Proteopedia - User contributions [en]

BASIL2022GV3R8E

2026-03-21T21:48:59Z

Bonnie Hall:

=Characterization of the 3r8e Protein, a Novel Glucose Kinase=

== 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 our protein of interest (POI) is a glucose kinase.
== 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.

[[Image:Workflow1.png|500px|]]
== Methods ==
As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.

[[Image:3R8EDali180.png|400px|]]
[[Image:3R8EDALIHMM.png|400px|]]

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.

== Experimental Results/Function ==

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: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.

[[Image:FINAL_gel.png|250px|]]

== Project Implications ==
The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.
== Conclusions/Future Direction ==
Conclusions:

After various experiments and discussion, we have concluded that the novel protein 3R8E is a <scene name='90/904995/Mesh_backbone_with_atp_glucose/3'>Glucose Kinase</scene>. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively.

Future Direction:

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.

== References ==
<references/>
1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)

2. BASIL. https://basilbiochem.github.io/basil/

3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.

4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.

5. Pfam: The Protein families database in 2021 J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913

6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.

[[Category: BASIL]]

BASIL2022GV3R8E

2026-03-21T21:48:40Z

Bonnie Hall:

=Characterization of the 3r8e Protein, a Novel Glucose Kinase=
<
== 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 our protein of interest (POI) is a glucose kinase.
== 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.

[[Image:Workflow1.png|500px|]]
== Methods ==
As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.

[[Image:3R8EDali180.png|400px|]]
[[Image:3R8EDALIHMM.png|400px|]]

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.

== Experimental Results/Function ==

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: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.

[[Image:FINAL_gel.png|250px|]]

== Project Implications ==
The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.
== Conclusions/Future Direction ==
Conclusions:

After various experiments and discussion, we have concluded that the novel protein 3R8E is a <scene name='90/904995/Mesh_backbone_with_atp_glucose/3'>Glucose Kinase</scene>. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively.

Future Direction:

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.

== References ==
<references/>
1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)

2. BASIL. https://basilbiochem.github.io/basil/

3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.

4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.

5. Pfam: The Protein families database in 2021 J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913

6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.

[[Category: BASIL]]

Proteopedia:News

2024-03-07T21:15:51Z

Bonnie Hall:

This page is for '''external''' news reports etc. about Proteopedia. For new pages and capabilities '''within''' Proteopedia, please see [[Proteopedia:What's New]].

==Add New Items at the Top of Each Section and Date Them, Please!==

News on this page is ordered '''newest first''', oldest last, under each subheading. Please include the <font color="red">month and year</font> at the end of each entry that you add below. Subheadings below ''Statistics'' are in alphabetical order.

==Statistics==

Today, Proteopedia has {{NUMBEROFUSERS}} registered users. For more information, please see [[Proteopedia:About]]. For the number of pages, page views, edits, etc. please see [[Special:Statistics]]. The number of times any page has been viewed is displayed at the bottom of the page.

==Publications==

*''Proteopedia - a scientific 'wiki' bridging the rift between 3D structure and function of biomacromolecules'', Eran Hodis, Jaime Prilusky, Eric Martz, Israel Silman, John Moult and Joel L Sussman. [http://genomebiology.com/2008/9/8/R121 ''Genome Biology'' 9:R121, August 2008] or [http://dx.doi.org/10.1186/gb-2008-9-8-r121 doi:10.1186/gb-2008-9-8-r121]

*''Proteopedia: A status report on the collaborative, 3D web-encyclopedia of proteins and other biomolecules'', Jaime Prilusky, Eran Hodis, David Canner, Wayne A. Decatur, Karl Oberholser, Eric Martz, Alexander Berchanski, Michal Harel and Joel L. Sussman. [http://www.sciencedirect.com/science/article/pii/S104784771100116X ''Journal of Structural Biology'' 175:244-52, August 2011] or [http://dx.doi.org/10.1016/j.jsb.2011.04.011 doi:10.1016/j.jsb.2011.04.011].

==Adoptions==

===Adoptions by Structural Bioinformatics Resources===
Some bioinformatics databases and resources have ''adopted'' Proteopedia. This means that they have chosen to offer links to Proteopedia for the benefit of their users.

* [http://www.expasy.org ExPASy Proteomics Server] (EXpert Protein Analysis SYstem) of the Swiss Institute of Bioinformatics offers links to Proteopedia on its 3D structure pages, e.g. [http://www.expasy.org/cgi-bin/get-pdb.pl?1A5H PDB code: 1A5H]. October, 2008.

* [http://topsan.org TOPSAN], The Open Protein Structure Annotation Network, a wiki designed to collect, share and distribute information about protein three-dimensional structures. October, 2008.

* [http://www.pxuniverse.com/ Protein Crystallography Universe], a website provided by Rigaku Life Sciences Group, links Proteopedia under Structure Analysis and Reference Shelf. October, 2008.

* [http://www.cathdb.info CATH], a hierarchical classification of protein domain structures [Class (C), Architecture (A), Topology (T) and Homologous superfamily (H)], provides links on its PDB pages to Proteopedia. September, 2008.

* [http://www.genecards.org GeneCards], a searchable, integrated database providing concise information on all known and predicted human genes, links relevant proteins to Proteopedia. September, 2008.

* [http://bioinformatics.org/jmol-tutorials Jmol Tutorial-Authoring Template] (JTAT) automatically provides a link to Proteopedia for each [[PDB file]] illustrated in its tutorials. August, 2008. See, for example, the bottom right (''Explore further ...'' under ''How To ...'') in the [http://www.bioinformatics.org/jmol-tutorials/jtat/jtatdemo/index.htm JTAT Demo Tutorial].

* [http://www.pdb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/index.html Molecule of the Month] by [[User:David S. Goodsell|David S. Goodsell]] provides links to Proteopedia in some recent articles. An example is linked at the end of the second page of the article on [http://www.pdb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb104_1.html Selenocysteine Synthase]. August, 2008. Here is the [[Teaching_Scenes%2C_Tutorials%2C_and_Educators%27_Pages|complete list]].

* [http://oca.weizmann.ac.il OCA Database and Browser] for 3D macromolecular structure has a link from each [[Protein Data Bank|PDB entry]] to Proteopedia. August, 2008.

* [http://www.ebi.ac.uk/pdbsum/ PDBsum] has a link to Proteopedia on every PDB entry page. August, 2008.

* [http://pdbwiki.org PDBWiki] has a link to Proteopedia on every PDB entry page. August, 2008.

* [http://www.pdb.org RCSB Protein Data Bank] (see Proteopedia's page on the [[Protein Data Bank]]), has a link from each [[Protein Data Bank|PDB entry]] to Proteopedia, but it can be a bit hard to find. To find it: At the ''Structure Summary'' tab/page for a given [[PDB code]], in the menu at left, click on ''External LInks''. On that page, you'll find a link to Proteopedia under ''Structure Summary''. If you think it would be useful to have a more prominent link to Proteopedia at RCSB-PDB, please email <email>info@rcsb.org</email>. In May, 2008, an international group of several structural bioinformaticians and crystallographers requested that a link to Proteopedia replace the present ''Jmol'' link under ''Display Options'' at the upper right of the main ''Structure Summary'' page for each entry, but that request was denied. [[User:Eric Martz|Eric Martz]] 03:19, 7 August 2008 (IDT)

* [http://pfam.janelia.org/ Pfam], a large database of protein families, each represented by multiple sequence alignments and hidden Markov models, will offer links to Proteopedia beginning with Pfam release 23. August, 2008.

* [http://kb.psi-structuralgenomics.org/ PSI Structural Genomics Knowledge Base] has a link to Proteopedia, on its Annotations tab, for every [[Protein Data Bank|PDB entry]]. August, 2008.

===Adoptions in College and University Classes===
See also [[Student Projects]], [[Teaching Scenes, Tutorials, and Educators' Pages]], and [[Teaching Strategies Using Proteopedia]].

