Identification and Characterization of Novel Iron-Sulfur Proteins in Mycobacterium tuberculosis

Amber Gadeken Author

08/02/2021 Added

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Analysis of Fe-S proteins and Biosynthesis proteins in M. tuberculosis.

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[00:00:00.660]Hello, and welcome to my summer 2021 UK research presentation.
[00:00:04.770]My name is Amber catechin and I conducted research the university of Nebraska
[00:00:08.430]Lincoln with funding from UCare under the mentorship of Dr. Lee majoring. Today,
[00:00:12.840]I will be presenting over my summer research project identification and
[00:00:16.260]characterization of novel iron sulfur proteins in microbacterium tuberculosis.
[00:00:20.940]My main focus for this research was identifying uncharacterized iron sulfur
[00:00:24.480]proteins and proteins involved in iron sulfur,
[00:00:26.610]biosynthesis and microbacterium tuberculosis,
[00:00:29.520]iron sulfur proteins are proteins that have the ability to bind to iron silver
[00:00:32.970]clusters,
[00:00:33.510]which are made by leaking sulfide ions two to three or four iron CAD ions in an
[00:00:37.740]assembly train of proteins that will synthesize a cluster of iron and sulfur.
[00:00:41.850]These clusters bind to iron sulfur proteins because the proteins have multiple
[00:00:45.300]high metal affinity, amino acids positioned close to each other.
[00:00:48.990]The figure on the right shows how these amino acids can be positioning to bind
[00:00:52.680]the iron sulfur clusters because iron sulfur clusters have a high rate offs
[00:00:56.700]potential. They have the ability to transfer electrons,
[00:00:59.370]which also allows proteins that bind to iron sulfur clusters to play unique and
[00:01:03.090]essential redox roles in cells,
[00:01:05.100]such as substrate binding and some activity and regulating gene expression.
[00:01:09.210]The figure on the left shows all the functions from our sulfur proteins.
[00:01:12.900]I have studied over the summer.
[00:01:14.580]The essential functions mentioned before fall under the intermediary metabolism
[00:01:18.690]and respiration, which make up the majority of iron sulfur proteins.
[00:01:22.770]These essential proteins are required for almost all organisms,
[00:01:25.710]including humans and pathogens like microbacterium, tuberculosis to survive.
[00:01:30.150]This is why research into potential iron sulfur proteins and iron sulfur
[00:01:33.420]biosynthesis is so important,
[00:01:35.340]but this research also is difficult because iron sulfur clusters and therefore
[00:01:39.450]iron sulfur proteins are known to be unstable.
[00:01:42.360]The iron sulfur clusters and iron sulfur proteins are stable under anaerobic
[00:01:45.990]conditions,
[00:01:46.800]which means if they are exposed to auction that iron sulfur clusters will be
[00:01:51.450]lost and therefore the protein will become non-functional.
[00:01:54.750]This makes it difficult to recognize iron sulfur proteins,
[00:01:57.330]which are more likely to be considered other types of metal binding proteins
[00:02:00.540]like zinc binding. If they are studied in aerobic conditions,
[00:02:03.960]when October proteins are recognized,
[00:02:05.850]it is still challenging to study and characterize their function and structure.
[00:02:09.960]But this also means there's a significant gap in research surrounding this
[00:02:14.730]summer. I narrowed my focus on potential irons over proteins in the pathogen,
[00:02:18.270]microbacterium, tuberculosis,
[00:02:20.100]and proteins that are part of the biosynthesis of iron sulfur clusters.
[00:02:23.580]I based my research off of a list of 50 genes that are found in tuberculosis and
[00:02:27.660]propose to be iron sulfur binding,
[00:02:29.370]which I cross-reference with other putative iron sulfur NIR.
[00:02:32.250]So for biosynthesis proteins,
[00:02:34.290]I compiled 109 potential iron sulfur and iron sulfur related proteins to analyze
[00:02:38.670]further.
[00:02:39.540]My goal was to find a central iron sulfur and iron sulfur for biosynthesis
