CD and Solid-State NMR Studies of Low-Order Oligomers of Transthyretin

Abstract

Characterization of oligomeric intermediate states populated at an early stage of misfolding and aggregation is essential to understanding molecular mechanism of pathogenic protein aggregation. Growing evidence also suggests that oligomeric species are more toxic than mature fibrillar counterparts. Here, we describe procedures for isolating oligomeric species of an aggregation-prone protein, transthyretin, associated with protein misfolding disorders, including cardiomyopathy and polyneuropathy. We also describe methods for structural studies of the oligomeric species using circular dichroism and solid-state NMR spectroscopy. These methods can be applied to structural characterization of oligomeric intermediates of other aggregation-prone proteins.

1. Introduction

Protein misfolding and amyloid formation are associated with a wide range of debilitating human diseases, including Alzheimer’s and Parkinson’s diseases, and amyloidosis [1,2,3]. Amyloid formation involves conformational changes from native polypeptides to misfolded intermediate states that self-assemble into β-structured fibrillar protein aggregates (amyloid). Recent studies suggested that a single protein can form distinct fibrillar aggregates with different biological activities, presumably through the formation of distinct oligomeric intermediate states [4,5,6]. In addition, small oligomeric species formed at an early stage of protein aggregation are believed to be real cytotoxic agents [7,8,9]. Thus, structural characterization of oligomeric intermediate states is critical to not only understanding the molecular mechanism of the pathogenic aggregation process but also to developing therapeutic interventions for the debilitating human diseases.

Investigation of the small oligomers has, however, been of a great challenge since oligomeric species are only transiently populated. In addition, multiple oligomeric species of different sizes co-exist in dynamic equilibrium with monomers and larger assemblies. The transient, heterogeneous nature of the oligomeric intermediate states hampers structural characterization of the elusive intermediates [8,9,10]. In this protocol, we describe protocols for the preparation of oligomeric intermediates of transthyretin (TTR) and their structural characterization using biophysical techniques such as circular dichroism (CD) and solid-state NMR. It is important to note that TTR is a natively folded protein that undergoes an aberrant conformational transition to misfolded aggregation-prone intermediates, subsequently self-assembling into β-structured amyloid. Thus, comparative structural analyses of the native state and oligomeric intermediates will help unravel critical regions undergoing aberrant structural changes during the oligomerization process, providing valuable insights into the molecular mechanism of protein misfolding.

2. Materials

Buffers used in this protocol are prepared using deionized water (18 MΩ-cm at 25 °C) and analytical-grade reagents. The buffer solutions are also filtered using 0.22 μm membrane filters and stored at 4 °C unless otherwise specified. Routine procedures for TTR expression and purification are not described in this protocol.

2.1. Preparation of TTR Oligomers

1. Recombinant human transthyretin (Creative BioMart, catalog number: TTR-1837H) (see Note 1)

2. Cytiva PD-10 desalting column (or similar)

3. Centriprep centrifugal filters, 10 kDa cutoff (or similar)

4. Ammonium sulfate

5. Guanidinium chloride

6. 2 N sodium hydroxide (store at 4 °C)

7. 20 mM sodium acetate buffer, pH 4.25 (store at 4 °C)

8. 5 mM and 10 mM sodium phosphate buffers, pH 7. 3

9. HiPrep^™ 26/10 desalting column (GE Healthcare) (store at 4 °C)

10. HiLoad^® 16/60 Superdex^® 200 column (GE Healthcare)

11. Ä KTA^™ pure 25 chromatography system (or similar)

12. Mettler Toledo InLab Micro Pro-ISM pH electrode (or similar)

13. UV-Vis spectrophotometer

14. Quartz cuvette for UV spectroscopy

15. 0.22 μm syringe filter (store at 4 °C)

16. Amicon^® stirred cell with 10,000 MWCO ultrafiltration disc membrane connected to a pressurized nitrogen gas tank (store at 4 °C)

