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==anti-Phosphohistidine Fab hSC44.ck with 3pHis peptide==
<StructureSection load='8uit' size='340' side='right'caption='[[8uit]], [[Resolution|resolution]] 2.20&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>[[8uit]] is a 6 chain structure with sequence from [https://en.wikipedia.org/wiki/Oryctolagus_cuniculus Oryctolagus cuniculus] 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=8UIT OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=8UIT 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]] 2.2&#8491;</td></tr>
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=NEP:N1-PHOSPHONOHISTIDINE'>NEP</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=8uit FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=8uit OCA], [https://pdbe.org/8uit PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=8uit RCSB], [https://www.ebi.ac.uk/pdbsum/8uit PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=8uit ProSAT]</span></td></tr>
</table>
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== Publication Abstract from PubMed ==
Histidine phosphorylation (pHis) is a non-canonical post-translational modification (PTM) that is historically understudied due to a lack of robust reagents that are required for its investigation, such as high affinity pHis-specific antibodies. Engineering pHis-specific antibodies is very challenging due to the labile nature of the phosphoramidate (P-N) bond and the stringent requirements for selective recognition of the two isoforms, 1-phosphohistidine (1-pHis) and 3-phosphohistidine (3-pHis). Here, we present a strategy for in vitro engineering of antibodies for detection of native 3-pHis targets. Specifically, we humanized the rabbit SC44-8 anti-3-pTza (a stable 3-pHis mimetic) mAb into a scaffold (herein referred to as hSC44) that was suitable for phage display. We then constructed six unique Fab phage-displayed libraries using the hSC44 scaffold and selected high affinity 3-pHis binders. Our selection strategy was carefully designed to enrich antibodies that bound 3-pHis with high affinity and had specificity for 3-pHis versus 3-pTza. hSC44.20N32F(L), the best engineered antibody, has an ~10-fold higher affinity for 3-pHis than the parental hSC44. Eleven new Fab structures, including the first reported antibody-pHis peptide structures were solved by X-ray crystallography. Structural and quantum mechanical calculations provided molecular insights into 3-pHis and 3-pTza discrimination by different hSC44 variants and their affinity increase obtained through in vitro engineering. Furthermore, we demonstrate the utility of these newly developed high-affinity 3-pHis-specific antibodies for recognition of pHis proteins in mammalian cells by immunoblotting and immunofluorescence staining. Overall, our work describes a general method for engineering PTM-specific antibodies and provides a set of novel antibodies for further investigations of the role of 3-pHis in cell biology.

Using phage display for rational engineering of a higher affinity humanized 3' phosphohistidine-specific antibody.,Martyn GD, Kalagiri R, Veggiani G, Stanfield RL, Choudhuri I, Sala M, Meisenhelder J, Chen C, Biswas A, Levy RM, Lyumkis D, Wilson IA, Hunter T, Sidhu SS bioRxiv [Preprint]. 2024 Nov 4:2024.11.04.621849. doi: 10.1101/2024.11.04.621849. PMID:39574610<ref>PMID:39574610</ref>

From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
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<div class="pdbe-citations 8uit" style="background-color:#fffaf0;"></div>
== References ==
<references/>
__TOC__
</StructureSection>
[[Category: Large Structures]]
[[Category: Oryctolagus cuniculus]]
[[Category: Synthetic construct]]
[[Category: Hunter T]]
[[Category: Kalagiri R]]
[[Category: Stanfield RL]]
[[Category: Wilson IA]]