*'''Amherst College, Amherst, Massachusetts, USA'''<ref>PMID: 24019219</ref>: A one-semester biochemistry course enrolling 40-45 students. Several professors introduce students to ''Proteopedia'' and ''FirstGlance in Jmol'' in laboratory, use them in lectures, require molecular images in homework tasks, and require their use in a structure-function analysis capstone project and a presentation of their results. February, 2014.

*'''Butler University, Indianapolis, Indiana, USA''': [https://www.butler.edu/arts-sciences/chemistry-biochemistry/faculty-staff/ Jeremy Johnson] has assigned projects in Proteopedia to his students for years. March, 2024.

*'''Grandview University, Des Moines, Iowa, USA''': Bonnie Hall. March, 2024. Students in CHEM 351 Biochemistry create a Proteopedia sandbox page highlighting protein structure and function relationships based on a current journal article. In CHEM 453 Biochemical Techniques, students use the BASIL curriculum to complete an authentic research project. They then publish their results on a Proteopedia page. After their results are reviewed, their pages are made available to all users of Proteopedia (search term: BASIL).

*'''Stony Brook University, Stony Brook, New York, USA''': Biology 207 taught by [http://www.stonybrook.edu/commcms/biology/contact.html Marvin H. O'Neal III]. Students work in groups of 2-4 to create a Proteopedia Workbench page. The [[Workbenches]] mechanism is used because it keeps the student project in one student's User: space (which [[Help:Protected_Pages|protects]] the page), and enables collaboration by using the ''workbench'' tab to permit other members of the group, and the instructors, read/write access.

*'''University of Massachusetts, Amherst, Massachusetts, USA''':
**Advanced Molecular Biology (Biochem 642, for graduate students) taught by Drs. Robert Zimmermann and Alice Cheung. A two-session, hands-on workshop introducing [http://firstglance.jmol.org First Glance in Jmol] and Proteopedia's [[Scene authoring tools]] was taught by [[User:Wayne Decatur]], who prepared [[User:Wayne Decatur/Biochem642 Molecular Visualization Sessions|Biochem642 Molecular Visualization Sessions]] and [[User:Wayne_Decatur/Teaching_Proteopedia|Teaching Proteopedia]]. Students were assigned problem sets that required authoring accompanying scenes in sandbox pages. November, 2009.
**[http://people.chem.umass.edu/jhardy/BMS2008.html Biomolecular Structure (Chem 791a, for graduate students)] taught by crystallographer Jeanne Hardy. A computer lab workshop introducing the [[Scene authoring tools]] was taught by [[User:Wayne Decatur]], who prepared [[User:Wayne_Decatur/Chem791A_Work_Session|Chem791A Work Session]] and [[User:Wayne_Decatur/Teaching_Proteopedia|Teaching Proteopedia]]. November, 2008.
**[[CBI_Molecules]] has involved a number students producing excellent pages for display on the [http://www.molecularplayground.org/ Molecular Playground]. [[User:Lynmarie K Thompson|Professor Lynmarie K. Thompson]] who has been involved with this has used Proteopedia for student projects in Chemistry 423 in [[Student Projects for UMass Chemistry 423 Spring 2011|2011]] and [[Student Projects for UMass Chemistry 423 Spring 2012|2012]].

*'''University of Sao Paolo, Sao Paolo, Brazil''': Luis Netto. March, 2024.

==Press==
* [http://www.pdb.org/pdb/general_information/news_publications/newsletters/2010q4/education_corner.html PDB Education Corner], Winter 2011
* [http://www.genengnews.com/best-of-the-web/proteopedia/2496/ GENnews Best Of The Web: Proteopedia ], May, 2010
* [http://www.the-scientist.com/article/display/57181/ The Scientist: "Structure Made Simple"]. March, 2010
* [http://seedmagazine.com/content/article/scientific_truth_in_the_age_of_wikipedia/ Seed Magazine: "Scientific Truth in the Age of Wikipedia"]. February, 2009
* [http://www.f1000biology.com/article/id/1121800 Proteopedia in Faculty of 1000]. October, 2008
* [http://www.nature.com/nrm/journal/v9/n10/full/nrm2512.html Nature Reviews Molecular Cell Biology] - October, 2008 Research highlights
* [http://www.nature.com/ncb/journal/v10/n10/pdf/ncb1008-1123.pdf Nature Cell Biology] - October, 2008, Proteopedia mentioned in Editorial
* [http://genomebiology.com/pressreleases/pressrelease22August08.asp GenomeBiology.com Press Release] August, 2008

==Blogs==
* [http://edfed.info/?p=4 edfed ] Proteopedia collects and disseminates information about molecules, February 8th, 2010
* [http://molecularmodelingbasics.blogspot.com/2009/10/proteopedia.html Molecular Modeling Basics] October, 2009. A blog related to the book of the same name.
* [http://beckerinfo.net/bioinformatics/2008/12/03/open-source-visualization/ BeckerInfo.net Open Source Visualization] December, 2008
* http:// car54.wordpress.com August, 2008 (Web site not longer available)

* [http://emmetcole.wordpress.com/2008/08/22/3d-protein-encyclopedia-aids-wikification-of-science-sort-of/#more-227 Wonder: Sci-Tech Department] August 2008
* [http://network.nature.com/blogs/user/rpg/2008/03/03/from-the-damn-but-thats-cool-department The Scientist -- Richard Grant's blog on Nature Network] March 2008
* [http://wwmm.ch.cam.ac.uk/blogs/murrayrust/?p=990 Peter Murray Rust's blog] March, 2008

==Press Release Re-Posts==
* [http://memagazine.asme.org/Articles/2008/November/Computing.cfm mechanical engineering] November, 2008
* [http://www.sciencedaily.com/releases/2008/08/080822120146.htm Science Daily] August, 2008
* [http://chronicle.com/wiredcampus/article/3277/proteopedia-an-online-encyclopedia-of-interactive-3-d-macromolecules The Wired Campus] August, 2008
* [http://esciencenews.com/articles/2008/08/22/life.isnt.2.d.so.why.should.our.encyclopedias.be e! Science News] August, 2008
* [http://google-sina.com/2008/08/23/life-isnt-2-d-so-why-should-our-encyclopedias-be/ Medical Health Articles] August, 2008
* [http://in.news.yahoo.com/43/20080823/982/tsc-scientific-texts-in-3d-with-interact.html YAHOO! News, India] August, 2008
* [http://webhosting.pl/Proteopedia..pierwsza.encyklopedia.z.trojwymiarowa.zawartoscia Proteopedia – pierwsza encyklopedia z trójwymiarową zawartością] August, 2008
* [http://news.webindia123.com/news/Articles/Health/20080823/1034770.html WEBINDIA123] August, 2008
* [http://pda.physorg.com/lofi-news-information-structural-pages_138620877.html pda.physorg.com] August, 2008
* [http://story.europesun.com/index.php/ct/9/cid/2411cd3571b4f088/id/398066/cs/1/ Europe Sun] August, 2008
* [http://www.aecomunicacioncientifica.org/portal/index.php?option=com_content&view=article&id=19118&catid=39:alphagalileo&Itemid=75 AECC] August, 2008
* [http://www.alphagalileo.org/index.cfm?_rss=1&fuseaction=readrelease&releaseid=531608 AlphaGalileo] August, 2008
* [http://www.bio-medicine.org/biology-news-1/Life-isnt-2-D--so-why-should-our-encyclopedias-be-3F-4582-1/ Bio-Medicine] August, 2008
* [http://www.feedzilla.com/news-archive/industry/2008-08-22-science.html FEEDZILLA] August, 2008
* [http://www.eurekalert.org/pub_releases/2008-08/bc-li2082208.php EurekAlert!] August, 2008
* [http://www.firstscience.com/home/news/breaking-news-all-topics/life-isn-t-2-d-so-why-should-our-encyclopedias-be-page-1-1_51454.html FirstScience News] August, 2008
* [http://www.freshnews.in/scientific-texts-in-3d-with-interactive-formats-developed-57558 Latest News] August, 2008
* [http://www.iconocast.com/00009/B3/News6.htm ICONOCAST] August, 2008
* [http://www.iconocast.com/S00009/B3/News6.htm ICONOCAST Spanish] August, 2008
* [http://www.physorg.com/news138620877.html PHYSORG.com] August, 2008
* [http://www.sciencecodex.com/life_isnt_2d_so_why_should_our_encyclopedias_be Science Codex] August, 2008
* [http://www.scientificblogging.com/news_releases/proteopedia_since_life_isnt_2d_why_is_your_encyclopedia www.scientificblogging.com] August, 2008
* [http://www.thaindian.com/newsportal/uncategorized/scientific-texts-in-3d-with-interactive-formats-developed_10087726.html Thaindian News] August, 2008
* [http://www.polit.ru/science/2008/08/26/proteopedia.popup.html polit.ru] August, 2008
* [http://infuture.ru/article/1024 infuture.ru] August, 2008
* [http://www.thesmarttechie.com/fullnews.php/45774 The Smart Techie] August, 2008
* Scientific texts in 3D with interactive formats developed [http://www.siliconindia.com/shownews/45774 Silicon India] August, 2008
* Scientific texts in 3D with interactive formats developed [http://www.newkerala.com/topstory-fullnews-14498.html newKerala.com] August, 2008
* 3D interactive text formats developed, [http://www.ndtv.com/convergence/ndtv/story.aspx?Id=NEWEN20080062682&ch=633555332445438750 NDTV.com] August, 2008
* [http://www.innovations-report.de/html/berichte/informationstechnologie/life_isn_039_t_2d_encyclopaedias_proteopedia_116571.html innovations report] August, 2008
* [http://www.medicalnewstoday.com/articles/119088.php Medical News Today] August, 2008
* Online wiki hosts interactive, 3D molecular structures, [http://www.itnews.com.au/News/83288,online-wiki-hosts-interactive-3d-molecular-structures.aspx iTnews] August, 2008
* Scientific texts in 3D with interactive formats developed, [http://sify.com/news/fullstory.php?id=14745946 SiFy news] August, 2008

