ADMINISTRACIÓN DE ORE CONTROL

Site directed mutagenesis was carried out on the mmfR gene to create mutants with either Y84 or Y144 converted to a phenylalanine residue in the MmfR protein using the Agilent QuikChange Lightening Site Directed Mutagenesis Kit. Figure 4.15 shows details of which atoms from the MmfR tyrosine residues are involved in MMF binding and how this will be effected in the phenylalanine mutants.

As can be seen in this figure and Figure 4.11, it is the OH group of Y85 which interacts directly with the MMF molecule and the NH2 group of Y144. The NH2 group will still be present in the same position for the Y144F mutant. The conversion of tyrosine to phenylalanine will also not considerably change the size of the binding pocket and so it is expected that little change will be seen from this mutation. The NH2 group will be present in all amino acids so it would be very hard to create a mutant that will properly check for the function of Y144 in ligand binding. The Y144F mutant therefore worked as a negative control compared to the results from the Y85F mutant. In the Y85F mutants, it is expected that a more considerable effect on ligand binding will be seen due to the absence of the key OH group. In this Y85F mutant, the similarity in size between tyrosine and phenylalanine and the presence of the benzene ring should minimize the effects of changing the size and conformation of the binding pocket, allowing the analysis of just the hydroxyl group and its role in ligand binding.

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Figure 4.15. Schematic highlighting the hydroxyl and amine groups of tyrosines 85 and 144 from MmfR involved in MMF binding

This schematic is based on the crystal structure of the MmfR/MMF2 complex. (73)

Once the mmfR sequence was mutated in E. coli to now code for phenylalanine instead of tyrosine, the sequence was inserted into the pCC4 vector where the wild type mmfR was normally inserted, creating pKMS85 and pKMS144. This could then be integrated into the

Streptomyces coelicolor M145 genome along with the L1 luxCDABE vector, allowing the

mutants to be analysed using the luciferase reporter assay. This assay was carried out as with the wild type MmfR and a final concentration of 100 µM or 200 µM MMF was added to cultures and the luminescence produced compared to no MMFs measured. The results collected from the initial trials with the Y85F and Y144F mutants can be found in Figure 4.16 and Figure 4.17. Figure 4.16 shows the luminescence produced at five time points over 72 hours by L1+WTmmfR,1 _{L1+mmfR Y85F and L1+mmfR Y144F in the presence and absence} of MMF4.2 _{Figure 4.17 is a bar chart of data from Figure 4.16 at the 48-hour time point only.} A t-test analysis of data can be found in Table 4.5 with L1+WTmmfR and 100 µM MMF4 being compared to the Y85F and Y144F mutants with MMF4 to look for significant differences in ligand binding properties.

1 _{This is the same strain as was referred to earlier as simply L1+mmfR.}

Figure 4.16. Luminescence produced by the lux operon when under the control of mmfLp and MmfR tyrosine mutants compared to wild type MmfR, in the presence and absence of MMF4

Luminescence produced is calculated as a ratio of the luminescence produced by the L1+WTmmfR with no MMFs. Strains used: L1+WTmmfR, L1+mmfR Y85F and L1+mmfR Y144F – luxCDABE under the control of mmfLp (L1) and mmfR under the control of

ermEp* producing either the wild type MmfR, a Y85F mutant or a Y144F mutant (pKMS01, pKMS85 and pKMS144 respectively).

Figure 4.17. Boxplot of luminescence produced by the lux operon when under the control of mmfLp and MmfR tyrosine mutants, compared to wild type MmfR measured in the presence of MMF4 at 48 hours

Luminescence produced is calculated as a ratio of the luminescence produced by the L1+mmfR with no MMFs, meaning that the luminescence produced by this strain has a value of one. Error bars are shown as the standard deviations of data with all data points collected also shown on the chart. Strain used: same as Figure 4.7

0 10 20 30 40 50 60 70 80 0 2 4 6 8 10 12 14 Time (hours) Ratio compared to WT MmfR WT+100 µM MMF4 Y85F+100 µM MMF4 Y85F+200 µM MMF4 Y144F+100 µM MMF4 WT noMMF Y85F no MMF Y144F no MMF

WT+100 µM

MMF4 Y85F+100 µM MMF4 Y85F+200 µM MMF4 Y144F+100 µM MMF4 0.0 0.5 1.0 1.5 2.0 2.5

Ratio of luminescence produced

Induction of luminescence in tyrosine mutants compared to WT MmfR with MMF4 at 48 hours

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Table 4.5. A t-test analysis luminescence produced by luxCDABE when under the control of mmfLp and the Y85F and Y144F MmfR mutants in the presence of 100 µM or 200 µM MMF4 to look for significant differences in the removal of repression

