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

Amber Gadeken Author

08/02/2021 Added

2 Plays

Description

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

Searchable Transcript

Search:

[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.

The screen size you are trying to search captions on is too small!

You can always jump over to MediaHub and check it out there.

Comments

0 Comments

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

Description

Searchable Transcript

Comments icon comment

Related Channels

Comments