Combining SPR with atomic-force microscopy enables single-molecule insights into activation and suppression of the complement cascade
| Autor: | John David Parkin, Elisavet Makou, Richard G. Bailey, Georg Hähner, Heather J. Johnston, Paul N. Barlow, Alison N. Hulme |
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| Přispěvatelé: | The Leverhulme Trust, University of St Andrews. School of Chemistry, University of St Andrews. EaSTCHEM |
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
QH301 Biology single-molecule biophysics Immunology NDAS chemical and pharmacologic phenomena molecular stretching Microscopy Atomic Force Complement factor B Biochemistry C3b immune response Protein–protein interaction protein-protein interaction 03 medical and health sciences QH301 Protein structure protein conformation Cleave Molecule Humans atomic force microscopy (AFM) Surface plasmon resonance Molecular Biology single-molecule analysis Complement Activation complement system 030102 biochemistry & molecular biology biology Chemistry Cell Biology Surface Plasmon Resonance factor H Complement system Kinetics 030104 developmental biology Immobilized Proteins inflammation Complement C3d self-assembling monolayer Complement Factor H Complement C3b biology.protein Biophysics Factor D surface plasmon resonance (SPR) Protein Binding |
| Zdroj: | The Journal of Biological Chemistry Makou, 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 single-molecule insights into activation and suppression of the complement cascade ', Journal of Biological Chemistry, vol. 294, no. 52, pp. 20148-20163 . https://doi.org/10.1074/jbc.RA119.010913 |
| ISSN: | 1083-351X 0021-9258 |
| DOI: | 10.1074/jbc.RA119.010913 |
| Popis: | This work was supported by Leverhulme Trust Grant RPG-2015-109. 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 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. Publisher PDF |
| Databáze: | OpenAIRE |
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