Role of Aromatic Residues in WhiB5 for Binding to the Primary Sigma Factor in Mycobacterium tuberculosis
Noah Mettler
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04/02/2021
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Mycobacterium tuberculosis uses Wbl transcriptional regulators to control virulent activity. This research specifically focuses on the role of WhiB5 residues, Trp12 and Phe13, in binding WhiB5 to the primary sigma factor (SigA). The importance of these residues in binding interactions was determined by the use of an in vitro pull-down assay. Furthermore, results were analyzed by spectroscopic and electrophoretic techniques.
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- [00:00:00.500]Hello, my name is Noah Mettler
- [00:00:03.020]and my project has focused on the Role of Aromatic Residues
- [00:00:06.160]in WhiB5 for Binding to the Primary Sigma Factor
- [00:00:09.500]in Mycrobacterium tuberculosis.
- [00:00:13.470]To begin, I would like to introduce my project
- [00:00:16.210]by providing details
- [00:00:17.320]on the background of mycobacterium tuberculosis
- [00:00:20.330]and Wbl transcriptional regulators.
- [00:00:22.380]Mycrobacterium tuberculosis remains a global threat
- [00:00:25.760]to public health.
- [00:00:27.200]Difficulty in treating this pathogen
- [00:00:29.220]is associated with powerful biological mechanisms
- [00:00:32.280]including the nullification of antibiotic treatments
- [00:00:35.330]and defense against immunological responses.
- [00:00:38.370]Such mechanisms are controlled
- [00:00:40.010]by a set of transcriptional regulators
- [00:00:42.010]known as the Wbl family,
- [00:00:44.250]which includes WhiB1 through WhiB7.
- [00:00:49.080]The structural and functional attributes of these proteins
- [00:00:51.610]are dependent on the binding
- [00:00:52.830]of a Four Iron-Four Sulfur cluster
- [00:00:55.160]which is visually presented in figure one.
- [00:00:58.520]Binding of Wbl proteins
- [00:01:00.070]through the primary Sigma factor, SigA,
- [00:01:02.680]is another important attribute
- [00:01:04.210]that enables transcriptional control.
- [00:01:06.630]As such interaction directs binding of RNA polymerase
- [00:01:09.300]to the promoter region.
- [00:01:11.180]My project focuses on the binding of WhiB5 to SigA
- [00:01:14.060]in particular.
- [00:01:15.390]Functionally, WhiB5 acts to regulate genes
- [00:01:17.990]associated with immunomodulation and reactivation
- [00:01:21.180]after chronic infection.
- [00:01:26.290]So what exactly is the overall goal of my project?
- [00:01:30.000]My research looked to determine what structural features
- [00:01:32.810]enable WhiB5 to bind SigA in order to perform
- [00:01:36.060]its transcriptional function.
- [00:01:38.110]As the functional properties of proteins
- [00:01:40.100]are dependent on the sequence of amino acids
- [00:01:42.320]that compose the structure,
- [00:01:43.960]I analyzed the role of two particular residues in WhiB5.
- [00:01:47.670]These residues tryptophan 12 and phenylalanine 13
- [00:01:51.520]were selected on the basis that their WhiB1 homologs
- [00:01:54.726]phenylalanine 17 and 18 have have been found to be essential
- [00:02:00.030]in binding WhiB1 to SigA.
- [00:02:02.150]The fact that these residues are aromatic
- [00:02:04.070]and play a designated role in creating
- [00:02:06.060]a hydrophobic environment to promote binding
- [00:02:09.910]between WhiB1 and SigA led me to believe that
- [00:02:12.240]WhiB5 tryptophan 12 and phenylalanine 13
- [00:02:14.920]would play a similar role.
- [00:02:20.760]This information led me to hypothesize that mutation
- [00:02:23.600]of tryptophan 12 and phenylalanine 13
- [00:02:26.020]to less hydrophobic residues, such as alanine,
- [00:02:29.340]would result in reduced binding between WhiB5 and SigA.
- [00:02:32.870]Again, this hypothesis was constructed based on the presence
- [00:02:35.980]of chemically similar aromatic residues
- [00:02:38.000]across these proteinaceous homologs.
- [00:02:41.060]Figure three shows the positioning of these residues
- [00:02:43.490]and an alignment between the predicted structure for WhiB5
- [00:02:46.937]and the experimentally result structure for WhiB1.
- [00:02:52.010]My experimental design for this project
- [00:02:53.970]consisted of three major steps.
- [00:02:56.200]First I overexpressed W12A and F13A WhiB5 mutants in E coli
- [00:03:01.400]along with WhiB5.
- [00:03:03.890]This is the step in which I am essentially producing
- [00:03:06.710]a large amount of the proteins that I seek
- [00:03:08.870]to isolate and analyze.
- [00:03:10.990]Next I purified wild-type WhiB5 and the mutants using
- [00:03:14.880]Ni-NTA affinity chromatography.
- [00:03:17.430]This is where I separate my mutants and wild-type proteins
- [00:03:20.090]from other cellular components
- [00:03:21.790]and conduct a pull-down assay,
- [00:03:23.770]which will demonstrate whether whether the mutants
- [00:03:25.940]are capable of binding SigA.
- [00:03:29.060]Lastly, I analyzed my results by using UV-Vis spectroscopy
- [00:03:32.990]and an SDS-PAGE.
