma-Aldrich, catalog number: P4333)

17. Reagents for transfection for in cellulo FRAP experiments (Sections J–L)

a. Opti-MEM I (Gibco, catalog number: 31985062)

b. Lipofectamine 2000 (Invitrogen, catalog number: 11668027)

18. Extrinsic dyes for fluorescence microscopy (Sections H and N)

a. SYPRO orange (5,000× stock in DMSO) (Sigma-Aldrich, catalog number: S5692)

b. AmyTracker 680 (Ebba Biotech AB)

19. Standard for calibration of q-angle for XPCS experiments (Sections E and F)

a. Silver(I) behenate (Thermo Scientific Chemicals, catalog number: 15452307)

Solutions

1. STE buffer (see Recipes)

2. Buffered sucrose (see Recipes)

3. Buffer A (denaturing buffer pH 8.0) (see Recipes)

4. Buffer B (refolding buffer pH 8.0) (see Recipes)

5. Elution buffer pH 5.0 (see Recipes)

6. 2× Physiological-like buffer (PhysB) pH 7.2 (see Recipes)

7. CuCl₂, 0.1 M (see Recipes)

8. 3% (or 0.88 M) H₂O₂ (see Recipes)

9. rPrP sample (see Recipes)

10. rPrP+CuCl₂ sample (see Recipes)

11. rPrP+CuCl₂+H₂O₂ sample (see Recipes)

12. Media for cell culture (complete DMEM) (see Recipes)

Recipes

1. STE buffer, pH 8 (100 mL)

a. Dissolve the solids in ~80 mL of ultrapure H₂O and stir until completely dissolved using a magnetic stirrer.

b. Monitor the pH of the solution (it should be ~7.0–7.5) and adjust to 8 by carefully adding NaOH.

c. Adjust the final volume to 100 mL.

d. Filter-sterilize the solution using a 0.22 μm syringe filter.

e. Store the prepared buffer at 4 °C for up to 1 month.

Note: It is recommended to always use freshly prepared buffers to ensure optimal performance.

2. Buffered sucrose, pH 7.2 (50 mL)

a. Dissolve the solids in ~30 mL of ultrapure H₂O and stir until completely dissolved using a magnetic stirrer.

b. Monitor the pH of the solution (it should be ~7.5–8.0) and adjust to 7.2 by carefully adding HCl.

c. Adjust the final volume to 50 mL with ultrapure H₂O.

d. Store the prepared buffer at 4 °C for up to 1 month.

Note: Filtration is not necessary for this buffer due to the high concentration of sucrose, which increases viscosity. Ensure the solution is fully homogeneous and all components are completely dissolved before verifying the pH.

3. Buffer A (denaturing buffer) pH 8.0 (500 mL)

a. Dissolve Na₂HPO₄ and NaH₂PO₄ in ultrapure water and stir until completely dissolved using a magnetic stirrer.

b. Add Tris to the solution and continue stirring until fully dissolved.

c. Add ultrapure guanidine hydrochloride (GdnHCl) and continue stirring until the powder is completely dissolved.

d. Monitor the pH of the solution. If necessary, adjust the pH carefully to 8.0 using HCl or NaOH.

e. Adjust the final volume to 500 mL with ultrapure water.

f. Filter-sterilize the solution using a 0.22 μm syringe filter to ensure sterility.

g. Store the prepared buffer at 4 °C for up to 1 month.

Notes:

1. Ultrapure guanidine (purity ≥99%) is required to avoid impurities that could interfere with the protein purification process.

2. Guanidine is highly hygroscopic and contributes significantly to the final volume.

*Note: Sodium phosphate buffer (monobasic + dibasic) has a final concentration of 100 mM.

4. Buffer B (refolding buffer) pH 8.0 (600 mL)

a. Dissolve Na₂HPO₄ and NaH₂PO₄ in ultrapure water and stir until completely dissolved using a magnetic stirrer.

b. Add Tris to the solution and continue stirring until fully dissolved.

c. Monitor the pH of the solution. If necessary, adjust the pH carefully to 8.0 using HCl or NaOH.

d. Adjust the final volume to 600 mL with ultrapure water.

e. Filter-sterilize the solution using a 0.22 μm syringe filter to ensure sterility.

f. Store the prepared buffer at 4 °C for up to 1 month.

*Note: Sodium phosphate buffer (monobasic + dibasic) has a final concentration of 100 mM.

5. Elution buffer pH 5.0 (500 mL)

a. Dissolve NaH₂PO₄ in ultrapure water and stir until completely dissolved using a magnetic stirrer.

b. Add imidazole to the solution and continue stirring until fully dissolved.

c. Monitor the pH of the solution using a calibrated pH meter. If necessary, adjust the pH carefully to 5.0 using HCl or NaOH.

d. Adjust the final volume to 500 mL with ultrapure water.

e. Filter-sterilize the solution using a 0.22 μm syringe filter to ensure sterility.

f. Store the prepared buffer at 4 °C for up to 1 month.

Note: A significant volume of HCl may be required to reach this pH due to the buffering capacity of imidazole.

