Dan Tawfik lab: Directed evolution: Difference between revisions
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< | <StructureSection load='3gkl' size='500' frame='true' align='right' scene='3gkl/Al/1' > | ||
Iterative rounds of random mutagenesis and selection of immunity protein 9 (colored yellow) toward higher affinity for ColE7, and selectivity (against ColE9 inhibition), led to significant increase in affinity and selectivity. Several evolved variants were obtained. The crystal structures of the two final generation <scene name='3gkl/Al/3'>variants</scene> <font color='lime'><b>R12-2</b></font> ('''3gkl'''; T20A, N24D, T27A, S28T, V34D, V37J, E41G, and K57E) and <font color='darkred'><b>R12-13</b></font> ([[3gjn]]; N24D, D25E, T27A, S28T, V34D, V37J, and Y55W) in complex with ColE7 were solved. | Iterative rounds of random mutagenesis and selection of immunity protein 9 (colored yellow) toward higher affinity for ColE7, and selectivity (against ColE9 inhibition), led to significant increase in affinity and selectivity. Several evolved variants were obtained. The crystal structures of the two final generation <scene name='3gkl/Al/3'>variants</scene> <font color='lime'><b>R12-2</b></font> ('''3gkl'''; T20A, N24D, T27A, S28T, V34D, V37J, E41G, and K57E) and <font color='darkred'><b>R12-13</b></font> ([[3gjn]]; N24D, D25E, T27A, S28T, V34D, V37J, and Y55W) in complex with ColE7 were solved. | ||
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<scene name='3gkl/Align/2'>Structural alignment</scene> of the immunity protein 9 (Im9, [[1bxi]], colored yellow), <font color='lime'><b>evolved variant R12-2 (lime)</b></font>, and <font color='blue'><b>immunity protein 7 (Im7, [[7cei]], colored blue)</b></font> reveals their structural identity. However, when the immunity proteins-bound <scene name='3gkl/Align/3'>colicins within their complexes were aligned</scene>, they demonstrate somewhat different picture. The Im9 and Im7 are differ more in their binding configurations (19°, with Tyr54-Tyr55 as the pivot), while the variant R12-2 is in an intermediate configuration between Im9 and Im7. Of note, in the variant R12-2 (3gkl) and Im9 ([[1bxi]]) there are Tyr54 and Tyr55, while in the Im7 ([[7cei]]) Tyr55 and Tyr56 are homologous to them. The most <scene name='3gkl/Align/4'>prominent differences</scene> are in the loop between helices α1 and α2 in Im9 (yellow, labeled in black) and <font color='lime'><b>evolved variant R12-2 (lime, labeled in black)</b></font>. This loop consists of three mutations: N24D, T27A, and S28T in variant R12-2. We can see the deviations in the relative position of helices α1 and α2, in the loop's backbone and in the side chains of residues 24, 26 and 28. | <scene name='3gkl/Align/2'>Structural alignment</scene> of the immunity protein 9 (Im9, [[1bxi]], colored yellow), <font color='lime'><b>evolved variant R12-2 (lime)</b></font>, and <font color='blue'><b>immunity protein 7 (Im7, [[7cei]], colored blue)</b></font> reveals their structural identity. However, when the immunity proteins-bound <scene name='3gkl/Align/3'>colicins within their complexes were aligned</scene>, they demonstrate somewhat different picture. The Im9 and Im7 are differ more in their binding configurations (19°, with Tyr54-Tyr55 as the pivot), while the variant R12-2 is in an intermediate configuration between Im9 and Im7. Of note, in the variant R12-2 (3gkl) and Im9 ([[1bxi]]) there are Tyr54 and Tyr55, while in the Im7 ([[7cei]]) Tyr55 and Tyr56 are homologous to them. The most <scene name='3gkl/Align/4'>prominent differences</scene> are in the loop between helices α1 and α2 in Im9 (yellow, labeled in black) and <font color='lime'><b>evolved variant R12-2 (lime, labeled in black)</b></font>. This loop consists of three mutations: N24D, T27A, and S28T in variant R12-2. We can see the deviations in the relative position of helices α1 and α2, in the loop's backbone and in the side chains of residues 24, 26 and 28. | ||