NMR method development for large proteins presented at ENC 2005

NMR structure/dynamics of apo- and holo-forms of heme chaperone ccmE

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

Development of NMR techniques - Longitudinal and transverse spin relaxation optimization: TROSY, XY-TROSY, LTROSY, CRINEPT, Poly-SPI Direct detection of H-bonds by polarization transfer Quantum chemical calculations (DFT) of NMR measurables: coupling across H-bonds, chemical shifts etc. 13 C detection spectroscopy for deuterated and paramagnetic systems: new strategy for backbone and side chain assignment, - 13 C- 13 C residual dipolar couplings etc. Cross-correlated relaxation for structure refinement Optimal control theory for construction of theoretically optimal NMR experiments: [ 1 H, 13 C  (Methyl) ]TROSY, COCAIN, diagonal-free NOESY-TROSY etc. - Automation in NMR: automatic assignment, AI knowledge communicating systems

Chemical shift correlations in protein backbone spin systems using TROSY

Remodelling of outer membrane protein A A NMR conformer of the N-terminal domain of OmpA A BBP NMR structure exterior outer membrane periplasmic space EF-hand loop III Tb 3+ Thr Ser Asp Lys Asp Gly Asn Gly Tyr Ile Ser Ala Ala Glu Ala Ser

Role of flexible C-terminal 15 amino acids of 44 kDa BsCM in catalysis Endo -oxabicyclic transition state analog, TSA Putative transition state

Refolding of HTH DBP protein in 6 M Urea by Hofmeister reagents HTH in 6 M Urea unfolded HTH in 6 M Urea, 1.7 M NaCl native structure refolding HTH in 6 M Urea unfolded HTH in 6 M Urea, 0.5 M NaTFA distorted structure, molten globule refolding

Engineered monomeric chorismate mutase lacking a preorganized structure 1

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

The primate erythrocyte/immune complex clearing mechanism

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 quantum 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

Definition of the optimization problem   C U H =  J  I z S z +  x (t) I x +  y (t) I y

Definition of the optimization problem for isolated 2 spin ½ system S I DD( IS ), CSA( S ) and CSA( I ) interactions max (Khaneja et al, PNAS, 2003, 100, 13162)

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

An overview Construction of optimal polarizationtransfer schemes for 220 kDa complex, CR1(SCR 15-17)/C3b - 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

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

A schematic model of intramolecular dynamics in FkpA

Chemical shift changes by complex formation with (1) reduced and carboxymethylated bovine  -lactalbumin, (2) RNAse AS

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 Atomic structure is 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

R ex by cross-correlated relaxation 0 20 40 60 80 100 120 20 30 40 50 60 70 80 90 100 110 120 Residue Number R 2 , s -1 R 2 R 2 - R ex fr. CCR

Ratio between peak volumes corresponding to oligomerization states of RNAse AS R = V oligomeric /V monomeric RNAse AS [mM]

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 at 600 MHz

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 Krystina Bromek Dr. Donghan Lee SNF and ETH for financial support Prof. Paul Barlow Prof. Ari Helenius Dr. Christiana Ritter

NMR method development for large proteins presented at ENC 2005

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NMR method development for large proteins presented at ENC 2005