Sandbox Reserved 1845: Difference between revisions

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=== Phe243 ===

<scene name='10/1075247/F243_original/1'>Phe243</scene> is located 3.6 Å from the ligand. Two mutations at this position, <scene name='10/1075247/F243i/1'>F243I</scene> and F243W, increase the catalytic activity of the enzyme. The F243I mutation inserts the smaller isoleucine whose side chain allows the ligand to sit closer. This reduces the ligand distance to 3.0 Å, improving substrate binding. The F243W mutation inserts the bulkier, nitrogen-containing aromatic aide chain. Trp brings the ligand slightly closer at 3.2 Å and introduces potential for new interactions, such as hydrogen bonding or [https://en.wikipedia.org/wiki/Pi-stacking#:~:text=In%20chemistry%2C%20pi%20stacking%20(also,interaction%22)%20is%20electrostatically%20repulsive. π-stacking]. Both mutations result in improved catalytic performance. The F243I mutant shows a 27.5% increase in activity, while the F243W mutant shows a 17.5% increase, compared to the wild-type enzyme.<ref name="Tournier"/>

<scene name='10/1075247/F243_original/1'>Phe243</scene> is located 3.6 Å from the ligand,<scene name='10/1075247/Original_15_mutation_structure/6'>Reference Phe243</scene>. Two mutations at this position, <scene name='10/1075247/F243i/1'>F243I</scene> and F243W, increase the catalytic activity of the enzyme. The F243I mutation inserts the smaller isoleucine whose side chain allows the ligand to sit closer. This reduces the ligand distance to 3.0 Å, improving substrate binding. The F243W mutation inserts the bulkier, nitrogen-containing aromatic aide chain. Trp brings the ligand slightly closer at 3.2 Å and introduces potential for new interactions, such as hydrogen bonding or [https://en.wikipedia.org/wiki/Pi-stacking#:~:text=In%20chemistry%2C%20pi%20stacking%20(also,interaction%22)%20is%20electrostatically%20repulsive. π-stacking]. Both mutations result in improved catalytic performance. The F243I mutant shows a 27.5% increase in activity, while the F243W mutant shows a 17.5% increase, compared to the wild-type enzyme.<ref name="Tournier"/>

=== Tyr 127 ===

The mutation of Tyr to Gly at <scene name='10/1075247/Y127/4'>Tyr127</scene> also increases the thermostability. The melting point of Y127G is increased to 87.0°C from the WT melting point of 84.7°C. Tyr has a bulky, rigid aromatic side chain that can cause structural strain, shown in <scene name='10/1075247/Y127_spacefill/1'>Y127 representation</scene>. Gly is the smallest amino acid and lacks a side chain, providing greater flexibility. The mutation <scene name='10/1075247/Y127g/2'>Y127G</scene> melting point is increased to 87.0°C. The mutation reduces [https://en.wikipedia.org/wiki/Steric_effects#:~:text=Steric%20hindrance%20is%20the%20slowing,as%20slowing%20unwanted%20side%2Dreactions. steric hindrance] and relieves strain in the protein structure, demonstrated in <scene name='10/1075247/Y127g_space_fill/1'>Y127G representation</scene>. By increasing flexibility, the Y127G mutation helps the protein maintain its folded structure under heat stress.<ref name="Tournier"/>

The mutation of Tyr to Gly at <scene name='10/1075247/Y127/4'>Tyr127</scene> also increases the thermostability,<scene name='10/1075247/Original_15_mutation_structure/6'>Reference Tyr 127</scene>. The melting point of Y127G is increased to 87.0°C from the WT melting point of 84.7°C. Tyr has a bulky, rigid aromatic side chain that can cause structural strain, shown in <scene name='10/1075247/Y127_spacefill/1'>Y127 representation</scene>. Gly is the smallest amino acid and lacks a side chain, providing greater flexibility. The mutation <scene name='10/1075247/Y127g/2'>Y127G</scene> melting point is increased to 87.0°C. The mutation reduces [https://en.wikipedia.org/wiki/Steric_effects#:~:text=Steric%20hindrance%20is%20the%20slowing,as%20slowing%20unwanted%20side%2Dreactions. steric hindrance] and relieves strain in the protein structure, demonstrated in <scene name='10/1075247/Y127g_space_fill/1'>Y127G representation</scene>. By increasing flexibility, the Y127G mutation helps the protein maintain its folded structure under heat stress.<ref name="Tournier"/>

