Sandbox Reserved 1852: Difference between revisions
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[https://www.pnas.org/doi/full/10.1073/pnas.1401073111 Impact of Scaffold Rigidity] | [https://www.pnas.org/doi/full/10.1073/pnas.1401073111 Impact of Scaffold Rigidity] | ||
The Diels-Alderase protein aims to create optimal reacting conditions between the diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital (LUMO) and the diene’s highest occupied molecular orbital (HOMO) in the transition state.<ref name="Siegel">PMID:20647463</ref> The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate (diene) and N,N- dimethylacrylamide (dienophile). The binding site contains an H-bond donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and an H-bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene. Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. | The Diels-Alderase protein aims to create optimal reacting conditions between the diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital (LUMO) and the diene’s highest occupied molecular orbital (HOMO) in the transition state.<ref name="Siegel">PMID:20647463</ref> The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate (diene) and N,N- dimethylacrylamide (dienophile). The binding site contains an H-bond donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and an H-bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene. Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. | ||
The Diels-Alderase enzyme was built using de novo enzyme design, which relies on computational modeling that is refined through programming and collaborative problem-solving from online users. The original protein was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges. | |||
==General Structure== | ==General Structure== | ||
====Scaffold==== | |||
After early Rosetta computational modelling, an ideal protein scaffold was found in the 6-bladed beta-propeller of Loligo vulgalis, or the Europoean Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. | |||
[[Image:BindingPocket.png|300px|right|thumb|Figure 1. Active Site]] | [[Image:BindingPocket.png|300px|right|thumb|Figure 1. Active Site]] | ||
====Active Site==== | ====Active Site==== | ||