2.2. Transmission Electron Microscopy (TEM)

1. Uranyl acetate (1%) in deionized water (filtered through 0.22 μm filter)

2. Formvar/carbon-coated copper 400 mesh grids

3. Pelco^® EMX reverse tweezers (or similar)

4. PELCO easiGlow^™ glow discharge system (or similar)

5. Transmission electron microscope (120 kV or higher)

6. Grade 1 Whatman filter paper

2.3. Circular Dichroism (CD) Spectroscopy

1. 1 mm pathlength quartz cuvette

2. JASCO J-815 CD spectrometer (or similar) equipped with a Peltier thermostat

2.4. Solid-State NMR

1. Thermo Sorvall Legend XTR Refrigerated Centrifuge (or similar)

2. 3.2 mm magic angle spinning (MAS) rotor

3. NMR spectrometer equipped with 3.2 mm MAS probe

3. Methods

3.1. Preparation and isolation of low-order TTR oligomers:

Carry out all the steps at 4 °C unless otherwise specified.

1. While stirring, gradually add 2 mL of TTR (10 mg/mL in 10 mM phosphate buffer, pH 7.3) to 18 mL of 20 mM sodium acetate buffer (pH 4.25) (see Note 2).

2. Incubate the protein solution at 4 °C for 5–9 days (see Note 3). Formation of low-order TTR oligomers at various incubation times can be monitored by size-exclusion chromatography (SEC) (see Section 3.2 and Fig. 1).

3. To slow down/halt further aggregation of TTR and to obtain relatively stable low-order TTR oligomers, increase pH of the TTR solution from 4.4 to 7.3 by adding 2 N NaOH. While increasing the pH of the TTR solution, care should be taken to avoid TTR aggregation at its isoelectric point (pI: ~ 5) (see Note 4).

4. Remove the added salts by desalting the TTR solution using desalting column. Equilibrate the HiPrep^™ 26/10 desalting column (two columns connected in series) with 5 mM sodium phosphate buffer (pH 7.3). Load 20 mL of the TTR solution into the desalting column and elute the protein with 5 mM phosphate buffer at a flow rate of 5 mL/min (see Note 5).

5. Concentrate the desalted TTR solution to 1.5–2 mL using 10,000 MWCO ultrafiltration disc membrane in an Amicon^® stirred cell under nitrogen gas. Filter the resultant TTR solution using a 0.22 μm syringe filter (see Note 6).

6. Use the size-exclusion gel filtration column to isolate TTR oligomers. Load 1.5 mL of the concentrated TTR solution into HiLoad 16/60 Superdex 200 SEC column, and elute the protein with 5 mM sodium phosphate buffer (pH 7.3) at a flow rate of 1 mL/min. Collect the TTR oligomer fractions and store the fractions at 4 °C (see Note 7) (see Fig. 2).

7. Use the TTR oligomers for structural analysis with CD spectroscopy or transmission electron microscopy (TEM), as soon as they are collected. Prolonged storage of the low-order TTR oligomers may induce the formation of bigger aggregates.

Fig. 1.

Size distribution analysis of TTR oligomers: TTR was incubated at pH 4.4 and 4 °C for different interval and the oligomers formation was monitored using HiLoad 16/60 Superdex 200 SEC column (GE Healthcare).

Fig. 2.

SEC analysis of TTR oligomers. TTR oligomers were isolated using HiLoad 16/60 Superdex 200 SEC column and the protein fractions were collected for CD spectroscopy analysis.

3.2. SEC Analysis of TTR Oligomers:

While the SEC can be carried out at room temperature or at 4 °C, sample preparation for the SEC should be done at 4 °C:

1. During the incubation of TTR at pH 4.4 and 4 °C, the formation of low-order oligomers is monitored by size-exclusion chromatography.

2. At various incubation times, retrieve 500 μL of TTR solution from the incubated stock solution into a microcentrifuge tube.

3. Increase pH of the 500 μL TTR solution to 7.3 by using 2 N NaOH (see Note 8).

   (Determine the required amount of NaOH to increase the pH as described in Note 4.)

4. Filter the neutralized TTR solution using a 0.22 μm syringe filter. Load ~300 μL of the filtered TTR solution into a HiLoad 16/60 Superdex 200 column and elute the protein with 5 mM sodium phosphate buffer (pH 7.3) at a flow rate of 1 mL/min (see Fig. 1).

3.3. Characterization of TTR oligomers by TEM:

TEM sample preparation for TTR oligomers can be performed at room temperature.

1. TTR oligomers are characterized by TEM. The below recommended sample volume, incubation time, and staining procedures of the protein sample are optimized for the TTR oligomers at a working concentration of 10–20 μM.

