For NMR experiments it is necessary for a protein to be stable for a minimum of 3 days and preferably a couple of weeks to allow time for data collection. Since FapD58-253 PA7 showed good expression, was the most stable and produced one of the better NMR spectra it was used for optimisation experiments. Additives were screened to test their effect on the protein stability, a broad range were initially added during the purification on a small scale and the amount of soluble protein measured qualitatively by SDS-PAGE (Data Not Shown). Additives were then selected from these results and DSF was used to test the quantitative effect on protein stability (Figure
3.29). The DSF results indicated that although the additives may improve the soluble yield of FapD
many did not improve the thermal stability of the protein and several had a detrimental effect; however high concentrations of Glycerol (10 % w/v), Potassium Phosphate (100 mM) or Sodium Chloride (425 mM) did improve the thermal stability.
Figure 3.29: FapD Thermal Stability – Differential Scanning Fluroimetry (DSF). Graphs
showing the normalised thermal denaturation curves of FapD in the presence and absence of a selection of additives. The results are separated into two separate graphs are included for clarity, despite the experiments being carried out concurrently. Notably Triton X-100 and Ethanol have a detrimental effect on the Stability of FapD as measure by the melting temperature (Tm) values (temperature at 0.5 relative fluorescence). While similarly Glycerol, Potassium Phosphate and a higher concentration of NaCl increased the Tm of the protein.
Since the best additives identified in the DSF screen were additional NaCl, potassium phosphate and glycerol. FapD was purified in the presence of these additives and tested for long term stability by 1D NMR. The potassium phosphate and increased NaCl samples precipitated before data could be collected but 1D data for glycerol was collected (Figure 3.30A). Glycerol appears to stabilise the protein over the period of several days, however it has an adverse effect on the spectra due to relaxation. Some alternative NMR buffers such as a Glutamine-Arginine buffer [177] were also tested (Figure 3.30B) but none produced a beneficial effect on stability.
Figure 3.30: Stability Trials of FapD. a, Overlay of 1D spectra of FapD58-253 PA7 in 10 % glycerol 250 mM NaCl, 20 mM HEPES pH 7.5 before (Blue) and after (Red) 5 day incubation at room temperature, showing no significant changes in signal. b, Overlay of 1D spectra of FapD58-253 PA7 in Glutamine-Arginine NMR Buffer [177] before (Blue) and after (Red) 3 day incubation at room temperature showing a significant loss of signal due to sample precipitation.
To improve the NMR spectra but retain the stabilising effect glycerol was compared at 10% (v/v) (Figure 3.30A) 5 % (v/v) (Figure 3.31A) and 2 % (v/v) (Figure 3.31B) and the effects on stability compared, 2 % (v/v) glycerol was insufficient to stabilise the protein for a 5 day period but both 10 % (v/v) and 5 % (v/v) effectively stabilised the protein and produced well dispersed 15N 1H 2D spectra (Figure 3.31C).
Figure 3.31: Stability of FapD in glycerol. a, Overlay of the amide proton region of 1D spectra of
FapD58-253 PA7 in 250 mM NaCl, 20 mM HEPES pH 7.5, 5 % (v/v) glycerol before (Blue), after 1 day (Red) and after 3 days (Green) incubation at room temperature, showing no significant changes in signal. b, Overlay of the amide proton region of 1D spectra of FapD58-253 PA7 in 250 mM NaCl, 20 mM HEPES pH 7.5, 2 % (v/v) glycerol before (Blue), after 1 day (Red) and after 3 days (Green) incubation at room temperature showing a perceptible (~30 %) loss of signal due to sample precipitation. c, 15N 1H HSQC 2D spectra of FapD58-253 PA7 in 250 mM NaCl, 20 mM HEPES pH 7.5, 5 % (v/v) glycerol showing the protein is stable and the spectra relatively well dispersed.
FapD contains a conserved cysteine residue in the active site which should be exposed and may affect the stability of the protein. Two approaches were adopted for dealing with this, the reducing agent DTT was used to reduce the cysteine and prevent potential intermolecular disulphide bridges forming and a C67A mutant version of the gene was made to eliminate any potential protease activity (although the precipitant showed no evidence of degradation (Figure 3.26D). 1D NMR was used to test the effect of the DTT and C67A mutant on the stability of FapD, some minor improvement was seen with the addition of DTT to glycerol, although DTT alone was insufficient to stabilise the protein for NMR (precipitated in hours) in the presence of 10 % (v/v) glycerol the protein was stable over a week at room temperature (Figure 3.32A). The FapD C67A mutant was also stable in the presence of 10 % (v/v) glycerol for at least 5 days (Figure 3.32B). The FapD C67A did not show any improvement in stability as the protein was still unstable over a time course of days in the absence of glycerol (Figure 3.33A). Comparison of the spectra of FapD C67A and FapD (Figure 3.33B) indicated that although there were some small differences the proteins displayed a similar degree of structure and probably retained the same fold. Since FapD C67A behaved similarly to the wild type protein this suggests the protease activity, if present, had little effect on FapD stability.
Figure 3.32: Stability Trials of FapD58-253 PA7 with DTT and C67A substitution. a, Overlaid 1D 1H NMR spectra of FapD58-253 PA7 250 mM NaCl, 20 mM HEPES pH 7.5, with 5 mM DTT and 10 % (v/v) glycerol showing the spectra before (blue) and after (red) one week incubated at room temperature (~20 ◦C). b, Overlaid 1D 1H NMR spectra of FapD58-253 PA7 with C67A substitution in 250 mM NaCl, 20 mM HEPES pH 7.5, 10 % (v/v) glycerol showing the spectra before (blue) and after (red) five days incubated at room temperature (~20 ◦C). The spectra both show almost no change in the peak intensities or positions indicating the proteins remain stable over the period of the incubation.
Figure 3.33: NMR of FapD C67A. a, Overlay of 1D spectra of FapD58-253 C67A in 250 mM NaCl, 20 mM HEPES pH 7.5, 0 % (v/v) glycerol before (Blue) and after (Red) 1 day incubation at room temperature showing a significant loss of signal, probably due to precipitation b, Overlay of FapD58- 253 15N (Blue) and FapD58-253 C67A (Red) 15N 13C 2H, 1H 15N Transverse Relaxation optimised spectroscopy (TROSY) 2D spectra in 250 mM NaCl, 20 mM HEPES pH 7.5, 10 % (v/v) glycerol showing the proteins possess similar structure.