==Abstract Re-Posts==
* [http://www.yourlabdata.com/index.php?option=com_content&task=view&id=25246&Itemid=75 Your Lab Data] August, 2008
* [http://www.find-health-articles.com/rec_pub_18673581-proteopedia-scientific-wiki-bridging-rift-3d-structure-function.htm find-health-articles.com]. November , 2008

==Miscellaneous Postings==
* [http://www.the-scientist.com/fragments/survey/videoawards2010/voting.jsp?campaignId=12 Nominated for Best Website in the Labbies Awards] by the [http://www.the-scientist.com TheScientist.com] June, 2010
* [http://digg.com/tech_news/Proteopedia_Since_Life_Isn_t_2D_Why_Is_Your_Encyclopedia digg.com/tech_news digg/Tech News] August, 2008
* [http://www.stumbleupon.com/url/www.scientificblogging.com/news_releases/proteopedia_since_life_isnt_2d_why_is_your_encyclopedia StumbleUpon] August, 2008
* [http://www.tiede.fi/keskustelut/viewtopic.php?f=3&t=32738&start=0&st=0&sk=t&sd=a&sid=3a2616e09f985ce2c5e2e7cd5657e10f TIEDE.fi] August, 2008

==Talks in Meetings and Seminars==

* [http://hgm2008.hugo-international.org/Abstracts/Publish/Symposia/Symposium03/hgm12.html Proteopedia] - a Scientific 'Wiki' Bridging the Rift Between 3D Structure and Function of Biomacromolecules, HUGO's 13th Human Genome Meeting, September, 2008.
* In ''[http://www.sdsc.edu/pb/Talks/3Dsig.ppt I am not a PDBid I am a Biological Macromolecule]'', the keynote talk at [http://www.ebi.ac.uk/~rafi/3dsig08/Home.html 3DSig 2008, Structural Bioinformatics and Computational Biophysics] (an ISMB satellite meeting Toronto, 18-19 July 2008), [http://www.sdsc.edu/pb Philip E. Bourne] discussed removing the barrier between the literature and the PDB, featuring his [http://www.sdsc.edu/pb/Talks/3Dsig.ppt BioLit] project. In this context, he listed Proteopedia as Possibility 1, "a completely new beginning", and discussed its advantages (anyone can contribute leading to wiki quality) and disadvantages (reward for authoring? limitations of wiki format).

==Training Workshops==

*[http://workshops.molviz.org Short courses and one-day workshops] are taught by [[User:Eric Martz|Eric Martz]] on macromolecular structure visualization and structural bioinformatics. These now include a segment on Proteopedia, including use of Proteopedia's Scene-Authoring Tools. In 2008, these have been at the Weizmann Institute of Science in Israel, and at Osaka University and the Okinawa Institute of Science and Technology in Japan. For curricula and upcoming dates, please see [http://workshops.molviz.org Workshops.MolviZ.Org]. August, 2008.

==References==
<references />

BASIL2023GVP30646

2024-02-04T03:37:03Z

Bonnie Hall:

'''Characterization of Novel Xylulokinase P30646'''
<Structure load='' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />

== Abstract ==
The Protein Data Bank (PDB) and UniProt contain a wealth of protein structures, many of which do not have a known function. One such protein is P30646, an uncharacterized sugar kinase. Sugar kinases play a role in a variety of metabolic processes within the body, so understanding their functions is of great importance. Our work aimed to further characterize P30646 through the identification of a substrate for this enzyme. Utilizing materials Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, our group combined in silico and in vitro data for a full analysis of P30646. Computation tools including BLAST, DALI, SPRITE, and InterPro allowed our group to compare the structural and sequential similarities between P30646 and proteins with known functions. In silico analysis indicated that P30646 had xylulokinase activity, and xylulose was tested as a substrate in vitro using a coupled kinase assay. The kinase assay indicated that P30646 does have strong enzymatic activity for xylulose. The combination of our in silico and in vitro results provide a strong indication that P30646 is a sugar kinase with affinity for xylulose breakdown.

== Background ==
The study of protein structure and function is essential, as the structure of a protein is often closely related to its function. This project investigated the protein P30646 (UniProt), a protein with a known structure but an unknown function. It is understood that P30646 is an uncharacterized sugar kinase that is involved in the metabolic processes of Caenorhabditis elegans. Through the analysis of results obtained through computational and laboratory methods, this work aims to identify the function of P30646 and gain insight into its role in the metabolism of C. elegans.

<scene name='95/957645/P30646/4'>Click to display 3D model</scene>

'''Workflow'''

[[Image:Worklow.jpg]]

'''Predicted Mechanism'''

[[Image:Mechanisms.jpg]]

== Methods ==

To determine the function of P30646 as well as a potential substrate for the enzyme, we first conducted an in silico exploration. We utilized tools including DALI, InterPro, PredictProtein, and BLASTp to gain insight into the potential function P30646. Each of these tools draws upon data for proteins of unknown functions to generate hypotheses about the function of the protein of interest. We then identified potential substrates for our protein of interest through docking tools including SwissDock and PyRx.

The hypotheses we developed through computational tools were tested in vitro by overexpressing and purifying P30646, followed by an experimental analysis of the protein. This analysis involved a Bradford assay, a coupled kinase assay, and SDS-PAGE.

[[Image:Structure-P30646.png]]

== Computational Exploration ==
Our in silico experiments focused on first determining a potential function for our protein of interest, and then on identifying an ideal substrate. We hypothesized about a function for P30646 through structural and sequential alignments with proteins of known function. A number of similar proteins were identified, including a 4BC3 (PDB), a human D-xylulokinase. 4BC3, along with other xylulokinases, is both structurally and sequentially similar to P30646.
Results for the protein family membership of P30646, as well as a sequential alignment with 4BC3 are provided below.

A combination of all in silico data collected led us to conclude that our protein of interest is a D-xylulokinase.

[[Image:InterPro.png]]
[[Image:DALI-Alignment.png]]

Our hypothesis that P30646 is a D-xylulokinase implies that D-xylulose would be an ideal substrate for the protein. In order to test this, our group utilized SwissDock and PyRx to conduct in silico docking experiments. Other substrates tested included ethylene glycol, 5-deoxy-5-fluoro-D-xylulose, and ammonium and sulfate ions. Docking of D-xylulose to P30646 is shown below.

