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==== | ==Engineered streptavidin variant (YNAFM) in complex with the Strep-tag II peptide== | ||
<StructureSection load='6tip' size='340' side='right'caption='[[6tip]]' scene=''> | <StructureSection load='6tip' size='340' side='right'caption='[[6tip]], [[Resolution|resolution]] 2.10Å' scene=''> | ||
== Structural highlights == | == Structural highlights == | ||
<table><tr><td colspan='2'>Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id= OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol= FirstGlance]. <br> | <table><tr><td colspan='2'>[[6tip]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Streptomyces_avidinii Streptomyces avidinii] and [https://en.wikipedia.org/wiki/Synthetic_construct Synthetic construct]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6TIP OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=6TIP FirstGlance]. <br> | ||
</td></tr><tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=6tip FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6tip OCA], [https://pdbe.org/6tip PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=6tip RCSB], [https://www.ebi.ac.uk/pdbsum/6tip PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=6tip ProSAT]</span></td></tr> | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.1Å</td></tr> | ||
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=NH2:AMINO+GROUP'>NH2</scene></td></tr> | |||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=6tip FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6tip OCA], [https://pdbe.org/6tip PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=6tip RCSB], [https://www.ebi.ac.uk/pdbsum/6tip PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=6tip ProSAT]</span></td></tr> | |||
</table> | </table> | ||
== Function == | |||
[https://www.uniprot.org/uniprot/SAV_STRAV SAV_STRAV] The biological function of streptavidin is not known. Forms a strong non-covalent specific complex with biotin (one molecule of biotin per subunit of streptavidin). | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
The affinity system based on the artificial peptide ligand Strep-tag(R) II and engineered tetrameric streptavidin, known as Strep-Tactin(R), offers attractive applications for the study of recombinant proteins, from detection and purification to functional immobilization. To further improve binding of the Strep-tag II to streptavidin we have subjected two protruding loops that shape its ligand pocket for the peptide - instead of D-biotin recognized by the natural protein - to iterative random mutagenesis. Sequence analyses of hits from functional screening assays revealed several unexpected structural motifs, such as a disulfide bridge at the base of one loop, replacement of the crucial residue Trp120 by Gly and a two-residue deletion in the second loop. The mutant m1-9 (dubbed Strep-Tactin XT) showed strongly enhanced affinity towards the Strep-tag II, which was further boosted in case of the bivalent Twin-Strep-tag(R). Four representative streptavidin mutants were crystallized in complex with the Strep-tag II peptide and their X-ray structures were solved at high resolutions. In addition, the crystal structure of the complex between Strep-Tactin XT and the Twin-Strep-tag was elucidated, indicating a bivalent mode of binding and explaining the experimentally observed avidity effect. Our study illustrates the structural plasticity of streptavidin as a scaffold for ligand binding and reveals interaction modes that would have been difficult to predict. As result, Strep-Tactin XT offers a convenient reagent for the kinetically stable immobilization of recombinant proteins fused with the Twin-Strep-tag. The possibility of reversibly dissociating such complexes simply with D-biotin as a competing ligand enables functional studies in protein science as well as cell biology. | |||
The Role of Changing Loop Conformations in Streptavidin Versions Engineered for High-affinity Binding of the Strep-tag II Peptide.,Schmidt TGM, Eichinger A, Schneider M, Bonet L, Carl U, Karthaus D, Theobald I, Skerra A J Mol Biol. 2021 Apr 30;433(9):166893. doi: 10.1016/j.jmb.2021.166893. Epub 2021 , Feb 24. PMID:33639211<ref>PMID:33639211</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 6tip" style="background-color:#fffaf0;"></div> | |||
==See Also== | |||
*[[Avidin 3D structures|Avidin 3D structures]] | |||
== References == | |||
<references/> | |||
__TOC__ | __TOC__ | ||
</StructureSection> | </StructureSection> | ||
[[Category: Large Structures]] | [[Category: Large Structures]] | ||
[[Category: | [[Category: Streptomyces avidinii]] | ||
[[Category: Synthetic construct]] | |||
[[Category: Eichinger A]] | |||
[[Category: Skerra A]] | |||
Latest revision as of 16:03, 24 January 2024
Engineered streptavidin variant (YNAFM) in complex with the Strep-tag II peptideEngineered streptavidin variant (YNAFM) in complex with the Strep-tag II peptide
Structural highlights
FunctionSAV_STRAV The biological function of streptavidin is not known. Forms a strong non-covalent specific complex with biotin (one molecule of biotin per subunit of streptavidin). Publication Abstract from PubMedThe affinity system based on the artificial peptide ligand Strep-tag(R) II and engineered tetrameric streptavidin, known as Strep-Tactin(R), offers attractive applications for the study of recombinant proteins, from detection and purification to functional immobilization. To further improve binding of the Strep-tag II to streptavidin we have subjected two protruding loops that shape its ligand pocket for the peptide - instead of D-biotin recognized by the natural protein - to iterative random mutagenesis. Sequence analyses of hits from functional screening assays revealed several unexpected structural motifs, such as a disulfide bridge at the base of one loop, replacement of the crucial residue Trp120 by Gly and a two-residue deletion in the second loop. The mutant m1-9 (dubbed Strep-Tactin XT) showed strongly enhanced affinity towards the Strep-tag II, which was further boosted in case of the bivalent Twin-Strep-tag(R). Four representative streptavidin mutants were crystallized in complex with the Strep-tag II peptide and their X-ray structures were solved at high resolutions. In addition, the crystal structure of the complex between Strep-Tactin XT and the Twin-Strep-tag was elucidated, indicating a bivalent mode of binding and explaining the experimentally observed avidity effect. Our study illustrates the structural plasticity of streptavidin as a scaffold for ligand binding and reveals interaction modes that would have been difficult to predict. As result, Strep-Tactin XT offers a convenient reagent for the kinetically stable immobilization of recombinant proteins fused with the Twin-Strep-tag. The possibility of reversibly dissociating such complexes simply with D-biotin as a competing ligand enables functional studies in protein science as well as cell biology. The Role of Changing Loop Conformations in Streptavidin Versions Engineered for High-affinity Binding of the Strep-tag II Peptide.,Schmidt TGM, Eichinger A, Schneider M, Bonet L, Carl U, Karthaus D, Theobald I, Skerra A J Mol Biol. 2021 Apr 30;433(9):166893. doi: 10.1016/j.jmb.2021.166893. Epub 2021 , Feb 24. PMID:33639211[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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