BASIL2022GV3R8E
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| - | =Characterization of the 3r8e Protein, a Novel Glucose Kinase | + | =Characterization of the 3r8e Protein, a Novel Glucose Kinase= |
<StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''> | <StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''> | ||
== Abstract == | == Abstract == | ||
| - | The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that | + | The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, coupled kinase activity assays, and SDS PAGE analyses were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Previous and subsequent analysis of protein 3r8e validated our initial in silico and in vitro results. Overall, we have strong preliminary evidence that our protein of interest (POI) is a glucose kinase. |
== Introduction == | == Introduction == | ||
As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions. | As apart of a research project under the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) consortium, our group was tasked with characterizing and identifying the function of this protein to provide further insight of the protein's relationship to the bacteria. Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Previous research has shown that there is relationship between our POI and bacteria found in soil. Current research techniques have made the role more apparent and below is the general workflow detailing how we generated our conclusions. | ||
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| - | From here, we were able to form the conclusion that our POI interacts with glucose based on the alignment with a known hexokinase. To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. Along with the PyRx docking, we visualized | + | From here, we were able to form the conclusion that our POI interacts with glucose based on the alignment with a known hexokinase. To validate that glucose actually binds and interacts with our protein of interest, we conducted a PyRx in silico docking experiment with a total of five hexose substrates. Other substrates tested include fructose, galactose, lactose, and ribose, however, experimental in silico docking results for those substrates were significantly less than glucose. Along with the PyRx docking, we visualized <scene name='90/904995/Glucose_and_atp/1'>ATP and glucose</scene> within the proposed active site in the PyMol visualization software tool. We also were then able to find which active site amino acid were crucial to binding, which are highlighted <scene name='90/904995/3r8e_amino_acids_updated/3'>here</scene>. The binding affinity of glucose was -5.1 kcal/mol, which strengthens our idea that glucose is phosphorylated by our protein of interest. The confidence behind our in silico results allowed us to move into testing our hypothesis in vitro and because ATP aids in the phosphorylation of glucose, an <scene name='90/904995/3r8ec_w_glc_and_atp/1'>interactive structure</scene> has been provided to represent interactions of glucose and ATP in the active site. |
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Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization. | Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization. | ||
| - | [[Image: | + | [[Image:SA_1.png|400px|]] |
For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein. | For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein. | ||
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After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. | After validating our results, we now look to take our findings to a micropublication website for undergraduate research. By doing this, not only will our work be forward facing and available to the science community, but it also allows for collaboration and further questions to be asked. After completing the micropublication, we look to continue to develop research strategies for putative kinases, as the PDB has thousands of proteins with unsolved functions. We will do this by combining machine learning, data science, and lab work to allow undergraduate students and scientist to effectively research and study putative kinase structures and functions. | ||
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== References == | == References == | ||
<references/> | <references/> | ||
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6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC. | 6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC. | ||
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| + | [[Category: BASIL]] | ||
Current revision
Characterization of the 3r8e Protein, a Novel Glucose Kinase
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Dalton Dencklau, Michel Evertsen, Bonnie Hall, Jaime Prilusky