Both tools supported our prediction that D-xylulose was an ideal substrate for P30646, and we moved forward to test our hypotheses in the laboratory.

[[Image:Swiss-Dock.png]]

== Laboratory Experiments ==
In order to test our hypotheses in vitro, we first performed an overexpression of our protein of interest followed by purification using affinity chromatography. Our group then tested D-xylulose for its fit as a substrate for P30646. The results of a coupled kinase assay for P30646 with D-xylulose as a potential substrate are shown below.

[[Image:KinaseData.png]]

The results of our coupled kinase assay showed us that P30646 aids in the phosphorylation of D-xylulose, as demonstrated in the predicted mechanism for our protein of interest. While it is clear that D-xylulose is a potential substrate for P30646, it is unclear whether or not this is the best substrate for P30646 without further testing.

== Conclusions ==

Through a combination of the data collected in vitro and in silico, our group concluded that P30646 is a D-xylulokinase. Computational tools aided in this determination by indicating that P30646 belongs to the carbohydrate kinase family, and more specifically to the D-xylulokinase family. Alignment with similar D-xylulokinases further supported this idea.

Evidence from the coupled kinase assay led our group to determine that D-xylulose is a possible substrate for P30646. The data collected indicates that P30646 has the capability to phosphorylate D-xylulose, though the specific activity of the enzyme is lower than expected. Increased testing using a variety of carbohydrates is necessary to make further conclusions regarding D-xylulose as a substrate for P30646.

== References ==
1. BASIL. https://basilbiochem.github.io/basil/

2. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)

3. Di Luccio, Eric et al. Journal of Molecular Biology vol. 365,3 (2007): 783-98. doi:10.1016/j.jmb.2006.10.068

4. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.

5. The use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref>.

BASIL2023GVP76586

2024-02-04T03:33:04Z

Bonnie Hall:

== Investigating the Function of Protein P76586 ==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>

== The Initial Search ==
We were given a Protein with a predicted structure from Uniport and an unknown function. We are trying to find the function of the protein. We did this by first using computational tools like Blast, Dali, and Interpro to help us find potential substrates.
The first computational tool we used was Blast the results of which are shown below on Table 1. (blast figure).
From the Blast results we saw that it was from the ROK family and that it was a putative kinase.
The next tool we used was Dali shown in table 2.(Dali figure).
From the Dali results the best results we saw were from a putative kinase, glucokinase and a DNA-binding kinase. These results are what showed us that there could be potential nucleosides though we weren't for sure which one it could be until we go to docking.
The next tool we used was InterPro which can be seen in Table 3 (InterPro figure).
The InterPro figure really help us narrow down that it could be either of those to substrates because again it shown a sugar kinase and a ATPase which would mean that there is potentially a nucloeside or something to with ATP.
== Molecular Docking ==
From the initial search throughout all of the computational tools, we decided that our putative kinase was potentially a glucokinase. We docked other sugars along with glucose in figure 1.

[[Image:Sugar_substrates.png]]

Taking in consideration of the DNA binding domain found in the InterPro results we docked DNA nitrogenous bases and nucleosides, the structures of the nitrogenous bases are shown in figure 2.
[[Image:Nitrogenous_Bases_Structures.png]]

From the results of the docked sugars, shown in table 4, nitrogenous bases, and nucleosides, we determined that guanosine was a strong potential substrate but still wanted to test glucose due to the computational tools results since glucokinase was a common output in all of our searches.
[[Image:Sugar_affinities.png]]

We used Pymol to visualize the intermolecular interactions in the active site with guanosine (figure 3) and glucose (figure 4).
[[Image:Docked_guanosine.png]]
[[Image:Docked_glucose.png]]
==Structural Highlights==
<scene name='95/957646/Globular_structure/1'>P76586 is a globular protein</scene> with 397 amino acids. It's <scene name='95/957646/Secondary_structure/1'>secondary structure</scene> is made up of mostly alpha helices, but also has beta sheets and random coil. Through docking we were able to identify possible amino acids involved in the <scene name='95/957646/Active_site/1'>active site</scene> of P76586. Potential amino acids in the active site are Tyr23, Asp81, Arg183, Gln393, and Tyr389.
== Results ==
Our protein of interest has a weight of ≈44.53kD. When analyzing SDS PAGE (figure 5) we were slightly concerned we weren't working with our protein of interest. We didn't get a great image out of SDS, if there were more time we would run again with more protein in the well so that we could see it better. We also made the mistake of not including our induction samples and our fractions from protein purification using Nickle affinity chromatography. The band at 75kD and 25kD we believe are there from the imaging and is not from our actual protein sample.
[[Image:SDS.png]]

We ran two coupled kinase assays, one with glucose and one with guanosine. After calculating the specific activity of our protein with glucose and guanosine we determined there was not enough activity for glucose or guanosine to be the substrate (table 5). We didn't have to modify the kinase assay protocol for Abl Kinase other than changing the substrate and ensuring we were using a high enough substrate concentration that was greater than the protein concentration.
[[Image:Kinase_assay.png]]

== Conclusions ==
Although we determined that neither glucose or guanosine were the substrate for P76586, we are unsure if this may have been from experimental error. But looking back at the docking results I think we should have tried a wider range of substrates. Maybe a substrate that had closer hydrogen bond interactions. If we were to continue our research we would spend much more time on the docking portion in order to really dissect the active site and its interactions with the substrates. Overall this research experience was really educational and we gained a lot of skills from working in the lab and working as a team.
</StructureSection>
== References ==
<references/>

The use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref>

UniProt data for protein P76586. https://www.uniprot.org/uniprotkb/P76586/entry

BASIL2023GVP76586

2024-02-04T03:30:44Z

Bonnie Hall:

BASIL2023GV1ZBS

2024-02-04T03:23:11Z

Bonnie Hall:

=='''''Inquiry of the Possible Function of Protein 1ZBS'''''==
==''' Abstract'''==
ZBS is a novel protein whose structure is solved but the function is unknown. This research was designed to attempt to uncover the function. Computational research indicated that N-acetylglucosamine (NAG) may be the potential substrate, and that the protein may phosphorylate NAG. This was determined using multiple computational tools, such as BLAST-P, DALI, SPRITE, InterPro. Molecular docking using NAG as a substrate was done with PyMol and Vina docking. After the computational research was completed, the protein was over-expressed and purified. The protein was used to test for activity with the substrate NAG. The kinase assay concluded that NAG is most likely not the substrate for 1ZBS due to a lack of specific activity.
== '''Introduction''' ==
<StructureSection load='1zbs' size='340' side='right' caption='1ZBS dimer as in its original state' scene=''>
1ZBS is a protein with unknown function discovered through a genomics project of predictive folding. The goal of this project was to determine what the function of protein 1ZBS was using a combination of computational analysis and physical lab work.
[[Image: FlowChart.png ]]
For this project, we started by doing our computational work, which includes BLAST, DALI, SPRITE, Net-GO, and the Docking analysis, while we started transforming our bacteria, and over-expressing and purifying our protein for the kinase assays. From the computational analysis, it was believed that 1ZBS could be a NAGK protein. Further analysis into the kinase activity did not fully support this conclusion, as the specific activity was too low.
== '''Sequence Alignments''' ==

=== BLAST-P ===

The first type of analysis that was done was using Protein Blast from NCBI. The analysis was first done as it only gave very broad information about the potential function of the protein, by matching domains across other proteins with known functions.
[[Image:BLASTP.png]]
These results for 1ZBS showed that it was a match to the superfamily BADF. BADF is the superfamily that NAGK proteins also belong in so this was one of the reasons that we believed that 1ZBS may potentially be a NAGK protein.

=== InterPro ===
InterPro is another sequence alignment software that we used to analyze the protein 1ZBS. This software gave similar results to. the Blast-P analysis, yet again giving matches to the BadF superfamily. The results showed a match to the general superfamily (green), as well as a match to the ATPase domain of the BadF family. These results further supported the idea that our protein should belong to this superfamily. On it's own, this is helpful for a starting place to determine the function but given the rest of the data from the analysis, it helps solidify the potential function of the 1ZBS protein.
[[Image:InterPro_Results_1ZBS.png]]
== '''Structural Alignments''' ==
=== DALI ===
DALI is a global alignment software that is used to match proteins up to possible structural matches within the Protein Data Bank (PDB).