- [00:03:34.810]UV-vis spectroscopy enabled me to determine
- [00:03:37.160]the concentration of proteins in my samples
- [00:03:39.710]while SDS-PAGE confirmed the presence of proteins
- [00:03:42.493]and demonstrated purity of the samples.
- [00:03:48.740]So here I included a concise representation
- [00:03:51.800]demonstrating the mechanism for a pull-down assay.
- [00:03:55.210]Essentially my SigA protein, which contains a 6-His tag
- [00:03:58.810]to bind the column Ligand is used as a bait.
- [00:04:02.240]This bait SigA binds the nickel matrix
- [00:04:04.810]in the into affinity column.
- [00:04:07.650]And it will also bind the WhiB5 prey.
- [00:04:10.860]If a mutation is severe enough, the WhiB5 prey
- [00:04:13.540]will not bind the SigA bait.
- [00:04:15.770]And if there is no a mutation
- [00:04:17.830]or the mutation does not affect binding, WhiB5 and SigA
- [00:04:21.260]will be alluded from the column together
- [00:04:23.240]and then both of them will show up on the SDS-PAGE.
- [00:04:28.970]As per my results, UV-Vis spectroscopy
- [00:04:31.890]presented me with evidence that illustrated reduced binding
- [00:04:34.590]between my W12A and F13A mutants.
- [00:04:38.620]Figure six shows the total concentration of protein
- [00:04:42.070]calculated from the absorbance at 280 nanometers
- [00:04:45.090]in my samples collected from the pull-down assay.
- [00:04:48.460]It is clear that the amount of protein collected
- [00:04:50.680]was significantly higher for wild-type
- [00:04:52.730]relative to mutants.
- [00:04:54.290]However, it is important to also take note
- [00:04:57.250]of the absorbance at 410 nanometers
- [00:05:01.120]to the absorbance at 280 nanometers,
- [00:05:03.774]the ratio between those two absorbances.
- [00:05:06.950]Due to the fact that the Wbl protein
- [00:05:09.170]contains an Iron-Sulfur cluster
- [00:05:11.020]that absorbs electromagnetic radiation at 410 nanometers,
- [00:05:15.460]increases in the absorbance at 410 nanometers
- [00:05:18.710]is indicative of the presence of WhiB5.
- [00:05:22.060]The lower ratios of W12A and F13A
- [00:05:24.860]demonstrate that these mutants have reduced binding
- [00:05:27.260]with SigA as they are not eluted together
- [00:05:30.290]in the final sample.
- [00:05:34.640]I performed an SDS-PAGE to confirm the mutants
- [00:05:37.460]did not effectively bind SigA and elute together.
- [00:05:40.740]As you can see in figure seven,
- [00:05:42.380]a band approximating 15 kilodaltons
- [00:05:46.220]which is the molecular mass of WhiB5
- [00:05:49.060]only shows prominence for the wild-type sample.
- [00:05:51.730]While all these samples contain SigA
- [00:05:53.690]which appears as the band round 20 kilodaltons,
- [00:05:56.615]the mutants lack WhiB5 bands.
- [00:05:59.190]This again supports the notion that the mutation
- [00:06:01.730]of these aromatic residues reduces the ability
- [00:06:04.360]of WhiB5 to bind SigA.
- [00:06:06.640]I would also like to note that there appeared
- [00:06:08.840]to be partial protein contamination
- [00:06:10.700]on the SDS-PAGE gel.
- [00:06:16.890]In conclusion, it was found that the mutation
- [00:06:19.300]of tryptophan 12 and phenylalanine 13
- [00:06:21.810]to a less hydrophobic residue,
- [00:06:23.660]reduces the ability of WhiB5 to bind SigA.
- [00:06:26.790]This conclusion is supported by the results presented
- [00:06:29.940]with UV-Vis spectroscopy as the protein concentrations
- [00:06:33.410]in the 410 to 280 ratio were lower
- [00:06:35.800]for the W12A and F13A mutants.
- [00:06:39.770]Additionally, the lack of bands on the SDS-PAGE
- [00:06:42.280]further confirms this finding.
- [00:06:44.210]These results do support my original hypothesis
- [00:06:46.830]pointing to the likelihood of a hydrophobic environment
- [00:06:50.010]contributing to successful binding between WhiB5 and SigA.
- [00:06:54.790]It is probable that tryptophan 12 and phenylalanine 13
- [00:06:58.190]are major contributors to the environment,
- [00:07:03.067]playing a similar role to phenylalanine 17 and 18 and WhiB1.
- [00:07:07.450]Overall findings such as these are important
- [00:07:09.940]as they may enable targeting of structural features
- [00:07:13.360]unique Wbl proteins that can help to reduce freelance
- [00:07:17.180]in mycobacterium tuberculosis.
- [00:07:19.630]Drug therapies could be developed
- [00:07:21.240]to obstruct transcriptional functions
- [00:07:23.150]in order to inhibit antibiotic resistance mechanisms,
- [00:07:26.340]immunomodulation or various other contributors to variance.
- [00:07:33.050]I would like to end by acknowledging my faculty mentor,
- [00:07:35.810]Dr. Limei Zhang, the Zhang lab as a whole
- [00:07:38.640]and the UNL UCARE Program.
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