=== Ser 283 & Asp 238 ===

Ser 283 and Asp 238, are located outside of the binding pocket in the <scene name='10/1075247/Start_material_for_s283_and_d2/2'>~~Original Protein~~</scene>.Two wild-type residues, <scene name='10/1075247/S283-d238/5'>Ser283 and Asp238</scene>, were engineered to form a disulfide bond by replacing them with Cys. This decision was based on their spatial proximity in the 3D structure and their location in a region that resembles metal-binding sites in homologous PET-degrading enzymes. Unlike those metal-dependent sites, the LCC structure lacked coordinated ions. For that reason, the researchers engineered a covalent linkage instead to increase thermal stability without requiring additives like calcium. The wild-type protein has a melting point of 84.7°C, while the <scene name='10/1075248/C283-c238/2'>S283C and D238C mutant</scene> increased the melting point to 94.5°C, a 9.8°C improvement, which is higher than any other mutations. However, this increase in stability was accompanied by a 28% decrease in enzymatic activity compared to the wild-type. This trade-off between stability and activity shows the balance in enzyme engineering, as increasing structural integrity can sometimes restrict the flexibility needed for catalytic function.

Ser 283 and Asp 238, are located outside of the binding pocket in the <scene name='10/1075247/Start_material_for_s283_and_d2/2'>Reference Ser283 and Asp238</scene>.Two wild-type residues, <scene name='10/1075247/S283-d238/5'>Ser283 and Asp238</scene>, were engineered to form a disulfide bond by replacing them with Cys. This decision was based on their spatial proximity in the 3D structure and their location in a region that resembles metal-binding sites in homologous PET-degrading enzymes. Unlike those metal-dependent sites, the LCC structure lacked coordinated ions. For that reason, the researchers engineered a covalent linkage instead to increase thermal stability without requiring additives like calcium. The wild-type protein has a melting point of 84.7°C, while the <scene name='10/1075248/C283-c238/2'>S283C and D238C mutant</scene> increased the melting point to 94.5°C, a 9.8°C improvement, which is higher than any other mutations. However, this increase in stability was accompanied by a 28% decrease in enzymatic activity compared to the wild-type. This trade-off between stability and activity shows the balance in enzyme engineering, as increasing structural integrity can sometimes restrict the flexibility needed for catalytic function.

