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Dan Tawfik lab: Directed evolution: Difference between revisions

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== Kemp eliminase ==
* [[2rkx]]
* [[2rkx]]
* [[3iio]]
* [[3iio]]
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== Colicin7 and Immunity proteins ==
{{ABSTRACT_PUBMED_19749752}}
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<applet 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.
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<applet load='3gkl' size='500' frame='true' align='left' scene='3gkl/Align/1' />
<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.
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<applet load='3gkl' size='500' frame='true' align='right' scene='3gkl/Active_site/2' />
Comparison of the different Im-colicin complexes reveals changes in the binding configuration of the evolved variants which increase affinity toward ColE7 by re-aligning pre-existing Im9 residues. Glu30 of Im9 ([[1bxi]], colored yellow) forms <scene name='3gkl/Active_site/3'>double salt bridge</scene> with Arg54 of <font color='orange'><b>ColE9 (orange)</b></font>, whereas Asp51 have not direct side chain–side chain interactions. <font color='blue'><b>Asp31 of Im7 (blue)</b></font> (corresponding to Im9 Glu30) is involved in <scene name='3gkl/Active_site/6'>cluster of salt bridge bonds</scene> to <font color='darkmagenta'><b>Arg520 and Lys525 of ColE7 (darkmagenta)</b></font>, while <font color='blue'><b>Asp52 of Im7</b></font> (corresponding to Im9 Asp51) is within hydrogen bond distance to <font color='darkmagenta'><b>Thr531 and Arg530 of ColE7</b></font>. <font color='lime'><b>Glu30 in the variant R12-2 (lime)</b></font> is shifted and forms a <scene name='3gkl/Active_site/8'>double salt bridge</scene> to <font color='magenta'><b>Arg520 of ColE7 (magenta)</b></font>. <font color='lime'><b>Asp51</b></font> is within hydrogen bond distance to <font color='magenta'><b>Thr531 of ColE7</b></font>. However, the side chains of <font color='magenta'><b>Lys525 and Arg530</b></font>, which are very important in salt bridge contacts with Glu30 and Asp51, respectively, in the structure of the ColE7–Im7 complex have a different conformation that eliminates these contacts in evolved variant R12-2.
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<applet load='3gkl' size='500' frame='true' align='left' scene='3gkl/Mut/1' />
In the <scene name='3gkl/Mut/2'>Im9</scene> <font color='magenta'><b>Val37 (colored magenta)</b></font> forms stabilizing hydrogen bond with Leu33. In the <scene name='3gkl/Mut/3'>evolved variant R12-2</scene>, <font color='darkmagenta'><b>Ile37 (colored darkmagenta)</b></font> interacts with two additional residues, Tyr54 and Ser50. Moreover, <font color='darkmagenta'><b>Ile37</b></font> also forms additional hydrogen bond with Gly41 and can thereby have enabled the appearance of the selectivity mutation E41G.
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<applet load='3gkl' size='500' frame='true' align='right' scene='3gkl/Align/5' />
In contrast to the <font color='lime'><b>evolved variant R12-2</b></font> (3gkl), the <font color='cyan'><b>evolved variant R12-13</b></font> ([[3gjn]]) carries the <scene name='3gkl/Align/10'>Tyr55Trp mutation</scene> in the conserved region. Both <font color='lime'><b>Tyr55 in R12-2</b></font> and <font color='cyan'><b>Trp55 in R12-13</b></font> could sustain the
hydrophobic core and create a <scene name='3gkl/Align/11'>hydrogen bond</scene> to Lys528 backbone (3gkl colicin residues are colored in <font color='magenta'><b>magenta</b></font>, 3gjn colicin residues are colored <font color='blueviolet'><b>blueviolet</b></font>). However, the additional bulkiness of the Trp contributes in expanding its  <scene name='3gkl/Align/9'>hydrophobic interactions</scene> to Phe541 and Phe513 also leading to the small shift in the alkyl chain of Arg530.
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<applet load='3gkl' size='500' frame='true' align='right' scene='3gkl/Align/5' />
The <scene name='3gkl/Ali/1'>overall conformation</scene> of the two evolved variants <font color='lime'><b>R12-2</b></font> (3gkl) and <font color='cyan'><b>R12-13</b></font> ([[3gjn]]) is very similar. The variant <font color='lime'><b>R12-2</b></font> carries <scene name='3gkl/Ali/2'>mutation E41G</scene>. In the bound wildtype Im9 (yellow) Glu41 makes a <scene name='3gkl/Ali/3'>salt bridge</scene> with the <font color='orange'><b>ColE9’s</b></font> Lys97 ([[1bxi]]). While in the <font color='blueviolet'><b>R12-13</b></font>/<font color='cyan'><b>ColE7</b></font> complex the <font color='blueviolet'><b>closest ColE7 residues</b></font> <scene name='3gkl/Ali/4'>contacting</scene> <font color='cyan'><b>R12-13 Glu41</b></font> are <font color='blueviolet'><b>Thr531 (3.37Å) and Lys528 (8.85Å)</b></font> ([[3gjn]]). In the <font color='lime'><b>R12-2</b></font>/<font color='magenta'><b>ColE7</b></font> complex the <scene name='3gkl/Ali/5'>closest</scene> <font color='magenta'><b>ColE7 residue</b></font> to <font color='lime'><b>R12-2 Gly41</b></font> is <font color='magenta'><b>Thr531 (9.48Å)</b></font> (3gkl).
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==References==
==References==
<ref group="xtra">PMID:18354394</ref> <ref group='xtra'>PMID:20036254</ref> <references group="xtra"/>
<ref group="xtra">PMID:18354394</ref> <ref group='xtra'>PMID:19749752</ref> <ref group='xtra'>PMID:20036254</ref> <references group="xtra"/>




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[[Category: Lyase]]
[[Category: Lyase]]
[[Category: Structural genomic]]
[[Category: Structural genomic]]
[[Category: Antibiotic]]
[[Category: Antimicrobial]]
[[Category: Bacteriocin]]
[[Category: Bacteriocin immunity]]
[[Category: Endonuclease]]
[[Category: Hydrolase]]
[[Category: Metal-binding]]
[[Category: Nuclease]]
[[Category: Plasmid]]
[[Category: Protein-protein complex]]
[[Category: Zinc]]

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

Alexander Berchansky, Karsten Theis
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