Part:BBa_K5172001

PafA 70.2

PafA 70.2 is a protein redesign of the naturally-occurring PafA enzyme (BBa_K5172000), which exhibits high activity towards phosphomonoesters, but is also active against phosphodiesters and phosphotriesters (Lidbury et al, 2022). It has 531 residues and a molecular weight of 58.1 kDa, and 70% of its most conserved residues (identified using ConSurf) were fixed during the redesign process. In particular, residues within 5Å of the docked ligand (glycerol 3-phosphate) and the two Zn2+ ions were fixed - for more details on how this was redesigned, see our AI redesign guide using LigandMPNN. See WT PafA for further details (BBa_K5172000).

Expression Results

PafA 70.2 was expressed in E. coli Lemo21 (DE3) with results visualised on an SDS (see Figure 1).

Figure 1: SDS-PAGE result of PafA 70.2 post-protein purification. The flow-through (FT), binding buffer (BB), wash buffer (WB), and elutions (E1-10) were all tested to see which elutions contained protein.

Due to extra banding present, size exclusion chromatography was done to remove contamination (see figure 2).

Figure 2: SDS-PAGE result of PafA 70.2 post-SEC.

Determining the Stability of the Protein

Imidazole used in IMAC protein purification may cause them to unfold, reducing the accuracy of the characterisation assays. Circular dichroism (CD) experiments were carried out to ensure the proteins were folded the same way as our active WT protein. It shows the stability of the proteins by determining the temperature at which they aggregate out of solution or unfold. Figure (3A) shows the CD results plotting ellipticity against wavelength for PafA 70.2 compared to the CD results for WT PafA, Figure 3B. In the plots of ellipticity against wavelength for PafA 70.2, the curves do not flatten out at the higher temperatures as observed on the corresponding WT PafA, therefore suggesting that PafA 70.2 does not aggregate or unfold at temperatures up to 95℃. When the ellipticity at 208nm and 222nm is plotted for PafA 70.2 against temperature (Figure 3G and 3H) the sigmoidal shape is not present which confirms that it is not aggregating or unfolding, furthermore, the ranges in ellipticity values are much smaller with more consistent values.

Figure 3: (A) CD results for PafA WT, (B) normalised CD results for PafA WT, (C) & (D) a comparison of ellipticity to temperature at each wavelength for alpha helices (208nm & 222nm) on PafA WT, (E) CD results for 70.2, (F) normalised results for PafA and 70.2 at 208nm, (G) & (H) a comparison of ellipticity to temperature at each wavelength (208nm & 222nm) for alpha helices on 70.2

Characterisation of Activity

The enzyme's activity was measured with p-nitrophenyl phosphate (pNPP) as a substrate. pNPP is hydrolysed by phosphatases to release inorganic phosphate and p-nitrophenol (pNP), a yellow product which can be measured using 405 nm absorbance. The absorbance data was collected using a Tecan Spark plate reader, which allowed for a quick assessment of enzyme activity at various pNPP concentrations. Unfortunately, compared to the WT PafA (BBa_K5172000) (see Figure 4), 70.2 exhibited very low activity (see Figures 6 and 7), signalling that during redesign key functional residues were lost. This could be improved in future cycles of the AI redesign with LigandMPNN by increasing the number of residues conserved as well as experimentally validating more sequences.

Figure 4: Absorbance at 405 nm for 70.2 and PafA at different concentrations of pNPP. (A) 70.2 at 10nM absorbance at 405 nm increases slowly. (B) At 100 nM of 70.2, the absorbance at 405 nm increases. (C) PafA WT at 1 nM has a large increase in absorbance at 405 nm with time. (D) Control with no enzyme.

Temperature Assays

When temperature assays were conducted on 70.2 to determine the optimum temperature, like the other redesigned enzymes (BBa_K5172002) and (BBa_K5172003), it appears to have an exceptionally high optimum temperature of 80℃, as shown on Figure 9. Initially we considered that this may be due to the high temperatures causing pNPP to completely hydrolyse leaving no pNPP for the enzymes to hydrolyse, thus we decided to heat pNPP to the high temperatures before conducting the assay again at a lower temperature. This yielded results that were not significantly different to the pNPP which had not been previously preheated, see Figure 8. Thus we were able to determine that there was still pNPP present for the enzyme to hydrolyse.

Figure 5: Temperature assay results for PafA WT and its variants with pNPP preheated to 80℃ and 90℃ compared to no preheating (0℃).

Figure 8 shows that PafA had a much greater rate of activity with its pNPP preheated to 0℃, 80℃, and 90℃ compared to 70.2. This suggests that even though variants may be still active, they have an extremely low activity and this should be considered in future redesigns. This finding is supported by Figure 9 below which provides data from the temperature assays, in which the entire reaction mixture (including enzyme) was maintained at a variety of temperatures. The results show that PafA WT’s activity remains high from 0℃ to 70℃, then rapidly decreases, finally reaching an absorbance of 0 at 90℃. However, 70.2 has very low activity from 0℃ to 70℃ then spikes at 80℃ before rapidly decreasing and also reaching inactivity at 90℃.

Figure 6: Temperature assay results for PafA WT and its variants at 0-90℃, showing that variants 30.1, 50.2 and 70.2 have the highest maximum absorbance percentage at ~80℃ but PafA has the highest maximum absorbance percentage at ~37℃.

Key Findings:

• The enzymes designed on LigandMPNN had increased thermal stability compared to the WT PafA (BBa_K5172000).
• Unfortunately, their activity was extremely low, meaning we were unable to characterise their activities at different pHs and temperatures.
• This means further cycles of redesign on LigandMPNN would need to be done, with increased number of residues fixed.