[00:02:42.720]proteins that had no significant research into function or a developed structure
[00:02:46.770]from the original 109 potential iron sulfur proteins.
[00:02:50.100]78 of them were well studied already.
[00:02:52.470]I analyzed the amino acid sequences of the remaining 31 fugitive iron sulfur
[00:02:56.640]proteins by comparing their amino acid sequence acid sequences with genes from
[00:03:00.910]other bacteria in a database called blessed from these home logs.
[00:03:04.570]The database showed I was able to determine a potential function and identify if
[00:03:08.530]a protein was likely or unlikely to be iron sulfur binding.
[00:03:11.980]I'd also look for any research into the function and structure of these
[00:03:14.860]homologues to discern whether the punitive function of the target protein was
[00:03:18.610]already well understood.
[00:03:20.260]Nine of the 31 putative iron sulfur proteins had a highly conserved Hom log.
[00:03:24.700]That was well understood.
[00:03:26.200]I then use databases called Swiss model alpha fold and wrapped directs to
[00:03:29.860]compare the potential 3d structures of my target proteins with the 3d
[00:03:34.480]models of their homologues. I looked for clusters of sixteens in history,
[00:03:38.170]deans that are known to bind iron sulfur clusters of the 22 remaining
[00:03:42.850]target proteins.
[00:03:43.690]There were nine fugitive Oriental for proteins that I found could not find iron
[00:03:47.260]sulfur clusters because they did not have the correct amino acid alignment.
[00:03:51.010]Once I had a putative function and structure based on homologs editor conserve
[00:03:54.770]domain search through blast and compared the conserve family domains with
[00:03:58.270]structures of the generated rector model and the Swiss model.
[00:04:01.000]Homologues I also compared the functions of the domains with the functions of
[00:04:04.720]any blast Han logs to connect a putative function or family of protein with
[00:04:08.650]every putative potential for protein. With this information,
[00:04:11.830]I narrowed my focus to only a few are sulfur proteins,
[00:04:14.860]and I am so for biosensors proteins that were very intrigued.
[00:04:18.160]These proteins are shown in figure one,
[00:04:19.900]along with their putative function family,
[00:04:21.910]and whether they are related to biosynthesis of iron silver clusters,
[00:04:25.360]one iron sulfur for protein not involved in biosynthesis is RV 3, 2, 4, 2.
[00:04:30.550]This protein was labeled as a nitrogenous and their original paper. However,
[00:04:34.000]all of the hallmarks of this protein point to it being a foster or a Basile
[00:04:37.300]transfer race,
[00:04:38.350]I was inclined to agree until I started looking into the function of known
[00:04:41.470]phospholipid Basile transferases and realized there were not any documented iron
[00:04:45.610]sulfur cluster binding proteins in that family figure two shows the punitive
[00:04:49.330]iron sulfur cluster binding site on [inaudible] it's four sustains
[00:04:54.250]all line in a way that would allow the iron sulfur cluster to bind to it,
[00:04:57.820]meaning it is very likely an iron tool for protein.
[00:05:00.760]This is very intriguing and will need further research to understand what this
[00:05:03.850]difference may mean to the function of the protein and others as well.
[00:05:07.390]Another protein that was identified through my analysis was not an iron sulfur
[00:05:10.720]protein, but rather an iron sulfur biosynthesis protein called [inaudible],
[00:05:14.800]which is not found in human cells, but is found in microbacterium tuberculosis.
[00:05:18.850]SFS is part of the SEF opera on which consists of six SEF proteins that make
[00:05:23.200]iron sulfur clusters to give to ISO for proteins in an iron sulfur biosynthesis
[00:05:27.940]pathway as displayed in the figure on the right Steph S in particular
[00:05:32.770]changes L 16 to L alanine and sulfur,
[00:05:35.650]which will then be bound to iron by other proteins in the opera to make iron