2. Before applying TTR sample to formvar /carbon-coated copper 400 mesh grids, grids must be glow discharged in order to make the EM grids hydrophilic which will allow the easy spread of aqueous solutions on the grids (see Note 9).

3. Using the fine tip tweezers, grasp the edge of the EM grid, and place the EM grids on a glass slide (with the shiny side of the grid up). Place the glass slide in the glow discharger unit. Negatively discharge the grids at a current of 25 mA for 2 min under vacuum (1 × 10¹ bar).

4. Hold the glow-discharged grid along its edge using fine point reverse tweezers, and place the tweezers on a working surface. Apply 5 μL of the TTR sample to the shiny side of the grid, and incubate the sample for 30 sec on the grid.

5. Blot the remaining sample solution from the grid by touching the edge of the grid to a grade 1 Whatman filter paper. Do not let the grid dry at this point.

6. Immediately rinse the grid with 20 μL of deionized water, and blot the water from the grid using the filter paper. Perform the additional rinsing step if necessary (see Note 10). Do not let the grid dry.

7. Immediately add 8 μL of 1% uranyl acetate solution to the grid, and allow the protein sample to stain for 30 sec.

8. Leaving a thin layer of the staining solution remain on the grid surface, blot the remaining staining solution from the grid using the filter paper. Let the grid air-dried for about 5–10 min.

9. Image the EM grid under the transmission electron microscope at an accelerating voltage minimum of 80 kV (see Fig. 3) [11].

Fig. 3.

TEM characterization of TTR oligomers. TEM images of TTR oligomers eluting at 53 mL (a) and 61 mL (b) in Fig.2.

3.4. CD spectroscopy for TTR oligomers:

1. Perform the CD measurements at room temperature using a CD spectrometer equipped with a Peltier thermostat. Set the CD measurement parameters as specified here: scanning speed, 100 nm/min; D.I.T, 2 sec; wavelength range, 190–250 nm; and number of scans, 10.

2. Using an extinction coefficient of 7.76 × 10⁴ M⁻¹ cm⁻¹ at 280 nm, adjust the concentration of TTR oligomer fractions to 10 μM (monomeric TTR concentration).

3. Load 200 μL of TTR oligomeric sample (10 μM) into a quartz cuvette with 1 mm pathlength, and place the cuvette in the cell holder of the CD spectrometer. Wait for about 5 min for the protein sample to reach equilibrium.

4. Collect ten replicate scans of CD spectra for the TTR oligomeric sample. Overlay the replicated scans, and make sure that the CD spectra are consistent to confirm the stability of the TTR oligomers (see Notes 11 and 12).

5. Record the averaged CD spectrum from ten scans. If necessary, collect additional scans and record the averaged spectrum.

6. To obtain a baseline spectrum, collect ten replicate scans for the buffer alone (5 mM sodium phosphate) using the same instrument parameters as used for the protein sample. Record the averaged baseline CD spectrum.

7. Subtract the averaged baseline spectrum from the averaged sample spectrum. Save the raw data in ASCII format for further use with protein secondary structure analysis programs.

8. Two or more CD spectra can be overlaid for the secondary structural comparison of protein samples (see Fig. 4). When comparing the two spectra, make sure to keep the protein concentrations identical while measuring the CD spectra.

9. Protein secondary structure analysis can be carried out using a variety of algorithms (e.g., CONTIN, SELCON, and CDSSTR [12, 13]). An online server DICHROWEB [14, 15] can be used to analyze the CD data.

Fig. 4.

CD spectral analysis of various states of TTR along the aggregation pathway. CD spectra of native tetrameric (black), dimeric (red), and oligomeric (green) states of TTR.

3.5. Preparation of low-order TTR oligomers for solid-state NMR

1. Incubate 40 mg of TTR (uniformly ¹³C, ¹⁵N-labeled TTR) as described in Steps 1 and 2 of Section 3.1. Incubating about 40 mg of TTR should yield a sufficient amount of TTR oligomers to fill a 3.2 mm MAS rotor.

2. After 5–9 days of TTR incubation, transfer the TTR sample solution into a centrifuge tube (see Note 13). Precipitate out the TTR oligomers by adding 15–20% (w/v) ammonium sulfate to the TTR solution in the centrifuge tube, and rock the solution gently for about 15–20 min at 4 °C on a rocking shaker.