Strain used p-value _difference? Significant compared with L1+WTmmfR Average induction at 48 hr and 100 µM MMF4 (R.R.) Y85F and 100 µM MMF4 1.60E-5 YES 0.576 Y85F and 200 µM MMF4 2.30E-1 NO 0.865 Y144F and 100 µM MMF4 3.36E-2 YES 1.284

The Y85F strain did show a lower level of de-repression by MMF4 compared to the wild type strain. Only at twice the level of MMF4 did the Y85F strain produce statistically similar levels of induction to those seen in the wild type. As predicted, the Y144F mutation did not cause a significant reduction in release of MmfR by MMF4. Interestingly it did actually appear to produce significantly higher levels of luminescence upon the addition of 100 µM MMF4, with more than 125% of luminescence of the wild type strain with MMF4 at 48 hours (see Figure 4.17). Whether this mutation has indeed ‘optimised’ the binding pocket and made it more sensitive is as yet unclear but would be an interesting idea to investigate further in the project. It was an artefact also identified by Shanshan Zhou when running an in vitro gel shift assay, further indicating that a version of MmfR with improved ligand binding has indeed been produced.

Another observation from Figure 4.16 is that neither the Y85F or Y144F mutants appear to be as good at repressing luminescence as the wild type MmfR at the mmfLR intergenic region with both L1+mmfR Y85F and L1+mmfR Y144F producing higher levels of luminescence L1+WTmmfR in the absence of the MMFs. Y144F appears to be a better repressor than Y85F however. Although the mutations were to the ligand binding pocket, they are close to the dimer interface and so could be also effecting the overall structure of the protein and therefore its DNA binding properties.

Data from Figure 4.17 in particular, reveals large standard deviations and huge overlaps in the error bars. A t-test did show that the variation in the different sets of results were statistically significant but it is unclear whether from this data alone, reliable conclusions can be made about the activities of the mutant MmfRs compared to the wild type. For this reason it was decided that a range of concentrations of MMF4 would be trialled and the Bmax and Kd values derived in the hope of achieving some more distinct differences between samples. To obtain the Bmax and Kd values, MMF4 was added at the same ranges of concentrations as were added to the wild type MmfR strains in Section 4.4.2.

The standard curves collected for MMF4 binding to L1+mmfR Y85F and L1+mmfR Y144F compared to earlier data collected for L1+WTmmfR with MMF4 are shown in Figure 4.18. Using this data, the Bmax, Kd and binding potential of MMF4 to each of the mutants was calculated. These values have been compared to those from the wild type MmfR in Table 4.6.

Figure 4.18. Standard curve for production of luminescence as a result of MMF4 binding to and releasing the wild type MmfR compared to tyrosine 85 and 144 mutants

Strain used: L1+WTmmfR, L1+mmfR Y85F and L1+mmfR Y144F – luxCDABE under the control of mmfLp (L1) and mmfR under the control of ermEp* producing either the wild type MmfR, a Y85F mutant or a Y144F mutant (pKMS01, pKMS85 and pKMS144).

0 100 200 300 400 500 0 5 10 15 20 Concentration of MMF4 (µM)

Ratio of luminescence in the presence of

MMFs compared to no MMFs

WT+MMF4

Y144F+MMF4

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Table 4.6. Binding kinetics data for MMF4 binding wild type MmfR compared to the tyrosine 85 and 144 MmfR mutants, including the Bmax, Kd and binding potential values for each at 48 hours growth

Binding potential = Bmax/Kd

L1+mmfR strain Bmax Kd (µM) Binding potential

WT and MMF4 18.3 69.42 0.26

Y85F and MMF4 2.8 37.51 0.07

Y144F and MMF4 5.8 11.13 0.52

As can be seen in Table 4.6, the binding potential of the Y85F MmfR mutant to MMF4 is much lower than the wild type binding to the same ligand. This data further confirms that the hydroxyl group of the tyrosine residue in position 85 of MmfR is likely to be necessary for ligand binding and the resultant conformational change causing its release from the MARE operator. The Y144F mutant on the other hand appears to have a binding potential of almost double that of L1+WTmmfR with MMF4. It again appears therefore that this mutation has optimised the repressor, reducing the amount of MMFs needed to achieve de-repression. Further investigation is needed to establish whether the same would be seen for all five MMFs.

It is also of note that both the Y85F and Y144F mutants have a lower Bmax than the wild type. This indicates that for both of these mutants, there were differences in the level of MmfR release from the MARE operator that can be achieved the presence of the MMFs at a saturating concentration. The differences in DNA binding properties of the mutants is still poorly understood however.