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dc.contributor.authorMakou, Elisavet
dc.contributor.authorBailey, Richard G.
dc.contributor.authorJohnston, Heather
dc.contributor.authorParkin, John D.
dc.contributor.authorHulme, Alison N.
dc.contributor.authorHähner, Georg
dc.contributor.authorBarlow, Paul N.
dc.identifier.citationMakou , E , Bailey , R G , Johnston , H , Parkin , J D , Hulme , A N , Hähner , G & Barlow , P N 2019 , ' Combining SPR with atomic-force microscopy enables singlemolecule insights into activation and suppression of the complement cascade ' , Journal of Biological Chemistry , vol. 294 , no. 52 , pp. 20148-20163 .
dc.identifier.otherPURE: 265752681
dc.identifier.otherPURE UUID: 088924ca-2baf-4fb6-959a-7cd398db4024
dc.identifier.otherScopus: 85077295881
dc.identifier.otherPubMed: 31719147
dc.identifier.otherORCID: /0000-0002-6765-344X/work/67525757
dc.identifier.otherWOS: 000505005800026
dc.descriptionThis work was supported by Leverhulme Trust Grant RPG-2015-109.en
dc.description.abstractActivation and suppression of the complement system compete on every serum-exposed surface, host or foreign. Potentially harmful outcomes of this competition depend on surface molecules through mechanisms that remain incompletely understood. Combining surface plasmon resonance (SPR) with atomic force microscopy (AFM), here we studied two complement system proteins at the single-molecule level: C3b, the proteolytically activated form of C3, and factor H (FH), the surface-sensing C3b-binding complement regulator. We used SPR to monitor complement initiation occurring through a positive-feedback loop wherein surface-deposited C3b participates in convertases that cleave C3, thereby depositing more C3b. Over multiple cycles of flowing factor B, factor D, and C3 over the SPR chip, we amplified C3b from ∼20 to ∼220 molecules·μm−2. AFM revealed C3b clusters of up to 20 molecules and solitary C3b molecules deposited up to 200 nm away from the clusters. A force of 0.17 ± 0.02 nanonewtons was needed to pull a single FH molecule, anchored to the AFM probe, from its complex with surface-attached C3b. The extent to which FH molecules stretched before detachment varied widely among complexes. Performing force-distance measurements with FH(D1119G), a variant lacking one of the C3b-binding sites and causing atypical hemolytic uremic syndrome, we found that it detached more uniformly and easily. In further SPR experiments, KD values between FH and C3b on a custom-made chip surface were 5-fold tighter than on commercial chips and similar to those on erythrocytes. These results suggest that the chemistry at the surface on which FH acts drives conformational adjustments that are functionally critical.
dc.relation.ispartofJournal of Biological Chemistryen
dc.rightsCopyright © 2019 Makou et al. Final version open access under the terms of the Creative Commons CC-BY license.en
dc.subjectSurface plasmon resonance (SPR)en
dc.subjectAtomic force microscopy (AFM)en
dc.subjectComplement systemen
dc.subjectProtein-protein interactionen
dc.subjectProtein conformationen
dc.subjectSingle-molecule biophysicsen
dc.subjectFactor Hen
dc.subjectImmune responseen
dc.subjectMolecular stretchingen
dc.subjectSelf-assembling monolayeren
dc.subjectSingle-molecule analysisen
dc.subjectQH301 Biologyen
dc.subjectMolecular Biologyen
dc.subjectCell Biologyen
dc.titleCombining SPR with atomic-force microscopy enables singlemolecule insights into activation and suppression of the complement cascadeen
dc.typeJournal articleen
dc.description.versionPublisher PDFen
dc.contributor.institutionUniversity of St Andrews.School of Chemistryen
dc.contributor.institutionUniversity of St Andrews.EaSTCHEMen
dc.description.statusPeer revieweden

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