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Figure 2
Stability and flexibility of TrxT and Dhd. (a) Thermal shift assay of TrxT and Dhd after incubation of natively folded proteins with SYPRO Orange dye in a 96-well PCR plate at two different pH values. As the proteins unfold with temperature, the SYPRO Orange fluorescence emission increases. TrxT is thermally stable with a Tm of >70°C in alkaline buffers, whereas acidic buffers are necessary for Dhd to reach the same stability. Values were obtained in triplicate and were collected under different conditions (Supplementary Table S2). (b) Thermal stability of full-length (FL) TrxT and TrxT_ΔC constructs in the presence or absence of DTT (duplicates). FL TrxT is ∼20°C more stable than the protein core. (c)15N–[1H] heteronuclear NOE experiment for Dhd in the absence of DTT. The same experiment with DTT is shown in Supplementary Fig. 2[link](f). (d) 15N–[1H] heteronuclear NOEs were measured as duplicates for Dhd (red). All values are positive, with the exception of a side-chain resonance. The missing bars correspond to prolines and residues for which amide resonances were not assigned. (e) 15N–[1H] heteronuclear NOE experiment of TrxT in the absence of DTT. Assignments corresponding to the flexible residues (all located in the C-terminal domain) are shown in Supplementary Fig. S2(d). The same experiment with DTT is shown in Supplementary Fig. S2(e). (f) 15N–[1H] heteronuclear NOEs were measured as duplicates for TrxT. Negative values are characteristic of highly flexible regions. Positive and negative values are shown in royal blue and light blue as in (c). The missing bars correspond to prolines and residues for which amide resonances were not assigned.

IUCrJ
Volume 8| Part 2| March 2021| Pages 281-294
ISSN: 2052-2525
https://doi.org/10.1107/S2052252521000221