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| | # Test designed proteins from FoldIt | | # Test designed proteins from FoldIt |
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| - | ='''Current Focus'''=
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| - | ===Custom Display Vector (CDV) ===
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| - | ====Problem====
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| - | The underlying design of the streptavidin display constructs in the registry has the displayed protein bound close to the cell membrane. This constraint could be preventing streptavidin on the surface of the cell from forming tetramers lowering its effectiveness at binding biotin. Furthermore these existing display systems prevent the addition of another protein into them, preventing the user from displaying other proteins.
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| - |
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| - | ====Idea====
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| - | The first goal of making a new display vector was to incorporate a GS linker between the displayed protein and the ompA protein anchoring in in the cell wall. However when reviewing the construct we decided to add some other useful features as well. Maintaining the Lpp tag (to direct the protein to the periplasm) and the OmpA trans-membrane regions (for anchoring the construct) of the original 2006 parts we added:
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| - | #GS Linker (Gly4Ser)4 - allowing for more space between the protein and the cell wall
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| - | #TEV (Tyrosine-Glutamate-Valine) - protease site allowing for cleavage of displayed proteins
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| - | #NheI restriction site - allowing for the insert of '''any BioBrick protein''' into the display construct
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| - | <br><br>
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| - | [[Image:PDS_design.png |600px |center]]
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| - | <br><br>
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| - |
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| - | ====Current Status====
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| - | We have built the Custom Display Vector and have inserted streptavidin into it, however we ran out of time and were not able to characterize this part. It was subbmited to the registry with streptavidin (BBa_K215210 and BBa_K215211) and without a displayed protein(BBa_K215200 and BBa_K215200).
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| - | ----
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| - |
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| - | ===[http://www.fold.it Fold-It]===
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| - | ====Problem====
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| - | Streptavidin in its native form exists as a homotetramer, where adjacent subunits interact allowing for a strong interaction with biotin. This interaction is strong (Kd = 1.5E-15 M at pH 5.0) and can withstand most strong denaturing agents [1]. However when in its monomeric form, streptavidin does not maintain this strong interaction and its usefulness as a strong binder diminishes. For our system we needed a protein that could: be easily displayed on the surface of the cell, specifically bind a ligand, and release this ligand in the presence of biotin. The ability to display a protein on the cell surface is trivial, however there is difficulty in trying to get a protein to be functional on the surface of the cell. In the case of streptavidin the ability of the protein to form tetramers on the cell surface seems to be hindered, due to the poor ability of cells displaying streptavidin to bind biotinylated fluorophore (observed above). From this issue the idea of using a monomeric protein to bind biotin arose.
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| - |
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| - | ====The Idea====
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| - | There are engineered forms of streptavidin that have mutations preventing the formation of tetrameric structures. However as mentioned before, as a monomer streptavidin has a weaker affinity to biotin than would be desired. Instead of screening proteins from the literature for ability to bind biotin our group approached the Baker lab at our university. After mentioning our problem, it was recommended that we design a biotin binding protein using the [http://boinc.bakerlab.org/rosetta/ Rosetta software] they developed. Rosetta in conjunction with [http://www.folt.it Fold-It] (also developed at the University of Washington) would allow use to design and optimize proteins for binding biotin.
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| - |
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| - | ====The Trench Work====
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| - | The first step in designing our protein was looking at the native biotin-streptavidin interaction and taking measurements between key amino acids and the biotin molecule. From here we entered the constraints into Rosetta where it matched our measurements into proteins from a protein scaffold library. This produced a large set of scaffolds with different ways each one could be used to bind biotin. These scaffolds must be screened manually, and the scaffolds that look the most promising can be placed into Fold-It. Once in Fold-It, the public has access to your protein design and can tweak and tune the protein to optimize its interaction with biotin. This allows anyone (with or without prior protein knowledge) to optimize your protein scaffold.
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| - | As can be seen below Fold-It uses an easily learned user interface and uses a score board to show the players who is the best folder.
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| - | -----
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| - | <br>
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| - | <html>
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| - |
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| - | <table><tr><td>
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| - | <object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/P-UR3G7TBb4&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/P-UR3G7TBb4&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object>
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| - | </td><td>
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| - | Here we see a video of Fold-It, showing one of our biotin puzzles "Hold Me Tightly". The protein is represented as a cartoon model, showing off its secondary structure as well as key amino acid groups. Steric clashes of the amino acid side chains show up as red balls and can also be observed in the video. These steric clashs can be removed with the Shake Function. The Shake function in Fold-It performs coarse sampling of the amino acid conformations, looking for a global-minima.
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| - | </td></tr></table>
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| - |
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| - | <table><tr><td>
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| - | <object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/_Ugmw69_94g&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/_Ugmw69_94g&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object>
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| - | </td><td>
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| - | The function we see here is the Mutate function. This allows the user to sample many amino acids at a particular site, or the whole protein. Mutate looks for global-minima while sampling amino acids. As is seen here Alanine is mutated to Asparagine. The blue and white striped band indicates that a hydrogen bond has been formed, which is a favorable interaction between two polar residues.
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| - | </td></tr></table>
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| - |
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| - | <table><tr><td>
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| - | <object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/3V2OpBGruzQ&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/3V2OpBGruzQ&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object>
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| - | </td><td>
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| - | Another nice feature of Fold-It is the ability to select a sphere of amino acids around your ligand, and optimize these amino acids based off of a fine sampling of conformations. Here we see the amino acids surrounding the ligand being selected and having the Wiggle function performed on them. The Wiggle function in Fold-It allows the user to fine tune the protein structure. Finding a local-minima for the amino acid conformations.
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| - | </td></tr></table>
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| - |
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| - | </html>
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| - | <br>
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| - | -----
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| - | This accessible format has allowed over 100,000 users to help design proteins. Currently we have published protein puzzles on Fold-It and are screening though the top scoring designs. An undergrad in our group will be active though out the next year testing the designs and looking for biotin binding proteins.
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| | '''Continue to [https://2009.igem.org/Team:Washington/Accomplishments Accomplishments and & Submitted BioBricks]''' | | '''Continue to [https://2009.igem.org/Team:Washington/Accomplishments Accomplishments and & Submitted BioBricks]''' |
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| - | === References ===
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| - | #Haugland RP, "Coupling of antibodies with biotin". http://www.ncbi.nlm.nih.gov/pubmed/18287646?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum
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| - | {{Template:Team:Washington/Templates/Footer}}
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