== Group Mutations ==

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@@ Line 40: / Line 40: @@
 === Phe243 ===
-<scene name='10/1075247/F243_original/1'>Phe243</scene> is located 3.6 Å from the ligand. Two mutations at this position, <scene name='10/1075247/F243i/1'>F243I</scene> and F243W, increase the catalytic activity of the enzyme. The F243I mutation inserts the smaller isoleucine whose side chain allows the ligand to sit closer. This reduces the ligand distance to 3.0 Å, improving substrate binding. The F243W mutation inserts the bulkier, nitrogen-containing aromatic aide chain. Trp brings the ligand slightly closer at 3.2 Å and introduces potential for new interactions, such as hydrogen bonding or [https://en.wikipedia.org/wiki/Pi-stacking#:~:text=In%20chemistry%2C%20pi%20stacking%20(also,interaction%22)%20is%20electrostatically%20repulsive. π-stacking]. Both mutations result in improved catalytic performance. The F243I mutant shows a 27.5% increase in activity, while the F243W mutant shows a 17.5% increase, compared to the wild-type enzyme.<ref name="Tournier"/>
+<scene name='10/1075247/F243_original/1'>Phe243</scene> is located 3.6 Å from the ligand,<scene name='10/1075247/Original_15_mutation_structure/6'>Reference Phe243</scene>. Two mutations at this position, <scene name='10/1075247/F243i/1'>F243I</scene> and F243W, increase the catalytic activity of the enzyme. The F243I mutation inserts the smaller isoleucine whose side chain allows the ligand to sit closer. This reduces the ligand distance to 3.0 Å, improving substrate binding. The F243W mutation inserts the bulkier, nitrogen-containing aromatic aide chain. Trp brings the ligand slightly closer at 3.2 Å and introduces potential for new interactions, such as hydrogen bonding or [https://en.wikipedia.org/wiki/Pi-stacking#:~:text=In%20chemistry%2C%20pi%20stacking%20(also,interaction%22)%20is%20electrostatically%20repulsive. π-stacking]. Both mutations result in improved catalytic performance. The F243I mutant shows a 27.5% increase in activity, while the F243W mutant shows a 17.5% increase, compared to the wild-type enzyme.<ref name="Tournier"/>
 === Tyr 127 ===
-The mutation of Tyr to Gly at <scene name='10/1075247/Y127/4'>Tyr127</scene> also increases the thermostability. The melting point of Y127G is increased to 87.0°C from the WT melting point of 84.7°C. Tyr has a bulky, rigid aromatic side chain that can cause structural strain, shown in <scene name='10/1075247/Y127_spacefill/1'>Y127 representation</scene>. Gly is the smallest amino acid and lacks a side chain, providing greater flexibility. The mutation <scene name='10/1075247/Y127g/2'>Y127G</scene> melting point is increased to 87.0°C. The mutation reduces [https://en.wikipedia.org/wiki/Steric_effects#:~:text=Steric%20hindrance%20is%20the%20slowing,as%20slowing%20unwanted%20side%2Dreactions. steric hindrance] and relieves strain in the protein structure, demonstrated in <scene name='10/1075247/Y127g_space_fill/1'>Y127G representation</scene>. By increasing flexibility, the Y127G mutation helps the protein maintain its folded structure under heat stress.<ref name="Tournier"/>
+The mutation of Tyr to Gly at <scene name='10/1075247/Y127/4'>Tyr127</scene> also increases the thermostability,<scene name='10/1075247/Original_15_mutation_structure/6'>Reference Tyr 127</scene>. The melting point of Y127G is increased to 87.0°C from the WT melting point of 84.7°C. Tyr has a bulky, rigid aromatic side chain that can cause structural strain, shown in <scene name='10/1075247/Y127_spacefill/1'>Y127 representation</scene>. Gly is the smallest amino acid and lacks a side chain, providing greater flexibility. The mutation <scene name='10/1075247/Y127g/2'>Y127G</scene> melting point is increased to 87.0°C. The mutation reduces [https://en.wikipedia.org/wiki/Steric_effects#:~:text=Steric%20hindrance%20is%20the%20slowing,as%20slowing%20unwanted%20side%2Dreactions. steric hindrance] and relieves strain in the protein structure, demonstrated in <scene name='10/1075247/Y127g_space_fill/1'>Y127G representation</scene>. By increasing flexibility, the Y127G mutation helps the protein maintain its folded structure under heat stress.<ref name="Tournier"/>
 === Ser 283 & Asp 238 ===
-Ser 283 and Asp 238, are located outside of the binding pocket in the <scene name='10/1075247/Start_material_for_s283_and_d2/2'>Original Protein</scene>.Two wild-type residues, <scene name='10/1075247/S283-d238/5'>Ser283 and Asp238</scene>, were engineered to form a disulfide bond by replacing them with Cys. This decision was based on their spatial proximity in the 3D structure and their location in a region that resembles metal-binding sites in homologous PET-degrading enzymes. Unlike those metal-dependent sites, the LCC structure lacked coordinated ions. For that reason, the researchers engineered a covalent linkage instead to increase thermal stability without requiring additives like calcium. The wild-type protein has a melting point of 84.7°C, while the <scene name='10/1075248/C283-c238/2'>S283C and D238C mutant</scene> increased the melting point to 94.5°C, a 9.8°C improvement, which is higher than any other mutations. However, this increase in stability was accompanied by a 28% decrease in enzymatic activity compared to the wild-type. This trade-off between stability and activity shows the balance in enzyme engineering, as increasing structural integrity can sometimes restrict the flexibility needed for catalytic function.
+Ser 283 and Asp 238, are located outside of the binding pocket in the <scene name='10/1075247/Start_material_for_s283_and_d2/2'>Reference Ser283 and Asp238</scene>.Two wild-type residues, <scene name='10/1075247/S283-d238/5'>Ser283 and Asp238</scene>, were engineered to form a disulfide bond by replacing them with Cys. This decision was based on their spatial proximity in the 3D structure and their location in a region that resembles metal-binding sites in homologous PET-degrading enzymes. Unlike those metal-dependent sites, the LCC structure lacked coordinated ions. For that reason, the researchers engineered a covalent linkage instead to increase thermal stability without requiring additives like calcium. The wild-type protein has a melting point of 84.7°C, while the <scene name='10/1075248/C283-c238/2'>S283C and D238C mutant</scene> increased the melting point to 94.5°C, a 9.8°C improvement, which is higher than any other mutations. However, this increase in stability was accompanied by a 28% decrease in enzymatic activity compared to the wild-type. This trade-off between stability and activity shows the balance in enzyme engineering, as increasing structural integrity can sometimes restrict the flexibility needed for catalytic function.
 == Group Mutations ==