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Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags

Abdelkader, Elwy; Yao, Xuejun; Feintuch, Akiva; Adams, Luke A; Aurelio, Luigi; Graham, Bim; Goldfarb, Daniella; Otting, Gottfried

Description

Pseudocontact shifts (PCS) induced by tags loaded with paramagnetic lanthanide ions provide powerful long-range structure information, provided the location of the metal ion relative to the target protein is known. Usually, the metal position is determined by fitting the magnetic susceptibility anisotropy (Δχ) tensor to the 3D structure of the protein in an 8-parameter fit, which requires a large set of PCSs to be reliable. In an alternative approach, we used multiple Gd(3+)-Gd(3+) distances...[Show more]

dc.contributor.authorAbdelkader, Elwy
dc.contributor.authorYao, Xuejun
dc.contributor.authorFeintuch, Akiva
dc.contributor.authorAdams, Luke A
dc.contributor.authorAurelio, Luigi
dc.contributor.authorGraham, Bim
dc.contributor.authorGoldfarb, Daniella
dc.contributor.authorOtting, Gottfried
dc.date.accessioned2018-10-18T03:08:11Z
dc.date.available2018-10-18T03:08:11Z
dc.identifier.issn0925-2738
dc.identifier.urihttp://hdl.handle.net/1885/148498
dc.description.abstractPseudocontact shifts (PCS) induced by tags loaded with paramagnetic lanthanide ions provide powerful long-range structure information, provided the location of the metal ion relative to the target protein is known. Usually, the metal position is determined by fitting the magnetic susceptibility anisotropy (Δχ) tensor to the 3D structure of the protein in an 8-parameter fit, which requires a large set of PCSs to be reliable. In an alternative approach, we used multiple Gd(3+)-Gd(3+) distances measured by double electron-electron resonance (DEER) experiments to define the metal position, allowing Δχ-tensor determinations from more robust 5-parameter fits that can be performed with a relatively sparse set of PCSs. Using this approach with the 32 kDa E. coli aspartate/glutamate binding protein (DEBP), we demonstrate a structural transition between substrate-bound and substrate-free DEBP, supported by PCSs generated by C3-Tm(3+) and C3-Tb(3+) tags attached to a genetically encoded p-azidophenylalanine residue. The significance of small PCSs was magnified by considering the difference between the chemical shifts measured with Tb(3+) and Tm(3+) rather than involving a diamagnetic reference. The integrative sparse data approach developed in this work makes poorly soluble proteins of limited stability amenable to structural studies in solution, without having to rely on cysteine mutations for tag attachment.
dc.description.sponsorshipFinancial support by the Australian Research Council (ARC) and an Australia-Weizmann Making Connections grant is gratefully acknowledged. B. G. thanks the ARC for a Future Fellowship.
dc.format.mimetypeapplication/pdf
dc.publisherSpringer Verlag
dc.rights© Springer Science+Business Media Dordrecht 2015. http://www.sherpa.ac.uk/romeo/issn/0925-2738/..."Author's post-print on any open access repository after 12 months after publication" from SHERPA/RoMEO site (as at 18/10/18). This is a post-peer-review, pre-copyedit version of an article published in Journal of biomolecular NMR. The final authenticated version is available online at: http://dx.doi.org/10.1007/s10858-015-0003-z
dc.sourceJournal of biomolecular NMR
dc.subjectdouble electron–electron resonance
dc.subjecte. coli aspartate/glutamate binding protein
dc.subjectintegrative structural biology
dc.subjectlanthanide tag
dc.subjectpseudocontact shift
dc.subjectalgorithms
dc.subjectmagnetic resonance spectroscopy
dc.subjectmodels, molecular
dc.subjectmutagenesis, site-directed
dc.subjectprotein conformation
dc.subjectproteins
dc.titlePulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags
dc.typeJournal article
local.identifier.citationvolume64
dc.date.issued2016-01
local.publisher.urlhttps://link.springer.com
local.type.statusAccepted Version
local.contributor.affiliationAbdelkader, E. H., Research School of Chemistry, The Australian National University
local.contributor.affiliationYao, Xuejun, Research School of Chemistry, The Australian National University
local.contributor.affiliationOtting, G., Research School of Chemistry, The Australian National University
local.identifier.essn1573-5001
local.bibliographicCitation.issue1
local.bibliographicCitation.startpage39
local.bibliographicCitation.lastpage51
local.identifier.doi10.1007/s10858-015-0003-z
dcterms.accessRightsOpen Access
CollectionsANU Research Publications

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