4.3 Functional studies of cell-free-produced VSDs
4.3.3 The refolding strategy
Previous screenings of the VSDs in detergent or NDs revealed a high aggregation propensity and allowed for speculations about misfolded protein samples. However, the activity of cell-free-produced VSDs in liposomes could be verified (4.3.2). A strategy was needed to combine a reconstitution step into liposomes and a post-treatment procedure allowing liquid-state NMR measurement. The refolding strategy was first invented by Dr. Valiyaveetil for the in vitro-synthesized K+ channel KcsA and later transferred to cell-free-produced proteins in a cooperation with our group (Valiyaveetil et al., 2002a; Valiyaveetil et al., 2002b;
Focke et al., 2016). First, the described method had to be established in our lab (3.2.15). To this end, cell-free-produced KcsA was refolded and analyzed by SDS-PAGE (Figure 37). After refolding into liposomes, KcsA could be detected as an SDS-stable tetramer, which was in agreement with published data (Focke et al., 2016). Hence, the refolding procedure worked.
Next, the P-CF-produced VSDs were analyzed (Figure 37, P-CF pellet). His-DrVSD and His-hHV1-VSD were solubilized in 1 % SDS and reconstituted into asolectin-containing liposomes without any purification step (Figure 37, refolding). Afterwards the proteoliposomes were treated with 1 % Fos14, incubated for 2 h at room temperature, and centrifuged at 30,000xg for 15 min and 4 °C to separate pellet and supernatant fraction (Figure 37, insoluble Fos14 and soluble Fos14).
The same migration pattern as described for the KcsA could be detected for the refolded VSDs. VSDs existed as monomers in the P-CF pellet fraction. After the refolding into asolectin liposomes, a size shift to higher molecular weights could be detected for the monomer fraction, which was due to SDS-stable lipid attachments at the protein. A second signal arose, which corresponds to a dimeric VSD species. The obtained native oligomeric state stayed intact after treatment of the proteoliposomes with Fos14. Moreover, 100 % of the protein could be recovered after liposome solubilization. Taken together, the refolding procedure for the VSDs was successful and led to the formation of SDS-stable native dimers.
RESULTS
Figure 37: Refolding of KcsA and VSDs. The proteins under investigation are indicated below the SDS-PAGE figures. All proteins showed an overexpression signal in the pellet fraction from P-CF expression (P-CF pellet).
The protein pellet was solubilized in 1 % SDS and reconstituted into asolectin liposomes (refolding). Additional attached lipids might explain the size shift in gel migration pattern for the monomeric (M) protein.
Furthermore, a tetrameric (T) species for KcsA (pink arrow) and dimeric (D) species for DrVSD (orange arrow) and hHV1-VSD (green arrow) could be detected in the proteoliposome fraction (“refolding”). The VSDs in liposomes were resolubilized with 1 % Fos14, followed by a centrifugation step at 30,000xg. In the soluble fraction, the tetrameric/dimeric states as well as the lipid attachment were still observable (soluble Fos14). No protein could be detected in the insoluble fraction after Fos14 treatment (insoluble Fos14). The protein marker is indicated by PM and molecular weights are listed on the left.
Subsequently, the activity of refolded VSDs was tested (3.2.21). The refolded, Fos14-solubilized VSDs were purified in Fos14 performing a Ni2+-IMAC purification step. The elution fractions were collected, concentrated in Amicon centrifugal filter units (MWCO 10 kDa), centrifuged (16,100xg, 10 min, 4 °C) and the concentration was determined with a NanoDrop instrument. Afterwards, the VSDs were reconstituted 1:100 into POPE/POPG (3:1 w/w) liposomes and the fluorescence-based activity assay was performed. Both VSDs were functional (data not shown).
Next, the stability of refolded VSDs resolubilized in detergent was under investigation.
Concentrated samples in Fos14 were loaded onto a SEC column (Figure 38 A). Additionally, the refolding was repeated with His-hHV1-VSD-Strep whereby the liposome solubilization step was performed with DPC and the final sample was loaded onto the SEC column too.