6. 2× Physiological-like buffer (PhysB) pH 7.2 (100 mL)

a. Dissolve the solids in ~80 mL of ultrapure H₂O and stir until complete dissolution using a magnetic stirrer.

b. Monitor the pH of the solution (should be acidic, ~5.5) and adjust to 7.2 by carefully adding KOH flakes (do not use NaOH since K⁺ is prevalent over Na⁺ in the cellular milieu; see Theillet et al. [13]).

c. Complete the volume to 100 mL.

d. Filter-sterilize using a 0.22 μm syringe filter.

e. Store at 4 °C for up to 1 month or freeze at -20 °C for up to 6 months.

f. Equilibrate the buffer for at least 30 min at room temperature before preparing samples for experiments.

7. CuCl₂ 0.1 M

a. Using a CuCl₂ powder stored in a sealed container kept inside a desiccator (hygroscopic), weigh the corresponding amount.

b. Completely dissolve the CuCl₂ powder.

c. Filter-sterilize and keep at room temperature.

8. 3% (or 0.88 M) H₂O₂

Add components in this order in a microcentrifuge tube (1.5 mL capacity).

Note: H₂O₂ decomposes quickly; make this diluted solution just before preparing the sample. Once open, keep the 30% w/v H₂O₂ stock container at 4 °C for up to 6 months.

9. rPrP sample

Add components in this order in a low protein binding microcentrifuge tube.

10. rPrP+CuCl₂ sample

Add components in this order in a low protein binding microcentrifuge tube.

11. rPrP+CuCl₂+H₂O₂ sample

Add components in this order in a low protein binding microcentrifuge tube.

Note: H₂O₂ decomposes quickly. Therefore, to have a 3% w/v (or 0.88 M) H₂O₂ solution, make a 1:10 dilution of 30% w/v H₂O₂ in ultrapure H₂O just before preparing the sample (e.g., 50 μL of H₂O₂ + 450 μL of H₂O) (Recipe 8). Once open, keep the 30% w/v H₂O₂ stock container at 4 °C for up to 6 months.

12. Media for cell culture (complete DMEM)

Filter-sterilize using a 0.22 μm membrane, store at 4 °C, and prewarm at 37 °C before culturing HEK293 cells.

Laboratory supplies

1. XPCS measurements:

a. Silanized quartz capillary, 1.5 mm outer diameter, wall thickness 0.01 mm (Hampton Research, catalog number: HR6-128)

b. Disposable syringe, 1 mL

c. 1.5 mL microcentrifuge protein LoBind tubes (Eppendorf, catalog number: 0030108116)

2. Imaging chambers:

a. 35 × 10 mm diameter with a hole punched and 1.33 cm² area glass coverslip (SPL, catalog number: 200350)

b. μ-slide 8-well confocal slides with lid (Ibidi, catalog number: 80806-90)

c. Coverslips #1 thickness size 22 × 22 mm (Knittel glass, catalog number: VD12222Y100A), microscope slides size 76 × 26 mm (Knittel glass, catalog number: VA21100001FKB), and double-sided tape (3-M Scotch 665-C, 19 mm × 32.9 m)

Equipment

1. XPCS beamline at a synchrotron facility. We used the Cateretê beamline at the Brazilian Synchrotron Light Laboratory (Sirius) [14]. The beamline parameters of the XPCS experiments are detailed in [12].

2. Confocal line-scanning inverted microscope (Carl Zeiss, model: LSM 710) equipped with a 100× objective (oil-immersion, NA 1.46, Carl Zeiss)

3. Confocal spinning disk inverted microscope (Nikon, model: Eclipse-Ti CSU-X) equipped with a 60× objective (oil-immersion, 60×, plan Apo, oil, NA 1.4, Nikon), perfect focus system (PFS), and coupled with temperature, humidity, and CO₂ hypoxia incubator (On Stage Incubator, Okolab)

4. Fluorescence microscope (Leica, model: LMD 7) equipped with a 100× objective (Leica Microsystems, model: oil-immersion, NA 1.25)

5. Centrifuge (Hitachi, model: Himac CR22 GII)

6. Centrifuge (Heraeus, model: Megafuge 8R)

7. Spectrophotometer (Shimadzu, model: UV mini-1240)

8. Water bath (Novatecnica)

9. Sonicator (Sonics & Materials, model: VCX 130)

10. Thermal mixer (Eppendorf, model: ThermoMixer F1.5)

11. Incubator shaker (New Brunswick Scientific, model: Excella E24 Incubator Shaker Series)

12. Incubator (Quimis)

13. ÄKTAprime^TM plus (GE Healthcare)

Software and datasets

1. Fiji (Image J) (https://imagej.net/software/fiji/downloads access date July 1, 2024)

2. GraphPad Prism 8.1.1 (https://www.graphpad.com access date July 1, 2024)

3. Excel (Microsoft)

4. XPCS data analysis Python package developed in-house for the calculation of the autocorrelation from the scattering intensity. The software generates multi-tau, one-time, and two-time g₂ modes based on the method described by [15]. The temporal autocorrelation function for a set of pixels in a region with an average wavevector q [q = (4π/λ) sin(θ/2) with θ the scattering angle] is given by the one-time correlation

5. SAXS data analysis: SAXS patterns were obtained from the averaged XPCS dataset using the PyFAI software package (https://www.esrf.fr/UsersAndScience/Publications/Highlights/2012/et/et3)

Procedure