Combining SPR with atomic-force microscopy enables single-molecule insights into activation and suppression of the complement cascade

Elisavet Makou, Richard G. Bailey, Heather Johnston, John D. Parkin, Alison N. Hulme, Georg Hähner, Paul N. Barlow

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

Activation 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 moleculesm2. 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 towhich 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.
Original languageEnglish
Pages (from-to)20148-20163
JournalJournal of Biological Chemistry
Issue number52
Publication statusPublished - 27 Dec 2019

Keywords / Materials (for Non-textual outputs)

  • surface plasmon resonance (SPR)
  • atomic force microscopy (AFM)
  • complement system
  • protein-protein interaction
  • protein conformation
  • inflammation
  • single-molecule biophysics
  • C3b
  • factor H
  • immune response
  • molecular stretching
  • self-assembling monolayer
  • single-molecule analysis


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