1 / 24

Applications

Applications. cbEGF32-33 with and without Ca 2+ c Refinement using T 1 /T 2 ratios in 4 F1- 5 F1 Peptide Binding in 1 F1- 2 F1. N. N. C. C. TB-6. cbEGF32-33. Domain Organisation of Human Fibrillin-1. TB-6. cbEGF32-33. Fibrillin is Part of the EM. Fibrillin Microfibril

lowri
Download Presentation

Applications

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Applications • cbEGF32-33 with and without Ca2+c • Refinement using T1/T2 ratios in 4F1-5F1 • Peptide Binding in 1F1-2F1

  2. N N C C TB-6 cbEGF32-33 Domain Organisation of Human Fibrillin-1 TB-6 cbEGF32-33

  3. Fibrillin is Part of the EM Fibrillin Microfibril Extracellular Matrix

  4. Model for Microfibril Organisation = putative MAB binding site

  5. Ca2+ modulates overall structure EM Micrographs of Microfibrils with Ca2+ with EDTA

  6. Measurement of 15N-T1 and T2

  7. tm= 5.1 ns D||/D = 1.55 sin2(a) Ca2+ bound cbEGF32-33 is elongated

  8. Modelfree analysis of Ca2+ bound

  9. Relaxation of cbEGF32-33:with and without Ca2+ cbEGF32 cbEGF33 • local motions dominate cbEGF32 cbEGF33

  10. Removal of Ca2+ Increases Fast Motion

  11. N Ca2+ bound Ca2+ free N C C Removal of Ca2+ Increases Slow Motion Rex > 8 Hz Rex 4-8 Hz Rex 2-4 Hz

  12. Model of the effect of removing Ca2+ Wobble model

  13. 10F3 1F2 1F1 Modular Structure and Binding Sites of Fibronectin Cross Linking EDA EDB Site IIICS COOH RGD s s NH 2 COOH Fibrin Cell Heparin Collagen Fibrin Gelatin S. aureus C1q Heparin

  14. family of refined structures (without T1/T2) for each structure (i) fit T1/T2 data to obtain a c2(i) high c2(i) “bad” structures intermediate c2(i) structures low c2(i) “good” structures Use of T1/T2 Data in Analysis rank structures according to c2(i) classify structures

  15. Ranking of Structures: 4F15F1 8 structures with high c2 8 structures with intermediate c2 8 structures with low c2 RMSD4F1= 1.04 Å RMSD5F1= 9.38 Å RMSD4F1= 1.10 Å RMSD5F1= 8.62 Å RMSD4F1= 1.07 Å RMSD5F1= 4.40 Å

  16. Refinement with T1/T2 Potential Tjandra et al. (1997) Nat.Struct.Biol4, 443 • simulated annealing (Xplor(v3.8) or CNS (0.9)): Epot(r)=Ebonded(r)+Enonbonded(r)+Eexp(r)experimental: Eexp(r)=ENOE +Edihedral+ET1/T2 • T1/T2 potential: ET1/T2= kDANI [(T1/T2calc)-(T1/T2exp)]2

  17. Optimisation of Parameters A) force constant: B) diffusion tensor C) SA protocol?

  18. In vacuo In vacuo & T1/T2 4F1 5F1 Structure Refinement of 4F15F1 Inclusion of T1/T2 restraints improves definition of structure

  19. 1F1 2F1 1F1 2F1 B3 peptide B3 peptide 1F1 2F1 • AUC • ITC B3 peptide B3 forms a 1:1 Complex with 1F12F1

  20. 1F1 1F1 2F1 2F1 B3 peptide B3 Peptide Increases Anisotropy

  21. 1F1 2F1 1F1 2F1 B3 peptide B3 peptide 1F1 2F1 • AUC • ITC B3 peptide B3 forms a 1:1 Complex with 1F12F1

  22. 1F1 1F1 2F1 2F1 B3 peptide Ligand induced tethering

  23. Ligand tethering: Structures Complex NOE,RDC,T1/T2 free Complex (NOE)

  24. Summary • Relation ship between relaxation and dynamics • T1, T2 etc depend on J(w) at specific frequencies • motional models (diffusion) provide theoretical J(w) • fit T1,T2 to motional models via J(w) • extract correlation times and amplitudes

More Related