[00:05:39.640]sulfur clusters. There are two other known biosynthesis opera,
[00:05:43.390]and besides the SAF opera, the Isaac opera,
[00:05:46.010]and found in human cells and microbacterium tuberculosis,
[00:05:49.000]and the NIF opera not found in human cells or microbacterium tuberculosis Cephus
[00:05:53.680]is so intriguing because it is one of the main differences separating separating
[00:05:57.590]the Steph opera from the Isaac opera.
[00:06:00.110]These two opera ons are very similar and for the most part perform the same job,
[00:06:03.980]but the Isaac opera is known to function as a housekeeping iron sulfur cluster
[00:06:07.670]assembly chain.
[00:06:08.660]Whereas the SEF opera and only operates when there is a low amount of iron in
[00:06:12.080]the cell as seen in the figure on the left,
[00:06:14.630]SFS is thought to work by transferring its sulfur product to E which then passes
[00:06:19.340]it further on to the rest of the opera and to make the ISO for cluster. However,
[00:06:23.450]I'm Isaac S catalyzes the same reaction CFS does while also transferring the
[00:06:28.410]sulfur product to the rest of the opera. There's little known about why has,
[00:06:32.270]has to form a complex with stuff E in order to function like as a guest does,
[00:06:37.190]which makes it interesting for further research?
[00:06:39.680]Once I had identified CFS as a protein of interest,
[00:06:42.440]I started cloning Cephus and Sophie for future comparison to Isaac guests using
[00:06:47.180]a method called a preliminary chain reaction PCR.
[00:06:50.210]I amplified the gene in coding CFS, and Seth,
[00:06:53.210]and then a new the primers to both Seth S and Sufi.
[00:06:56.210]I checked my PCR product under UV light, and the Agros gel shown in figure two,
[00:07:01.040]after I'd amplified both genes,
[00:07:02.630]I then purified the plasma that the genes were going to be cloned into called
[00:07:05.960]pet 19 B and digested it with the enzymes NDE one and X H O one.
[00:07:11.450]I then ligated my gene into the plasma and the primer sealed the ends together.
[00:07:15.800]The final product looks like the plasma and shown in figure two.
[00:07:18.980]I then transform the plasma into [inaudible] cells and grew them on
[00:07:23.960]an Agra plate with ampicillin.
[00:07:25.850]I sent the transformed colonies of Ecole I for sequence analysis to make sure my
[00:07:29.300]gene did not mutate throughout the process.
[00:07:31.310]And when the sequence was confirmed to be correct,
[00:07:33.230]I really transformed and made frozen cultures at both Cephus and stuffy for
[00:07:36.860]future overexpression and purification.
[00:07:39.260]I then re transformed Isaac S for my culture that was previously cloned and
[00:07:43.010]transformed in the Jang lab.
[00:07:44.660]I overexpressed the Ecola cells and purified the protein from other DNA and
[00:07:48.380]protein within the cells. I ran an SDS page,
[00:07:51.110]seen in figure three to make sure my protein in my sample,
[00:07:55.250]and to examine if there were other contamination in the sample,
[00:07:58.730]my protein can be seen in the thick band in column B and C.
[00:08:02.900]I am currently overexpressing and purifying Seth S and we'll start
[00:08:06.740]overexpressing and purifying Stephie. In the coming month,
[00:08:09.770]I plan to run an assay with the protein collected,
[00:08:11.870]to test the enzyme activity between the SEP S sappy complex and Isaac S
[00:08:16.880]as well as continuing my research into putative, iron, sulfur, proteins,
[00:08:20.450]and iron. So for biosynthesis protein,
[00:08:23.060]that is the conclusion of my presentation. I hope you all enjoyed it.
[00:08:26.360]I would like to acknowledge the amazing mentorship under Dr. Lee,
[00:08:30.080]as well as the members of the, in the Jane lab.
[00:08:32.750]I look forward to answering any questions.

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Identification and Characterization of Novel Iron-Sulfur Proteins in Mycobacterium tuberculosis

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