Oligomeric State

A 50µL sample of His6-MmyJ in IMAC wash binding buffer was run through the gel filtration column. It was decided that the initial run should be done in this buffer with glycerol still present, rather than the gel filtration buffer, for the sake of the stability of the protein. In order to determine whether this would have any effect on the peak positions, another run was performed with albumin and aprotinin added to the His6-MmyJ sample to observe any shifts due to the inclusion of glycerol when compared to the calibration data. These data are shown

4.2 Protein Aggregation 4 STABILITY DETERMINATION

Figure 4.5: UV A280 chromatograms of 50 µL samples of His6-MmyJ with and without addition of calibration proteins to observe peak shift due to presence of glycerol. Monomeric His6-MmyJ has a molecular weight of 15.9 kDa, and so dimeric His6-MmyJ is expected to have a peak corresponding to approximately 32 kDa.

alongside the original calibration data in Figure 4.5.

The first thing to notice about the His6-MmyJ trace is that there is a large amount of material eluted in the dead volume peak. It was initially considered that the two peaks were the monomer and dimer of His6-MmyJ, and that they had simply been shifted by the different buffer conditions. However, the second trace proves that this is not the case as while the albumin and aprotinin peaks are shifted slightly compared to the calibration peaks, the degree of difference is negligeable compared to the shift that would be needed for peaks of around 15 kDa and 30 kDa to appear where the two His6-MmyJ peaks are. Also, the dead volume peak appears as a shoulder on the albumin peak at 66 kDa, demonstrating that it is not buffer conditions that have caused this peak to appear where it is. Due to this, the idea that the two peaks represented the monomer and dimer was discarded, and it was then assumed that the right hand peak corresponded to the dimer and the left hand peak corresponded to an unknown oligomeric state that was forming in solution from monomers and dimers, such that it was larger than 70 kDa and hence above the resolving range of the column7_{. In this case there}

7_{Both native and SDS-PAGE gels were attempted with fractions collected from both peaks but, due to small}

initial sample size and the protein being diluted further upon elution, neither type of gel offered conclusive visualisation of the contents of these peaks.

4.2 Protein Aggregation 4 STABILITY DETERMINATION

Figure 4.6: A280 chromatograms of 50 µL samples of His6-MmyJ C49S with and without addition of calibration proteins, run under identical conditions as previous wild type sample.

was no observed peak assigned to the monomer, and so it was assumed that His6-MmyJ does not appear in its monomeric form while in a non-denaturing solution.

The experiment was then repeated with a sample of His6-MmyJ C49S prepared in the same manner, as shown in Figure 4.6. As can be seen, the His6-MmyJ peaks appear to be in the same place, and so the mutation appears not to have altered the formation of oligomers. However, the dead volume peak is much larger, indicating that more protein has aggregated in this instance. This was not expected as the C49S mutation was introduced to minimise what was thought to be non-biologically relevant binding between monomers. Also, the dead volume peak is shifted significantly this time between the samples with and without added calibration proteins, yet the buffer conditions were identical for both runs, and so it can be inferred that this deviation may simply be due to a systematic error within the apparatus. If this is indeed the case then the minor aberrations apparent in Figure 4.5 may also be due to the same systematic error. From this and other runs where dead volume peaks were shifted slightly (a total of 22 separate experiments), the average shift in dead volume peak position was found to be ±0.0255 when compared to the calibration value of Ve/Vo= 1. This error could be introduced into the system at a number of points; for example if a small air bubble was injected into the column along with the 50µL sample, or if a slightly different length of tubing was used to attach the column

4.2 Protein Aggregation 4 STABILITY DETERMINATION

to the FPLC machine (although this was actively avoided by attempting to use the same piece of tubing for each experiment).

With this uncertainty in mind, the gel filtration data was then used to approximate the mass of the putative dimeric state corresponding to the right hand elution peak of both Figures 4.5 and 4.6. The position of this peak was recorded over 16 identical samples to give a mean peak position of 1.25±0.04, where the uncertainty is the standard deviation across the 16 values. This is higher than the previously estimated systematic error, possibly due to there being more concordance between the elution time of large molecules that never enter the matrix, and so the actual deviation in elution time may in fact increase as molecule size decreases. Nevertheless, using this value and standard error propagation techniques [148] [149], along with the equation of the trend line from Figure 4.4b, the peaks were found to correspond to an approximate mass of 53±11 kDa. This is not the expected mass of a His6-MmyJ dimer, which should be 31.8 kDa. This has led to some speculation that His6-MmyJ may form either a trimer or tetramer (as masses of 45 kDa and 60 kDa do fall within the error boundaries of this calculated value). However, despite there being some instances of tetrameric structures being reported [150], almost all ArsR family proteins are reported to form homodimers [51], and so more precise data would be needed to confidently describe the oligomeric state of His6-MmyJ.