Labeling Membrane Proteins Using Proximity-Induced or Affinity-Guided Approaches to Identify New Receptors
Makayla Gill
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04/05/2021
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This is a poster presentation for a project completed through the UCARE Program. The goal of this project is to develop methods in which the receptor proteins can be labeled in living cells with the membranes still intact. We aim to develop and implement new chemical reactions to form covalent bonds between ligands and their receptor proteins. Our strategy utilizes a chemical reaction called azo-coupling, which allows for the formation of bonds between ligands and the tyrosine amino acid residue, which is abundant in protein binding sites. Our project hopes to prove that this proposed chemical reaction is feasible, the transfection of our created labeling reagent occurs in proficient abundance, and the labeling reagent only binds to the intended protein receptors.
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- [00:00:00.960]Hello. My name is Makayla Gill.
- [00:00:03.330]I'm finishing the third year of my bachelor of science in chemistry at the
- [00:00:06.690]university of Nebraska Lincoln. And my project for the UCARE 2020 2021.
- [00:00:11.610]Academic term is titled "Labeling Membrane Proteins Using Proximity Induced or
- [00:00:15.990]Affinity Guided Approaches to Identify New Receptors."
- [00:00:19.980]This project was conducted through the Checco Research Group led by Dr.
- [00:00:23.340]James Checco in the Department of Chemistry at the University of Nebraska
- [00:00:26.850]Lincoln.
- [00:00:27.840]Sheryl Sharma was the UNL graduate student who trained me and guided me during
- [00:00:31.650]the project.
- [00:00:32.820]I'll start with some background. Cell-to- cell communication is a major focus of
- [00:00:37.410]chemical biology research.
- [00:00:39.390]Cell surfaces contain an abundance of membrane-bound proteins that play an
- [00:00:43.050]important role in these interactions.
- [00:00:45.600]A portion of these transmembrane proteins act as receptor molecules by binding
- [00:00:49.890]to a variety of Ligands extracellularly.
- [00:00:51.780]Understanding the ligand receptor binding interactions can be,
- [00:00:55.770]can provide useful insight to the field of pathology.
- [00:00:59.520]Transmembrane proteins make up to 30% of the total genome and are difficult to
- [00:01:03.750]identify due to their low abundance and low aqueous solubility.
- [00:01:08.310]Neurotensin is a 13 amino acid neuropeptide that functions as both a
- [00:01:12.000]neurotransmitter and a hormone through the activation of the neurotensin
- [00:01:15.240]receptor NTSR1, which is a GPCR, GPCRs,
- [00:01:20.250]or G protein.
- [00:01:21.420]Coupled receptors are the largest and most diverse group of membrane receptors.
- [00:01:25.470]in eukaryotes.
- [00:01:27.210]Neurotensin was chosen as the initial neuropeptide to explore because its
- [00:01:30.960]structure and the structure of its membrane bound receptor are known and easy to
- [00:01:35.730]produce. NTSR1 can be expressed in an active form in E. coli,
- [00:01:40.770]be purified, and analyzed in vitro,
- [00:01:43.680]which means it can be analyzed outside of a living organism. While this
- [00:01:48.000]hypothesis is not limited to neurotensin,
- [00:01:50.760]That is the initial peptide chosen for the first few fundamental stages of this
- [00:01:54.450]project and it is used for experiments discussed later in this presentation,
- [00:01:59.100]the neurotensin amino acid sequence and structure have been provided.
- [00:02:04.650]This research study will uncover a better understanding of how to identify
- [00:02:07.770]transmembrane proteins,
- [00:02:09.330]a protein that is integral to the membrane and spans across the entire surface
- [00:02:12.840]of the cell. While
- [00:02:14.250]most possible membrane sequences were identified when scientists sequence the
- [00:02:17.720]human genome,
- [00:02:18.540]we haven't been able to fully understand all the ligand receptor interactions
- [00:02:22.110]that occur in living systems.
- [00:02:24.360]There are plenty of proteins in ligands with unidentified purposes,
- [00:02:27.840]which we aspire to explore.
- [00:02:30.060]The goal of this project ... is to develop methods in which the
- [00:02:33.540]receptor proteins can be labeled in living cells with the membrane still intact.
- [00:02:38.370]However,
- [00:02:38.790]there are many obstacles to maneuver around such as the fragility of living
- [00:02:42.030]cells and irregular pH conditions and temperatures.
- [00:02:45.750]Our goal is to develop and implement new chemical reactions to form covalent
- [00:02:49.410]bonds between ligands and the receptor proteins.
- [00:02:53.550]Our strategy utilizes a chemical reaction called azo-coupling,
- [00:02:57.240]which allows for the formation of bonds between ligands and the tyrosine amino
- [00:03:00.910]acid residue, which is abundant in protein binding sites.
- [00:03:04.570]This project hopes to prove that this proposed chemical reaction is feasible.
- [00:03:08.560]The transfection of our creative labeling region occurs in proficient abundance
- [00:03:13.030]in the ... labeling region only binds to the intended protein receptors.
- [00:03:17.740]This is all diagrammed in figure one in the top middle of the poster.
- [00:03:23.170]Most of my time spent in the lab was dedicated to the synthesis and purification
- [00:03:26.740]of neurotensin analogs.
- [00:03:28.480]The synthesis of peptides was ... conducted in two different ways,
- [00:03:32.320]manual synthesis or use of the automated solid phase peptide synthesizer.