3. Collect the precipitated TTR oligomers by centrifuging at 18,000 × g for 1 hr at 4 °C and remove the supernatant.

4. Wash the precipitates with deionized water to remove the soluble native TTR. To do this, add about 10 mL of deionized water to TTR precipitates in the centrifuge tube, and shake the tube gently for 10–15 sec. Remove the supernatant. Repeat this step one more time.

5. Dry the TTR precipitates with inert gas and pack the TTR oligomeric sample into a 3.2 mm MAS rotor.

6. In order to rehydrate the dried oligomeric sample, add 5 μL of water to the oligomeric sample in the rotor. Place the MAS rotor in a microcentrifuge tube and centrifuge at 20,000 × g for 10 min (see Note 14). Place the end-cap to seal the rotor.

7. Record solid-state NMR spectra using a 600 MHz (or higher) spectrometer equipped with a 3.2 mm MAS probe.

4. Notes

1. Buffer exchange TTR into 10 mM phosphate buffer (pH 7.3) using a PD-10 desalting column. Concentrate the resultant TTR solution to 10 mg/mL using a Centriprep centrifugal filter unit and store at 4 °C.

2. Slow addition of TTR to acetate buffer with gentle agitation is important to maintain the uniform pH throughout the solution. The final pH of the TTR solutions should be 4.4.

3. Incubation of TTR at pH 4.4 for more than nine days will result in the formation of relatively bigger oligomers. To monitor the relative size distribution of oligomers at different incubation periods, refer to Section 3.2.

4. Predetermine the amount of 2 N NaOH required to increase the pH from 4.4 to 7.3 using the buffer mix (18 parts of 20 mM sodium acetate, pH 4.25, and 2 parts of 10 mM phosphate buffer, pH 7.3). Add the exact amount of NaOH, all at a time, to the TTR solution incubated at pH 4.4. It is critical to stir the protein solution while adding the NaOH to avoid the localization of low/high pH spots in the solution and to prevent the exposure of TTR to its isoelectric point. This procedure should result in the pH of the TTR solution to fall between 6.5 and 7.3. Adjust the final pH of TTR solution to 7.3 using NaOH.

5. If only one HiPrep^™ 26/10 desalting column is being used, load about 10 mL of protein solution every time, and the protein can be eluted at higher flow rates (~10 mL/min).

6. Filter the TTR solution through a 0.22 μm filter just before injecting it into the SEC column to remove any insoluble aggregates.

7. SEC can be performed at either room temperature or 4 °C. If the gel filtration is being done at room temperature, minimize the exposure of the concentrated TTR solution to room temperature while loading into the SEC column.

8. In order to avoid the localization of low pH spots in the TTR solution while changing the pH from 4.4 to 7.3, place the required amount of 2 N NaOH in a microcentrifuge tube. Add 500 μL of the TTR solution to the microcentrifuge and immediately mix the solution by pipetting in and out 4–5 times. Measure the pH.

9. It is ideal to glow discharge the grids right before using them for sample preparation.

10. Uranyl acetate can interact with the salts in the phosphate buffer and forms uranium phosphate salt crystals. For the cleaner TEM images, it is important to rinse away the phosphate salts from the EM grid before staining the protein with uranyl acetate.

11. When the TTR solution at 4 °C is transferred to a cuvette at room temperature, it tends to form air bubbles in the cuvette over the time during the data collection. Remove the air bubbles by tapping the cuvette.

12. Monitoring the HT voltage signal during the CD measurement is essential. HT voltage cutoff limit for the Jasco J-815 spectrometer is 700 V. In order to record a reliable CD spectrum, keep the HT voltage value below the cutoff limit. Using a 1 mm pathlength quartz cuvette and the TTR oligomers at a concentration of around 10 μM in 5 mM phosphate buffer should keep the HT voltage well below 700 V.

13. To minimize the loss of TTR oligomeric precipitates when transferring the suspension to a centrifuge tube, carry out the ammonium sulfate precipitation directly in the centrifuge tube.

14. To avoid the damage to the drive tip of the MAS rotor while centrifuging, insert a piece of Kimwipe in the microcentrifuge tube, then place the MAS rotor in the microcentrifuge tube.