Fos14 was used as the primary detergent because it was described in the literature to be favored for the hHV1-VSD stability (Li et al., 2015). However, its micelle size of 41 kDa could have possibly hindered liquid-state NMR measurements as it would have drastically increased the overall molecule size and therewith the tumbling rate. Contrary, DPC micelles with a size of 19 kDa are more suited for NMR studies with membrane proteins. For that reason, hHV1-VSD in DPC was additionally analyzed. Compared to previous obtained elution
RESULTS
profiles of the VSDs in DPC (Figure 23), the ones after refolding in Fos14 and DPC looked quite promising (Figure 38 A). Although a little shoulder for each peak could be detected, representing higher oligomeric species, the main peak was well-defined with an elution volume matching perfectly for dimeric hHV1-VSD in Fos14 and dimeric hHV1-VSD in DPC. The elution volume of the main peak of DrVSD referred to a monomeric protein species in Fos14 micelles. Additional small peaks at 2.05 ml (Fos14) and 2.15 ml (DPC) belonged to detergent molecules due to a higher detergent concentration in the samples compared to the running buffer caused by the beforehand performed concentration procedure of the VSDs.
Figure 38: Stability screening of refolded VSDs by SEC and NMR analyses. The SEC runs were performed by injecting 50 µl protein to an analytical Superdex200 PC 3.2/30 column with a flow rate of 0.05 ml/min at 16 °C with either 20 mM Tris-HCl pH 8.0 @ 4 °C, 150 mM NaCl and 0.07 % Fos14 or 20 mM HEPES-NaOH pH 7.0, 200 mM NaCl and 0.1 % DPC as running buffer. Black arrows indicate the void (0.89 ml) and the column volume (2.4 ml). Prior to loading, the samples were centrifuged at 18,000xg for 15 min. A The quality screen of VSDs refolded, solubilized and purified in Fos14 and DPC showed evaluated peak maxima for hHV1-VSD in Fos14 (green line) at around 140 kDa (1.35 ml), for DrVSD in Fos14 (orange line) at around 70 kDa (1.49 ml) and for
15N,2H-labeled hHV1-VSD in DPC (black line) at around 46 kDa (1.57 ml). B The sample of 33 µM 15N,2H-labeled hHV1-VSD in DPC was separated into three individual samples. They were incubated over night at the indicated temperatures and analyzed the next day by SEC runs. The profiles were identical. C The [15N,1H]-BEST-TROSY spectrum of 15N,2H-labeled hHV1-VSD was measured in 20 mM HEPES-NaOH pH 7.0, 20 mM NaCl and 0.1 % DPC and recorded at 318 K and 800 MHz (NS = 512, TD1 = 288). D A 15N,2H- labeled hHV1-VSD sample of around 76 µM in DPC was analyzed by NMR, centrifuged and loaded again onto the column (post-measurement) and compared with the sample of 33 µM (B) stored at 4 °C (pre-measurement). Shifts to smaller elution volumes could be detected indicating the presence of aggregates.
RESULTS Next, the stability of the refolded hHV1-VSD in DPC micelles (33 µM) was tested by incubation of the same sample at different temperatures overnight and reapplication to the SEC column as described previously (Figure 23, Figure 38 B). No changes in the elution profiles could be observed for any tested temperature even at 42 °C. Refolded VSDs seemed to be more stable than VSDs without any prior lipid contact. Finally, a NMR sample of refolded His-hHV1-VSD-Strep in DPC was prepared to a concentration of 76 µM. No gain in resolution and dispersion could be observed for the refolded VSD construct (Figure 38 C).
The data were in agreement with all other tested conditions. The spectrum from 7.5 to 8.8 ppm in the 1H-dimension pointed towards an unfolded or even a very large protein sample. Only a few signals could be observed above 8.5 ppm or below 7 ppm that would have suggested folded protein structures. To test whether the results support the theory of an aggregation-induced effect, the NMR sample was centrifuged (30,000xg, 30 min, 4 °C) after the measurements were finished. The supernatant was analyzed again by SEC (Figure 38 D). The complete elution profile was shifted to lower elution volumes. A large peak could be recognized at 0.98 ml corresponding to high molecular weight compounds. The incubation at 45 °C for a longer time than 16 h and in a higher sample concentration caused the formation of soluble aggregates of refolded VSDs. In conclusion, the refolding procedure was successful, but failed for studying dynamics of VSDs by liquid-state NMR due to the same reasons as observed before. High sample concentrations and high incubation temperatures induced aggregation of the cell-free-produced, reconstituted VSDs.
DISCUSSION
5 Discussion
In the next chapter, the obtained results will be discussed and compared with current literature data.