[[Image:DALI_alignment.png]]

This is the DALI alignment of 1ZBS and 2CH5, where 1ZBS is colored in green and <scene name='95/957643/2ch5/1'>2CH5</scene> is colored in yellow. The best results from the DALI alignment were mainly proteins with unknown functions, which makes it difficult to deduce a possible function off of. Because of these results, 2CH5, while only matching parts of the sequence was the best result for this analysis as it's function is known. The function of 2CH5 is a NAG kinase, which would further support the thought that 1ZBS could also be a NAG kinase.
== ''' Additional Tools''' ==
=== NetGO ===

We used NetGo 2.0 to help visualize the potential function of 1ZBS. This software uses predictive modeling to determine potential functions of the protein in query, and produces a set of gene ontology(GO) terms that best aligns with the protein's possible function. It starts with the most general information and builds to become more specific. These results came back in a list form. We then used AmiGo to visualize the connection between the terms. The final results landed us with a protein that has a purine ribonucleotide triphosphate binding domain, and could have some NAG kinase activity.

[[Image: NetGo.png]]

==''' Docking Analysis'''==

The docking analysis was started by using the software POCASA to help visualize the potential binding pockets in 1ZBS, as there was no literature available. These results, as shown below, helped us to better determine where we would limit the binding area to in PyRx.

From there we used PyRx to do molecular docking into the 1ZBS protein. This was completed using various different substrates in combination with ATP, which is needed for the kinase to be able to function. The control used was Imidazole which had a binding affinity of -2.9 kcal/mol. By completing this first, we had a better idea of what our binding affinity should be. Then we used <scene name='95/957643/Nag/1'>NAG</scene> to better determine if it was our substrate, as well as other similar structures found using the PDB. NAG had a binding affinity of -5.54kcal/mol. This was a better result than our control, and this helped further the belief that 1ZBS could be a NAG kinase.
The docking results did also give us a substrate with much more desirable binding affinity, which was N-(carboxycarbonyl)-glucosylamine, otherwise referred to as <scene name='95/957643/4gp/1'>4GP</scene>. This substrate had a binding affinity of -6.08 kcal/mol.

==''' Substrate Possibilities '''==
[[Image:Substrate_image_2.PNG]]

This figure compares the structures of NAG(A) and 4GP(B). All of the components of NAG are present in 4GP but they have been rearranged and an additional carbonyl group has been added to 4GP. We choose NAG as our substrate over 4GP because it is an inhibitor. Due to this, it is expected that 4GP will have better binding as it will bind to the transition state of the enzyme, however it will stop the function of the enzyme. This fact made it more ideal to test NAG over 4GP because NAG will give insight into the actual function of the substrate.
== '''Kinase Assays''' ==

We used NAG as our final substrate for the 1ZBS kinase assay, but before the kinase assay could be run, a Bradford Assay was needed to determine the concentration of the 1ZBS protein that was over-expressed and purified in the lab. The Bradford Assay gave us the graph below with an R squared value of 0.99, and the equation listed on the graph. Because this R squared value is fairly close to 1.0, we decided to use this equation to solved for the concentration of the 1ZBS that we over-expressed.

[[Image:1ZBS_Concentration.png]]

After we had run the Bradford Assay, we then ran a kinase assay with 1ZBS and NAG as the substrate. From this assay, we were able to determine that the specific activity was 0.0743 U/mg which is fairly low.
[[Image:1ZBS_CHART.png]]
== '''Conclusion''' ==
The goal of this research was to determine the function of 1ZBS. We did this by using a series of computational techniques to limit down the possibilities and better focus the research. The first tools allowed us to start general with the superfamily, and grow more specific into similar sequences/structures. The results of the matches lead us to believe that NAG was the substrate for 1ZBS, and docking analysis also showed promise for this hypothesis.
The downfall comes from the results from the kinase assay. This showed that the specific activity of 1ZBS with NAG was only 0.0743 U/mg which is much lower than what is expected from the intended substrate. Because the specific activity was lower than intended, we do not believe that NAG is the correct substrate for 1ZBS, and that 1ZBS is not a N-acetylglucosamine kinase.

== References ==
<ref>DOI:10.2210/pdb1zbs/pdb </ref>
<references/>

BASIL2023GVQ8DN35

2024-02-04T03:20:38Z

Bonnie Hall:

Q8DN35 Protein

The predicted function of the Q8DN35 protein was studied during the spring 2023 semester at Grand View University by Lucas Kramer and Anie Rehbein in CHEM 453 - Biochemical Techniques.

<Structure load='' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />

== Computational Structure ==

<scene name='95/957644/Q8dn35_structure/2'>Click to display 3D model of Q8DN35</scene>

== Introduction ==
Q8DN35 is an enzyme with a computational structure but no known function. Our enzyme was predicted to be a kinase. The hypothesized function of this protein was that of a glucokinase, and through in silico research, two substrates were selected for further testing. Fructose-6-Phosphate (F6P) and N-acetyl-D-glucosamine (NAG) were selected based on binding affinity during PyRx docking analysis and based on correct binding site location. NetGO machine learning algorithms predicted the function of Q8DN35 to be a glucokinase, with N-acetylglucokinase activity also predicted deeply. Kinetic activity was analyzed to determine whether F6P or NAG were correct substrates. Bradford standards and gel electrophoresis were also conducted to determine concentration of Q8DN35 and to ensure the protein was present.

== Function ==
Through computational docking results, the predicted function for the Q8DN35 protein is that of a glucokinase. The use of tools such as NetGO, BLAST, DALI, InterPRO, PyRX and SPRITE allowed for this predicted function. Q8DN35's sequence is related to that of the ROK Kinase family. NetGO predicted that this protein will have glucokinase activity as it's deepest result. PyRX docking data showed us that NAG (N-acetyl-D-glucosamine) and F6P (fructose-6-phosphate) had high binding affinity in the potential binding pocket of Q8DN35.
== Binding site ==

[[Image:Docking site Q8DN35.png]]

This image displays potential binding sites to the Q8DN35 protein. Each grey "X" indicates a potential binding site. These binding sites were taken into consideration when looking at PyRx docking tools.

== Experimental process ==

[[Image:Experimental procedure Q8DN35 smaller.png]]

== Q8DN35 DNA analysis ==
To confirm presence of Q8DN35 DNA, plasmid digestion was performed to observe correct DNA. Sizes were determined from looking at the plasmid map provided for our kinase enzyme DNA. Cutting with both of the chosen restriction enzymes yielded a smaller band containing our insert and a larger band containing the rest of the plasmid. Using only one restriction enzyme gives one band with all of the plasmid DNA present.

[[Image:Q8DN35 DNA Gel.png]]

== Protein purification analysis ==
Through the use of a gravity purification column and Ni-NTA resin, Q8DN35 was purified and a Bradford analysis was conducted to determine the amount of protein in the purified sample. This purification resulted in 0.055 mg/mL of protein being purified. Through SDS-PAGE analysis, observation of purified protein was hard to see in the fraction column but large banding occurred in the pre- and post-induction samples around 31.88 kDa, which is the predicted molecular weight for Q8DN35. Further analysis with more concentrated protein sample will need to be done to confirm correct protein positioning.

[[Image:Q8DN35 initial SDS-PAGE.png]]
== Kinase activity analysis ==
To observe Q8DN35's activity, a kinase activity was ran with 2 substrates to observe potential substrate usage. The 2 substrates that were tested are NAG and F6P. After kinetics calculations were finished, we conclude that NAG has a negative effect on Q8DN35 activity and F6P has a positive effect. The magnitude of increase in activity was not ideal, so further exploration into substrates and kinetic assay protocols will need to be done to determine the correct substrate for Q8DN35.