- [00:03:37.180]After the peptides were synthesized,
- [00:03:38.920]peptides were purified via the Agilent prep,
- [00:03:41.020]high performance liquid chromatography instrument,
- [00:03:43.780]or HPLC individual methods on the HPLC were developed for each analog
- [00:03:48.700]synthesized in order to separate identify and quantify the peptides properly.
- [00:03:53.830]Any necessary fractions from the HPLC were then collected, frozen, and lyophilized.
- [00:03:59.200]Lyophilized peptides were analyzed via matrix assisted laser resorption
- [00:04:03.370]ionization,
- [00:04:04.450]or MALDI to confirm proper synthesis and identify errors or contamination.
- [00:04:10.750]Concentration of peptide samples was found either through finding the mass after
- [00:04:14.770]a second round of lyophilization or calculated using a UV-Vis
- [00:04:18.690]spectrophotometer at a determined wavelength.
- [00:04:22.240]Once the concentration of the stock samples was known,
- [00:04:25.030]I was able to use the analytical Agilent HPLC for purification,
- [00:04:30.340]the synthesis of the neurotensin analogs is a preliminary step to any further
- [00:04:34.150]experimentation. In addition to peptide synthesis,
- [00:04:37.180]things I been working on in the lab that are integral to the experimental
- [00:04:40.210]procedure, include handling the Chinese Hamster Ovary K1,
- [00:04:44.170]or CHO-K1 cells such as passaging the cells
- [00:04:48.550]transfection of the cells in vitro incubation, et cetera.
- [00:04:52.720]SDS page in Western blotting will be able to indicate the presence of the
- [00:04:56.110]protein we're trying to identify at the time.
- [00:04:58.840]Assays are analytical in vitro procedures, used to detect, quantify,
- [00:05:02.710]and study the binding or activity of a biological molecule such as an enzyme.
- [00:05:07.630]Well, biological activity is important to consider throughout our experiment.
- [00:05:11.140]The priority is to ensure that binding is occurring.
- [00:05:15.940]Figure one.
- [00:05:16.510]Shows the HPLC confirming the formation of an azo-bond between the neurotensin
- [00:05:20.920]analog and phenol. The reaction was performed at a pH of approximately seven
- [00:05:25.780]and at room temperature formation of a new peak at 350 nanometer wavelength
- [00:05:30.760]was observed at one and a half hours.
- [00:05:33.730]And the reaction was almost complete at six hours.
- [00:05:38.230]Figure two shows a Western blot that confirms the formation of a covalent bond
- [00:05:41.740]between neurotensin and the neurotensin receptor.
- [00:05:45.490]The experiment was carried out using our neurotensin D 5 analog by Dr.
- [00:05:49.660]James Checco. In lane one of the anti-FAM blot, the top row,
- [00:05:54.610]there was a covalent bond observed additional confirmation of the covalent bond
- [00:05:58.580]between neurotensin and its receptor was provided by the absence of that band in
- [00:06:02.840]the control lanes, where we didn't expect there to be covalent bonding.
- [00:06:06.620]The low levels of NTSR1 in Lanes 1 and 3
- [00:06:09.740]compared to link four of the anti-NTSR1 blot hint towards the
- [00:06:14.270]increased internalization of the receptor and the presence of an agonist,
- [00:06:18.650]which means that the receptors are moving from the plasma membrane to the inside
- [00:06:22.250]of the cell.
- [00:06:23.870]Figure three shows a Western blot that suggests the reduction in azo-coupled
- [00:06:27.470]bond between neurotensin and the neurotensin receptor.
- [00:06:31.820]The experiment in Western blot were performed by Sheryl Sharma,
- [00:06:35.450]as you can see in lanes one and two NTSR1 is well expressed reduction in
- [00:06:39.860]the intensity of the band containing sodium dithionate is observed,
- [00:06:43.910]and the identity of the covalent bond observed maybe the hypothesized azo-coupled
- [00:06:48.420]bond,
- [00:06:50.450]some conclusions we're able to draw based on figure two, three,
- [00:06:54.710]and four is first the proposed chemical reaction,
- [00:06:58.040]the modification with your aryl-diazonium salt works
- [00:07:03.220]Second,
- [00:07:03.880]There is the formation of an azo-bond between neurotensin and the phenol.
- [00:07:08.080]And third, there is a covalent bond between neurotensin and NTSR1,
- [00:07:12.220]which may be the hypothesized azo-coupled bond. In the future,
- [00:07:16.990]we will apply our findings to new peptides chosen for their important
- [00:07:21.070]similarities to neurotensin. Fortunately,
- [00:07:23.830]I will be continuing this project this summer and during the academic year with
- [00:07:27.130]Chekhov research group, until my graduation in May of 2022,
- [00:07:31.390]I will be continuing this project through the UCARE program,
- [00:07:33.880]as well as continuing it as a university honors program senior thesis. Below,
- [00:07:38.380]I have listed all the references I used to build the background section of
- [00:07:41.500]this presentation. This work was supported in part by gifts from the Pepsi,
- [00:07:46.270]Quasi Endowment and Union Bank and Trust through the UNL UCARE program.
- [00:07:51.040]Special thanks to Sheryl Sharma, Dr.
- [00:07:53.200]Checco, and the Checco Research Group for their continued support and guidance
- [00:07:56.920]throughout this project. Thank you for listening.
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