Application Notes

1 3 A P P L I C A T I O N N O T E The inflection points of unfolding (equivalent to the melting temperature, T m ) for WbaP are listed in Table 2. Samples 1, 2, 9, 10 and 16, did not exhibit a distinct inflection point which can be attributed to the aforementioned reasons. However, for samples 11, 12, 13 and 15, a clear unfolding transition was observed by monitoring the fluorescence intensity changes at 350 nm, with the three highest inflection points highlighted in red in Table 2. In these samples, WbaP unfolds at around 55°C. However, the nanodisc disassembles at a temperature above 65°C, as evidenced by the r H during the thermal gradient. By taking SDS-PAGE, isothermal DLS, and nanoDSF into account, AASTY 6-55 was identified as the optimal copolymer to solubilize and stabilize WbaP. The initial set of copolymers used for WbaP was significantly smaller than that used here and did not include any of the AASTY copolymers (2) . The results of this expanded copolymer panel demonstrate the utility of revisiting targets for screening with newly developed copolymers for high-value targets. Unlike WbaP, which has minimal soluble domains, PglB has a large soluble acetyltransferase domain. A thermal ramp was also applied to PglB samples. The nanoDSF data for this group of samples yielded inconclusive results when analyzed using fluorescence intensity at a single wavelength or the ratio 350 nm/330 nm. However, the turbidity signal provides an effective and reliable method for examining the thermal stability of the protein. An increase in turbidity indicates the formation of large aggregates (>12.5 nm and >100 µg /mL). In this case, the unfolding of the soluble domain triggered the aggregation of the entire nanodisc, making it a suitable indicator for assessing the thermal stability of the protein.

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