[[Image:Kinase assay table for Q8DN35.JPG]]

== References ==
1.Miyazono, K., Tabei, N., Morita, S., Ohnishi, Y., Horinouchi, S., & Tanokura, M. (2012). Substrate recognition mechanism and substrate-dependent conformational changes of an ROK family glucokinase from Streptomyces griseus. Journal of bacteriology, 194(3), 607–616. https://doi.org/10.1128/JB.06173-11
2. The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.

<references/>

BASIL2022GV3HDT

2023-10-21T16:28:05Z

Bonnie Hall:

==Characterizing Putative Kinase 3HDT and its Potential Substrates==
<StructureSection load='3hdt.pdb' size='340' side='right' caption='Structure of putative kinase 3HDT' scene='90/904996/3hdt_structure_1st_pic_on_pg/5'>
Putative kinase, PDB ID 3HDT, shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands.

== Introduction ==
Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. The goal of this research is to study a protein (PDB ID 3HDT) with unknown function and elucidate its potential substrates. This work uses ''in silico'' techniques to characterize the protein before biochemical assays were used to test for kinase activity. This research is part of the BASIL project that involved performing ''in silico'' and ''in vitro'' modules to make predictions and study the function of this protein.

== ''In Silico'' Anaylsis ==
We used a variety of ''in silico'' tools to find similarities between 3HDT and other amino acid sequences, protein families, and 3-dimensional structures of known proteins in the PDB. Below are the recorded results and information from each database. From this information, a function was hypothesized for 3HDT and potential substrates such as dCMP were selected for kinase assays.

===='''BLASTp'''====
Beginning with BLASTp<ref>National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [cited 2022 April 23].</ref>, we queried the FASTA sequence for our protein, PDB ID 3HDT. Our query sequence aligned with the NK superfamily and showed a high degree of overlap indicating that this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our hypothesis that 3HDT is a cytidylate kinase. Additionally, the query hits showed several cytidylate kinase-like family proteins almost completely aligning with our protein, 3HDT.

[[Image:BLASTp image 1.png |600px| left | thumb | BLASTp alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.]]

[[Image:BLASTp image 2.png |600px| left | thumb | Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.]]

===='''Pfam'''====
We used 3HDT's FASTA sequence to search in Pfam<ref>Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman
Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913</ref> for protein families that 3HDT may belong to. Pfam predicted that 3HDT is part of the cytidylate kinase-like family which concurs with the BLASTp results shown previously. Also shown below are domains commonly associated with the sequence queried.

[[Image:Pfam search resultsJRAF2022.png |800px| center | thumb | Pfam query aligned with a cytidylate kinase-like family.]]

[[Image:Pfam domainsJRAF2022.png |700px| center | thumb | Top domain results from Pfam.]]

===='''DALI'''====
Two of the top results from our DALI<ref>Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.</ref> query (both cytidylate kinases) show a high structural resemblance with 3HDT when overlaid in DALI’s viewer. It is also worth noting that the majority of the results from our DALI query consisted of cytidylate kinases. This information helped us hypothesize the function (cytidylate kinase) of 3HDT due to the structural similarity between the proteins (structure of the protein = function) indicating a similarity in function between these kinases and our protein.

[[Image:3hdtDALI.png | 400px|center|thumb| Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).]]

== Docking==
We used POCASA<ref>J. Yu, Y. Zhou, I. Tanaka, M. Yao, Roll: A new algorithm for the detection of protein pockets and cavities with a rolling probe sphere. Bioinformatics, 26(1), 46-52, (2010) [PMID: 19846440]</ref> to determine potential binding pockets within 3HDT before using PyRx<ref>Small-Molecule Library Screening by Docking with PyRx. Dallakyan S, Olson AJ. Methods Mol Biol. 2015;1263:243-50.</ref> to dock dCMP into the protein. PyMOL<ref>The PyMOL Molecular Graphics System, Version 1.7.4.5 Edu Schrödinger, LLC.</ref> was then used to visualize the binding pockets with dCMP and other various ligands docked in 3HDT. This <scene name='90/904996/Binding_pockets/1'>binding pocket</scene> (in purple) is a region within 3HDT where the substrate dCMP could bind as indicated by POCASA. This area was also where dCMP bound with the highest affinity in PyRx. The <scene name='90/904996/Hydrophobic_interactions/1'>amino acids</scene> (Gly21, Ser22, Gly23, Val27, Thr 142, Gln149, Arg150, Thr197, Leu200, Thr201) interact within that area to help hold the substrate in place with hydrophobic interactions. Also, there are two residues, <scene name='90/904996/Dcmp_with_3hdt_active_site/1'>Lys146 (purple) and Leu 202 (yellow)</scene> within the active site containing dCMP and cofactor ATP that are important in substrate binding. They interact with the substrate forming hydrogen bonds with the oxygens on the phosphate groups and the 6-membered ring. The program LIGPLOT<ref>Wallace, A C et al. “LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions.” Protein engineering vol. 8,2 (1995): 127-34. doi:10.1093/protein/8.2.127</ref> was instrumental in discovering these specific interactions between various residues and ligands. However, the PyRx and LIGPLOT results may be inaccurate due to an issue in the docking process where we were unsuccessful in getting PyRx to recognize where the ATP cofactor was bound before docking our substrate into the protein.

[[Image:POCASA 3hdt image 2.png | 500px| center | thumb| Predicted binding pockets for 3HDT represented by the white stippling.]]
Some of the predicted binding pockets were areas where ATP (red arrow) and dCMP (yellow arrow) bound with the highest affinity in PyRx.

[[Image:DCMP 2.png | 400px| left| thumb| dCMP (purple) docked with 3HDT and cofactor ATP (pink). Visualized in PyMOL. ]]
[[Image:DCMP reaction.png |275px| center|thumb | Binding affinity was increased when a hydroxyl group was removed from the ribose ring on CMP to make dCMP.]]

[[Image:DockingtableJRAF2020.PNG | 250px| left| thumb| Binding affinities for compounds docked using PyRx.]]

== Laboratory Experiments ==

===='''Plasmid Analysis'''====
BL21-Gold E.Coli were transformed with a plasmid containing the gene for 3HDT to produce our protein of interest. A pMCSG19 vector with ampicillin resistance was inserted into the cells via heat shock and incubated on a plate containing carbenicillin. To ensure we inserted the gene of interest the plasmid was digested with Xho1 and Nde1 before gel electrophoresis was run to verify its identity. The vector was 6,441 bp with the insert being an additional 660 bp for a total of 7,101 bp. This is approximately the size of the combined bands in lane five, demonstrating that we had the correct plasmid and could move forward with protein over-expression and purification.

[[Image:PlasmiddigestIMGJRAF.PNG |350px|left| thumb | Lane one contains ladder. Lane two contains pDNA without enzyme. Lane three contains pDNA + Xho1. Lane four contains pDNA + Nde1. Lane 5 contains pDNA + Xho1 + Nde1. Image has been modified to only show lanes of interest.]]

[[Image:Plasmidmap3hdt.PNG |300px|center| thumb | Vector map for pMCSG19.]]

===='''Coupled Kinase Assay'''====
Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. Various concentrations of dCMP between 5-10mM were used in a total of 8 assays. In the first round of assays (3 assays) 45.6ng of 3HDT and increasing concentrations of 5.0, 7.5, and 10.0mM of dCMP were added to wells each containing 100μL total. A concentration of 7.5mM dCMP resulted in the highest specific activity (0.377 U/mg) while increasing the substrate concentration to 10mM slightly lowered specific activity to 0.348 U/mg. When we repeated the first three kinase assays (5.0, 7.5, 10.0mM dCMP), there were discrepancies in the results indicating a potential experimental error such improper mixing, bubbles being present in the well, or too much time passing between adding substrate to the well and measuring absorbance in the plate reader. Additionally, the protein in the second round of assays was older (original protein but approximately a week had passed from time of over-expression) which could have affected the specific activity in those trials due to the protein starting to expire/decrease in function.

[[Image:Coupled assay.png |400px|left| thumb | Coupled kinase assay diagram with enzymes shown in color and phosphates in yellow.]]

[[Image:Updated Kinetics Table 5.4.22 3.PNG‎ |500px|center| thumb | Specific activity values and other data from all coupled kinase assays with 3HDT and dCMP.]]

Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH was measured and subtracted from the conversion rate in the presence of dCMP before calculating specific activity values.

===='''SDS-PAGE'''====
SDS-PAGE results for our purified protein sample are shown below. The first lane (left) contains a size standard and the second lane (right) contains our protein sample. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a metal binding protein. The mass of 3HDT is approximately 25.79 kD which matches the faint band at ~26kD, indicating that our protein of interest was present.

[[Image:3hdt sds page.png |225px| center | thumb | Results from SDS-PAGE with purified protein sample.]]

== Conclusion & Future Experiments ==

Our protein, 3HDT, was confirmed to be present using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate is likely not dCMP due to the low specific activity values demonstrated in this work. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT. Additionally, in the future we would like to improve upon our protein purification process to try and get a higher protein concentration than what we achieved as the low concentration may have affected our coupled kinase assay results.

== References ==
<references/>
[[Category: BASIL]]

BASIL2022GV3R8E

2023-10-21T16:22:20Z

Bonnie Hall:

=Characterization of the 3r8e Protein, a Novel Glucose Kinase=
<StructureSection load='3r8e' size='340' side='right' caption='Caption for this structure' scene=''>
== 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 our protein of interest (POI) is a glucose kinase.
== 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.

[[Image:Workflow1.png|500px|]]
== Methods ==
As you can see in the workflow portion above, we used a variety of in silico tools such as BLASTp, Pfam, DALI, PyRx, and PyMol to help us generate a hypothesis for our uncharacterized proteins function. Using the FASTA sequence found in the Protein Data Bank file, we then were able to find similarities between 3r8e and other characterized proteins. While exploring the DALI database, a significant structural alignment hit was found with protein 3vov. Structural and sequence alignment analysis with protein 3vov, a hexokinase, is provided below.

[[Image:3R8EDali180.png|400px|]]
[[Image:3R8EDALIHMM.png|400px|]]

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.

== Experimental Results/Function ==

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: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.

[[Image:FINAL_gel.png|250px|]]

== Project Implications ==
The goal of this project is to explore the techniques it takes to characterize a putative kinase. To do this, we became familiar with online alignment, structure, and function tools, paired with a variety of in vitro lab experiments, including bacterial protein overexpression, affinity chromatography, coupled kinase assays, and SDS PAGE. These techniques can be used to help characterize further putative kinases discovered in the future that do not have a defined function. This project is of importance because proteins are biomolecules responsible for as organisms' survival and understanding their unique function is essential for advances in modern medicine, scientific research, and agriculture.
== Conclusions/Future Direction ==
Conclusions:

After various experiments and discussion, we have concluded that the novel protein 3R8E is a <scene name='90/904995/Mesh_backbone_with_atp_glucose/3'>Glucose Kinase</scene>. By using an in-vitro assay, we obtained results comparing the phosphorylation rates of five sugars in the presence of our putative kinase. From the assay results, we were able to calculate an average specific activity for all experimental sugars, and by comparing these numbers we can clearly see that glucose is being phosphorylated in the presence of protein 3R8E. To get to this conclusion, we ran triplicates of the experiment for each sugar, and then followed a calculation procedure to get specific activity numbers to quantify how active our protein is with a given substrate. By doing this, we were able to validate and further support our hypothesis, which now can allow others to replicate or continue our research. Glucose provided us with a specific activity value of 0.214 +/- 0.893 and 1.223 +/- 2.575 for prep one and two respectively.

Future Direction:

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.

== References ==
<references/>
1. Blastp [Internet]. Bethesda (MD): Natiobal Library of Medicine (US), National Center for Biotechnology Information; 2004- [cited 2022 March]. Available from: (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)

2. BASIL. https://basilbiochem.github.io/basil/

3. Holm L (2020) Using Dali for protein structure comparison. Methods Mol. Biol. 2112, 29-42.

4. Small- Molecule Library Screening by Docking with PyRx. .Dallakyan S, Olson AJ Methods Mol Biol. 2015;1263:243-50. The full-text is available at https://www.researchgate.net/publications/2739554875. Small-Molecule Library Screening by Docking with PyRx.

5. Pfam: The Protein families database in 2021 J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G.A. Salazar, E.L.L. Sonnhammer, S.C.E. Tosatto, L. Paladin, S. Raj, L.J. Richardson, R.D. Finn, A. Bateman Nucleic Acids Research (2020) doi: 10.1093/nar/gkaa913

6. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.

[[Category: BASIL]]

Sandbox bh 12345

2023-07-20T19:07:00Z

Bonnie Hall:

==My Cool Page==
<StructureSection load='1iep' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page '''Sandbox bh 12345'''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Introduction ==

== DALI ==

[[Image:DALI_screenshot.png|300px|left|thumb| DALI Results]]

== Relevance ==

This is the <scene name='97/977759/Test_1/1'>ligand for this structure</scene>.

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

Sandbox bh 12345

2023-07-20T18:43:37Z

Bonnie Hall:

==My Cool Page==
<StructureSection load='1iep' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page '''Sandbox bh 12345'''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Introduction ==

== DALI ==

[[Image:DALI_screenshot.png|300px|left|thumb| DALI Results]]

== Relevance ==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

Sandbox bh 12345

2023-07-20T18:26:11Z

Bonnie Hall: New page: ==My Cool Page== <StructureSection load='1iep' size='340' side='right' caption='Caption for this structure' scene=''> This is a default text for your page '''Sandbox bh 12345'''. Click abo...

==My Cool Page==
<StructureSection load='1iep' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page '''Sandbox bh 12345'''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Introduction ==

== Disease ==

== Relevance ==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

Sandbox Reserved 1811

2023-07-19T22:15:18Z

Bonnie Hall:

{{Sandbox_Reserved_CHEM351_Spring2023}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

this is a citation for BASIL <ref>PMID:31381264</ref>

[[Image:DALI_screenshot.png|500px|left|thumb| Figure 2. DALI Data]]

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

File:DALI screenshot.png

2023-07-19T21:33:59Z

Bonnie Hall:

File:DALI figure.png

2023-07-19T21:31:01Z

Bonnie Hall:

File:DALI figure.pdf

2023-07-19T21:29:41Z

Bonnie Hall: uploaded a new version of "Image:DALI figure.pdf"

Dali from IJ

File:DALI figure.pdf

2023-07-19T21:22:18Z

Bonnie Hall: Dali from IJ

Dali from IJ

Sandbox Reserved 1799

2023-04-27T17:30:13Z

Bonnie Hall:

{{Sandbox_Reserved_CHEM351_Spring2023}}
==Pyrrolysyl-tRNA synthase (PylRS)==
<StructureSection load='2q7h' size='340' side='right' caption='PylRS' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==
Pyrrolysyl-tRNA synthase (PylRS) is a protein found in anaerobic archaea and bacteria. PylRS facilitates the installation of pyrrolysine. It binds to the pyrrolysine substrate. This protein has two functional domains. It has a C-terminus, which is the catalytic domain (PylSc) and has a Rossmann fold, which is a tertiary fold that binds to nucleotides. The other functional domain is the N-terminus that does the tRNA binding (PylSn). These two functional domains can work together to install two noncanonical amino acids at the same time.

There are three types of ligands in this protein, <scene name='95/954096/Yly_ligand/1'>YLY</scene>, POP (Pyrophosphate 2-) and EDO (1,2-ethanediol).
== Biological relevance and broader implications ==

== Important amino acids==
<scene name='95/954096/Amino_acids/1'>Important amino acids highlighted.</scene>

== Structural highlights ==
PylRS has two domains, an N-terminal and a C-terminal. The N-terminal is responsible for tRNA-binding, PylSn. The C-terminal is the catalytic domain, PylSc.

== References ==
<references/>

Sandbox Reserved 1806

2023-04-25T22:39:07Z

Bonnie Hall:

{{Sandbox_Reserved_CHEM351_Spring2023}}
==Pyrrolysine (2Q7H) Structure==
<StructureSection load='2Q7H' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

<scene name='95/954103/Protein_2q7h/1'>Pyrrolysine tRNA Synthetase</scene>, its function is to install noncanonical amino acids into proteins in living cells.

