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==4m5.3 anti-fluorescein single chain antibody fragment (scFv)== | |||
<StructureSection load='1x9q' size='340' side='right'caption='[[1x9q]], [[Resolution|resolution]] 1.50Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[1x9q]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1X9Q OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1X9Q FirstGlance]. <br> | |||
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 1.5Å</td></tr> | |||
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=ACT:ACETATE+ION'>ACT</scene>, <scene name='pdbligand=FLU:2-(6-HYDROXY-3-OXO-3H-XANTHEN-9-YL)-BENZOIC+ACID'>FLU</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=1x9q FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1x9q OCA], [https://pdbe.org/1x9q PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1x9q RCSB], [https://www.ebi.ac.uk/pdbsum/1x9q PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1x9q ProSAT]</span></td></tr> | |||
</table> | |||
== Evolutionary Conservation == | |||
[[Image:Consurf_key_small.gif|200px|right]] | |||
Check<jmol> | |||
<jmolCheckbox> | |||
<scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/x9/1x9q_consurf.spt"</scriptWhenChecked> | |||
<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked> | |||
<text>to colour the structure by Evolutionary Conservation</text> | |||
</jmolCheckbox> | |||
</jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=1x9q ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Here, we compare an antibody with the highest known engineered affinity (K(d)=270 fM) to its high affinity wild-type (K(d)=700 pM) through thermodynamic, kinetic, structural, and theoretical analyses. The 4M5.3 anti-fluorescein single chain antibody fragment (scFv) contains 14 mutations from the wild-type 4-4-20 scFv and has a 1800-fold increase in fluorescein-binding affinity. The dissociation rate is approximately 16,000 times slower in the mutant; however, this substantial improvement is offset somewhat by the association rate, which is ninefold slower in the mutant. Enthalpic contributions to binding were found by calorimetry to predominate in the differential binding free energy. The crystal structure of the 4M5.3 mutant complexed with antigen was solved to 1.5A resolution and compared with a previously solved structure of an antigen-bound 4-4-20 Fab fragment. Strikingly, the structural comparison shows little difference between the two scFv molecules (backbone RMSD of 0.6A), despite the large difference in affinity. Shape complementarity exhibits a small improvement between the variable light chain and variable heavy chain domains within the antibody, but no significant improvement in shape complementarity of the antibody with the antigen is observed in the mutant over the wild-type. Theoretical modeling calculations show electrostatic contributions to binding account for -1.2 kcal/mol to -3.5 kcal/mol of the binding free energy change, of which -1.1 kcal/mol is directly associated with the mutated residue side-chains. The electrostatic analysis reveals several mechanistic explanations for a portion of the improvement. Collectively, these data provide an example where very high binding affinity is achieved through the cumulative effect of many small structural alterations. | |||
Substantial energetic improvement with minimal structural perturbation in a high affinity mutant antibody.,Midelfort KS, Hernandez HH, Lippow SM, Tidor B, Drennan CL, Wittrup KD J Mol Biol. 2004 Oct 22;343(3):685-701. PMID:15465055<ref>PMID:15465055</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 1x9q" style="background-color:#fffaf0;"></div> | |||
== | ==See Also== | ||
*[[Antibody 3D structures|Antibody 3D structures]] | |||
[[ | *[[Monoclonal Antibodies 3D structures|Monoclonal Antibodies 3D structures]] | ||
[[ | *[[3D structures of human antibody|3D structures of human antibody]] | ||
[[ | == References == | ||
[[Category: | <references/> | ||
[[Category: | __TOC__ | ||
[[Category: | </StructureSection> | ||
[[Category: | [[Category: Homo sapiens]] | ||
[[Category: | [[Category: Large Structures]] | ||
[[Category: | [[Category: Drennan CL]] | ||
[[Category: | [[Category: Hernandez HH]] | ||
[[Category: | [[Category: Lippow SM]] | ||
[[Category: Midelfort KS]] | |||
[[Category: Tidor B]] | |||
[[Category: Wittrup KD]] | |||
Latest revision as of 09:43, 23 August 2023
4m5.3 anti-fluorescein single chain antibody fragment (scFv)4m5.3 anti-fluorescein single chain antibody fragment (scFv)
Structural highlights
Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedHere, we compare an antibody with the highest known engineered affinity (K(d)=270 fM) to its high affinity wild-type (K(d)=700 pM) through thermodynamic, kinetic, structural, and theoretical analyses. The 4M5.3 anti-fluorescein single chain antibody fragment (scFv) contains 14 mutations from the wild-type 4-4-20 scFv and has a 1800-fold increase in fluorescein-binding affinity. The dissociation rate is approximately 16,000 times slower in the mutant; however, this substantial improvement is offset somewhat by the association rate, which is ninefold slower in the mutant. Enthalpic contributions to binding were found by calorimetry to predominate in the differential binding free energy. The crystal structure of the 4M5.3 mutant complexed with antigen was solved to 1.5A resolution and compared with a previously solved structure of an antigen-bound 4-4-20 Fab fragment. Strikingly, the structural comparison shows little difference between the two scFv molecules (backbone RMSD of 0.6A), despite the large difference in affinity. Shape complementarity exhibits a small improvement between the variable light chain and variable heavy chain domains within the antibody, but no significant improvement in shape complementarity of the antibody with the antigen is observed in the mutant over the wild-type. Theoretical modeling calculations show electrostatic contributions to binding account for -1.2 kcal/mol to -3.5 kcal/mol of the binding free energy change, of which -1.1 kcal/mol is directly associated with the mutated residue side-chains. The electrostatic analysis reveals several mechanistic explanations for a portion of the improvement. Collectively, these data provide an example where very high binding affinity is achieved through the cumulative effect of many small structural alterations. Substantial energetic improvement with minimal structural perturbation in a high affinity mutant antibody.,Midelfort KS, Hernandez HH, Lippow SM, Tidor B, Drennan CL, Wittrup KD J Mol Biol. 2004 Oct 22;343(3):685-701. PMID:15465055[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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