NMR studies of Complement complex and FkpA chaperone with substrate
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NMR studies of Complement complex and FkpA chaperone with substrate

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It is my presentation in Hoor Sweden in August 2005. Really new NMR methods have been developed to tackle the problem of big proteins in NMR

It is my presentation in Hoor Sweden in August 2005. Really new NMR methods have been developed to tackle the problem of big proteins in NMR

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NMR studies of Complement complex and FkpA chaperone with substrate NMR studies of Complement complex and FkpA chaperone with substrate Presentation Transcript

  • Protein/Protein and Protein/Ligand interactions in large and dynamically disordered systems studied by NMR in solution 65 Å Substrate
  • Protein/Protein and Protein/Ligand interactions in large and dynamically disordered systems studied by NMR in solution Prof. K. Pervushin, BioNMR group , LPC, D-CHAB, ETH Zürich
  • An overview
    • Construction of optimal polarization transfer schemes for
    • 220 kDa complex, CR1(SCR 15-17)/C3b
    - 54 kDa dimeric chaperone FkpA and FkpA/substrate complexes View slide
  • The primate erythrocyte/immune complex clearing mechanism View slide
  • Human complement receptor type 1 (CR1)
  • INEPT-based HSQC of 220 kDa CR1/C3b complex  2 ( 1 H) [ppm]  1 ( 15 N) [ppm]
  • Fundamental bounds associated with polarization/coherence transfer imposed by qunatum spin dynamics   C 1. Maximum transfer bound, U 2. Minimal spin-evolution time required for the transfer,  min 3. Suppression of spurious transfers,   Q 4. Combined use of more source operators,   C
  • Differential driving of the manifolds I  and I  by selective rf-pulse I z = I  z  + I  z -> I  z   I  z = 2 I z S z I  i  = I i  (1/2 E + S z ) I  i  = I i  (1/2 E  S z ) I  z  I  z
  • Excitation profile of polychomatic pulse
  • Polychomatic pulse wave-form and spin trajectory
  • Polarization transfer using polychromatic irradiation  2 ( 1 H) [ppm]  1 ( 15 N) [ppm] CRINEPT POLY-C
  • PC-SPI spectra of free CR1 and CR1/C3b complex
  • CR1/C3b complex CR1 22 kDa CR1/C3b complex 220 kDa
  • 54 kDa dimeric chaperone FkpA and FkpA/substrate complexes
  • 54 kDa „moonlight“ chaperone with PPIase activity 65 Å Substrate
  • 54 kDa „moonlight“ chaperone with PPIase activity
  • 15 N relaxation measurements of free FkpA at 600 MHz R 1 ,R 2 rates of ( 15 N) is function of local (as well as global) mobility
  • 15 N relaxation measurements with FkpA at 600 MHz
  • 1 H- 15 N RDCs measurements in the presence of Pf1 phages
  • Histogramm of RDCs values in two media C 12 E 5 / hexanol/H 2 O L  n -Alkyl-poly(ethylene glycol)/ n -alkyl alcohol and glucopone/ n -hexanol mixtures Phages Pf1
  • RDCs values in 2 alignment media C-domain in dimer C-domain in monomer N-domain in monomer
  • A schematic model of intramolecular dynamics in FkpA
  • Chemical shift changes by complex formation with (1) reduced and carboxymethylated bovine  -lactalbumin, (2) RNAse AS
  • Mapping of chemical shift changes induced by interactions with substrate
  • 15 N relaxation measurements of FkpA in free and complex with RNAse AS R 1 ,R 2 rates of ( 15 N) is function of local (as well as global) mobility
  • Equilibrium binding of FkpA to substrates: (1) reduced and carboxymethylated bovine  -lactalbumin, (2) RNAse AS K d = 540  m
  • Protein Quality Control in the ER
  • Substrates recognized by GT RNase B RNase BS RNase B S protein alkylated RNase B - + - GT: RNase BS” S peptide 15-mer scrambled RNase B small glyco- peptides + - - -
  • RNase A
    • Crystal and NMR
    • structure available
    • 124 amino acids
    • 4 disulfide bonds
  • RNase A S protein ? Ratnaparkhi, G. S., and Varadarajan, R. (2001) J Biol Chem 276 , 28789-98. Chakshusmathi, G., Ratnaparkhi, G. S., Madhu, P. K., and Varadarajan, R. (1999) PNAS 96 , 7899-7904. “ NMR … is not possible due to aggregation at millimolar concentration”
    • No structural data
    • available
    • 124 amino acids
    • 4 disulfide bonds
  • RNase A 15 N- 1 H HSQC RNase A: complete assignment is available
  • Assignment of S-Protein 74 98 99 62 94 96 41 91 124 60 61 72 70 68 112 123 77 65 97 40? 76 109 124 100 75 71 44? 83 120? 63 95 111 79 64 56 57 90 21 69 28? 30? 78 67 113 58 59 `39? 46 110 1 H (ppm) 15 N (ppm)
    • RNase S Protein:
    • Line broadening
    • Resonance doubling
    RNase S: an additional set of resonances is observed RNase A: complete assignment is available S peptide cleavage 6.00 7.00 8.00 9.00 10.00 105.00 110.00 115.00 120.00 125.00 130.00 conformational exchange
  • Chemical Shift Difference between S protein and RNase A
  • Fast Amide Proton Exchange Highest: < 2 s -1 (by magnetization transfer) Lowest: 35 min -1 (by 1 H/ 2 D – exchange)
  • 15 N-Relaxation measurements
  • R ex by cross-correlated relaxation red - second resonance set observed ct-XY-TROSY CCR Model Free
  • Model free analysis
  • RNase S Protein - Concentration Scan 1.06 mM H (ppm) 1 15 N (ppm)
  • 0.2 mM RNase S Protein - Concentration Scan H (ppm) 1 15 N (ppm)
  • 0.08 mM RNase S Protein - Concentration Scan 1 H (ppm) 15 N (ppm)
  • Ratio between peak volumes corresponding to different oligomerization states of RNAse A S protein K d =1.2±0.08 mM 2 S-Prot [S Prot] 2 RNase S Protein - Concentration Scan 15 N (ppm) H (ppm) 1
  • Yanshun Liu et al. Protein Sci 2002; 11: 1285-1299 Fig. 2. Ribbon diagrams of the structures of the RNase A monomer (2.0 A, Wlodawer et al)
  • Yanshun Liu et al. Protein Sci 2002; 11: 1285-1299 Fig. 3. Ribbon representations of hypothetical models of RNase A tetramers
  • Mapping of “dimer” cross-peaks to monomeric and dimeric RNAse structures
  • High pressure NMR with S-protein M D Lys60
  • Dilution versus titration with chaperone Dilution of S protein Titration with FkpA Chaperone Lys 60 Leu 91
  • S Protein titration by the chaperone
  • Conformational dynamics in S Protein S Protein N S Protein U k u k f [S Protein] n >30ms ~80 Hz   k c
  • 15 N relaxation measurements of FkpA/S-protein complex
  • A „mother‘a arms“ model of chaperone activity of FkpA
  • Thanx a lot! Alexander Eletski Prof. Donald Hilvert Beat Vögeli Prof. Linda Thöny-Meier Dr. Osvaldo Moreira Prof. Andreas Plückthun Kaifeng Hu Dr. Helena Kovac (Bruker AG) Alexander Kienhoffer Dr. Maria Johansson Simon Alioth Katherina Vamvaca Dr. Krystina Bromek Veniamin Galius SNF and ETH for financial support Prof. Paul Barlow Prof. Ari Helenius Dr. Christiana Ritter