<scene name='95/954103/Protein_2q7h/2'>Ligand</scene>

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

Pyrrolysine has 2 domains, one called the N-terminal and the other being the C-terminal. Pyrrolysine is a modified lysine with a 4-methylpyrroline-5-carboxylate group linked by an amide to the ɛ-amino group. Pyrrolysine is lysine in which has a pyrroline ring linked to the end of the lysine side chain.

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

== Other important features ==

</StructureSection>
== References ==
<ref> PMID:17592110 </ref>
<references/>

Sandbox Reserved 1811

2023-04-03T21:20:18Z

Bonnie Hall:

{{Sandbox_Reserved_CHEM351_Spring2023}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

Sandbox Reserved 1810

2023-04-03T21:20:02Z

Bonnie Hall:

{{Sandbox_Reserved_CHEM351_Spring2023}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

Sandbox Reserved 1809

2023-04-03T21:19:46Z

Bonnie Hall:

{{Sandbox_Reserved_CHEM351_Spring2023}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

Sandbox Reserved 1808

2023-04-03T21:19:31Z

Bonnie Hall:

{{Sandbox_Reserved_CHEM351_Spring2023}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

Sandbox Reserved 1807

2023-04-03T21:19:18Z

Bonnie Hall:

{{Sandbox_Reserved_CHEM351_Spring2023}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

Sandbox Reserved 1806

2023-04-03T21:19:02Z

Bonnie Hall:

{{Sandbox_Reserved_CHEM351_Spring2023}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

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{{Sandbox_Reserved_CHEM351_Spring2023}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

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{{Sandbox_Reserved_CHEM351_Spring2023}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

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Bonnie Hall:

{{Sandbox_Reserved_CHEM351_Spring2023}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

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Bonnie Hall:

{{Sandbox_Reserved_CHEM351_Spring2023}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

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Bonnie Hall:

{{Sandbox_Reserved_CHEM351_Spring2023}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

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{{Sandbox_Reserved_CHEM351_Spring2023}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

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{{Sandbox_Reserved_CHEM351_Spring2023}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

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Bonnie Hall:

{{Sandbox_Reserved_CHEM351_Spring2023}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

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{{Sandbox_Reserved_CHEM351_Spring2023}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

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{{Sandbox_Reserved_CHEM351_Spring2023}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

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{{Sandbox_Reserved_BHall_F22}}
==Human Ornithinine Aminotransferase (hOAT)==
<StructureSection load='7T9Z' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

==(hOAT')==

=== Function of your protein ==
The protein Ornithinine Aminotransferase (OAT), in humans (hOAT), is an enzyme that catalyzes the reaction between carbamoyl phosphate and orthinine to form citrulline and phosphate. Its ligand is pyridoxal-5'phosphate (PLP) which is a cofactor of the reaction. An amino group from L-Orn is transferred to PLP which turns it into pyridoxine phosphate (PMP) and L-Orn is converted to L glutamate-y-semialdehyde. PLP is regenerated when the amino group PMP is transferred to alpha-KG. It has 3 ligands pyridoxal-5' phosphate (PLP) which is a cofactor in the reaction. An amino group from L-Ornithinine is transferred to PLP which converts it to pyridoxamine phosphate (PMP) and L-Orn is converted to L-glutamate-y-semialdehyde. PLP is regenerated when PMP is transferred to alpha-KG.

== Biological relevance and broader implications ==
hOAT is a ubiquitous enzyme found in almost all organisms and has been found to be overexpressed in hepatocellular carcinoma cells (HCC), which is a type of liver cancer. It is found predominantly in the liver and kidney. The liver, where it is an integral part of the urea cycle, and the intestine, where it synthesizes citrulline for export and plays a major role in amino acid homeostasis, particularly of L-glutamine and L-arginine. Studying this protein could be a potential drug to target cancer as a different therapy and radiation. hOAT has been a target mechanism-based inactivator (MBIs) in drug design efforts. HCC has diagnosed an advanced type of cancer which makes it more resistant to chemotherapy. hOAT inhibitors were created as fragmented-sized alternative substances such as GABA and 5-aminovaleric acid (AVA). hOAT was soaked with GABA and AVA and the new substrates prevented original interactions with catalytic amino acids and the ligand (PLP) which creates a tighter to hOAT instead of L-ornithine. GABA and AVA had a stronger binding affinity and slower turnovers which makes them potential drug targets for hOAT.

== Important amino acids==
<scene name='93/933999/Amino_acid_300-304/1'>Amino Acids 300-304</scene> are an important part of protein binding <ref>PMID:35460691</ref>.

== Structural highlights ==
<scene name='93/933999/Helix/4'>An image of the helix in HOAT protein.</scene>

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.
An image of the alpha carbon.

</StructureSection>
== References ==
<references/>

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{{Sandbox_Reserved_BHall_F22}}
==Human Ornithinine Aminotransferase (hOAT)==
<StructureSection load='7T9Z' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==
The protein Ornithinine Aminotransferase (OAT), in humans (hOAT), is an enzyme that catalyzes the reaction between carbamoyl phosphate and orthinine to form citrulline and phosphate. Its ligand is pyridoxal-5'phosphate (PLP) which is a cofactor of the reaction. An amino group from L-Orn is transferred to PLP which turns it into pyridoxine phosphate (PMP) and L-Orn is converted to L glutamate-y-semialdehyde. PLP is regenerated when the amino group PMP is transferred to alpha-KG.

== Biological relevance and broader implications ==
hOAT is a ubiquitous enzyme found in almost all organisms and has been found to be overexpressed in hepatocellular carcinoma cells (HCC), which is a type of liver cancer. It is found predominantly in the liver and kidney. The liver, where it is an integral part of the urea cycle, and the intestine, where it synthesizes citrulline for export and plays a major role in amino acid homeostasis, particularly of L-glutamine and L-arginine. Studying this protein could be a potential drug to target cancer as a different therapy and radiation. hOAT has been a target mechanism-based inactivator (MBIs) in drug design efforts. HCC has diagnosed an advanced type of cancer which makes it more resistant to chemotherapy. hOAT inhibitors were created as fragmented-sized alternative substances such as GABA and 5-aminovaleric acid (AVA). hOAT was soaked with GABA and AVA and the new substrates prevented original interactions with catalytic amino acids and the ligand (PLP) which creates a tighter to hOAT instead of L-ornithine. GABA and AVA had a stronger binding affinity and slower turnovers which makes them potential drug targets for hOAT.

== Important amino acids==
Amino Acids in PLP binding site are ,Lys 292</scene> Asp 263, Phe 177 and Arg 180</scene>.<ref> 35460691 </ref>. They are essential to the active site. PLP is covalently bonded to the amino acid lysine. The phosphate group interacts with the positively charged nitrogen of the arginine side chain. There is also a pi-stacking interaction between its ring and the ring of PLP.

== Structural highlights ==
hOAT is a protein with a <scene name='93/934007/Secondary_structure/1'>secondary structure</scene> consisting of mainly alpha-helices but it also has parallel and anti-parallel beta-sheets and random coil. It's a polymer with a <scene name='93/934007/Globular_module/1'>globular structure</scene> that has three subunits held together by non-covalent interactions such as hydrogen bonds and salt bridges hidden in the protein. The <scene name='93/934007/Ligands_of_interest/1'>ligand</scene> has a phosphate group surrounded by polar amino acids, and carbons are surrounded by non-polar amino acids to satisfy the needs of the ligand and active site. Interactions like hydrogen bonding and pi-stacking stabilize and bind the ligand in the active site.
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

Title

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Bonnie Hall: New page: {Sandbox_Reserved_BHall_Chem351_F19}} ==Your Heading Here (maybe something like 'Structure')== <StructureSection load='1stp' size='340' side='ri...

{Sandbox_Reserved_BHall_Chem351_F19}}

==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page '''Title'''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function ==

== Disease ==

== Relevance ==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

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{{Sandbox_Reserved_BHall_F22}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

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{{{Sandbox_Reserved_BHall_F22}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function of your protein ==

== Biological relevance and broader implications ==

== Important amino acids==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

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