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1. Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins

   https://doi.org/10.1371/journal.pone.0258531  

   Boyko, Kristina V.; Rosenkranz, Erin A.; Smith, Derrick M.; Miears, Heather L.; Oueld es cheikh, Melissa; Lund, Micah Z.; Young, Jeffery C.; Reardon, Patrick N.; Okon, Mark; Smirnov, Serge L.; et al (October 2021, PLOS ONE)

   van der Wel, Patrick (Ed.)

   A significant number of proteins possess sizable intrinsically disordered regions (IDRs). Due to the dynamic nature of IDRs, NMR spectroscopy is often the tool of choice for characterizing these segments. However, the application of NMR to IDRs is often hindered by their instability, spectral overlap and resonance assignment difficulties. Notably, these challenges increase considerably with the size of the IDR. In response to these issues, here we report the use of sortase-mediated ligation (SML) for segmental isotopic labeling of IDR-containing samples. Specifically, we have developed a ligation strategy involving a key segment of the large IDR and adjacent folded headpiece domain comprising the C-terminus of A . thaliana villin 4 (AtVLN4). This procedure significantly reduces the complexity of NMR spectra and enables group identification of signals arising from the labeled IDR fragment, a process we refer to as segmental assignment . The validity of our segmental assignment approach is corroborated by backbone residue-specific assignment of the IDR using a minimal set of standard heteronuclear NMR methods. Using segmental assignment, we further demonstrate that the IDR region adjacent to the headpiece exhibits nonuniform spectral alterations in response to temperature. Subsequent residue-specific characterization revealed two segments within the IDR that responded to temperature in markedly different ways. Overall, this study represents an important step toward the selective labeling and probing of target segments within much larger IDR contexts. Additionally, the approach described offers significant savings in NMR recording time, a valuable advantage for the study of unstable IDRs, their binding interfaces, and functional mechanisms. 

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2. Histone H2B Deacylation Selectivity: Exploring Chromatin’s Dark Matter with an Engineered Sortase

   https://doi.org/10.1021/jacs.1c13555  

   Wang, Zhipeng A.; Whedon, Samuel D.; Wu, Mingxuan; Wang, Siyu; Brown, Edward A.; Anmangandla, Ananya; Regan, Liam; Lee, Kwangwoon; Du, Jianfeng; Hong, Jun Young; et al (February 2022, Journal of the American Chemical Society)
3. Sequence variation in the β7–β8 loop of bacterial class A sortase enzymes alters substrate selectivity

   https://doi.org/10.1016/j.jbc.2021.100981  

   Piper, Isabel M.; Struyvenberg, Sarah A.; Valgardson, Jordan D.; Johnson, D. Alex; Gao, Melody; Johnston, Katherine; Svendsen, Justin E.; Kodama, Hanna M.; Hvorecny, Kelli L.; Antos, John M.; et al (August 2021, Journal of Biological Chemistry)

   null (Ed.)
4. Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes

   https://doi.org/10.1002/pro.4266  

   Gao, Melody; Johnson, D. Alex; Piper, Isabel M.; Kodama, Hanna M.; Svendsen, Justin E.; Tahti, Elise; Longshore‐Neate, Frederick; Vogel, Brandon; Antos, John M.; Amacher, Jeanine F. (March 2022, Protein Science)
5. User‐Controlled 4D Biomaterial Degradation with Substrate‐Selective Sortase Transpeptidases for Single‐Cell Biology

   https://doi.org/10.1002/adma.202209904  

   Bretherton, Ross_C; Haack, Amanda_J; Kopyeva, Irina; Rahman, Fariha; Kern, Jonah_D; Bugg, Darrian; Theberge, Ashleigh_B; Davis, Jennifer; DeForest, Cole_A (March 2023, Advanced Materials)

   Abstract Stimuli‐responsive biomaterials show great promise for modeling disease dynamics ex vivo with spatiotemporal control over the cellular microenvironment. However, harvesting cells from such materials for downstream analysis without perturbing their state remains an outstanding challenge in 3/4‐dimensional (3D/4D) culture and tissue engineering. In this manuscript, a fully enzymatic strategy for hydrogel degradation that affords spatiotemporal control over cell release while maintaining cytocompatibility is introduced. Exploiting engineered variants of the sortase transpeptidase evolved to recognize and selectively cleave distinct peptide sequences largely absent from the mammalian proteome, many limitations implicit to state‐of‐the‐art methods to liberate cells from gels are sidestepped. It is demonstrated that evolved sortase exposure has minimal impact on the global transcriptome of primary mammalian cells and that proteolytic cleavage proceeds with high specificity; incorporation of substrate sequences within hydrogel crosslinkers permits rapid and selective cell recovery with high viability. In composite multimaterial hydrogels, it is shown that sequential degradation of hydrogel layers enables highly specific retrieval of single‐cell suspensions for phenotypic analysis. It is expected that the high bioorthogonality and substrate selectivity of the evolved sortases will lead to their broad adoption as an enzymatic material dissociation cue and that their multiplexed use will enable newfound studies in 4D cell culture. 

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