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/Ligandinteractions in large and dynamically

disordered systems studied by NMR insolution

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.

- 13C detection spectroscopy for deuterated and paramagneticsystems: new strategy for backbone and side chain assignment, - 13C-13C residual dipolar couplings etc.

- Cross-correlated relaxation for structure refinement

- Optimal control theory for construction of theoretically optimalNMR experiments: [1H,13C(Methyl)]TROSY, COCAIN,diagonal-free NOESY-TROSY etc.

- Automation in NMR: automatic assignment, AI knowledgecommunicating 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

outermembrane

periplasmicspace

EF-hand loop III

Tb3+

ThrSerAsp

LysAspGly

AsnGlyTyrIleSer

AlaAla

GluAla

Ser

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

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 Ureaunfolded

HTH in 6 M Urea, 1.7 M NaClnative structure

refolding

HTH in 6 M Ureaunfolded

HTH in 6 M Urea, 0.5 M NaTFAdistorted structure, molten globule

refolding

1

Engineered monomeric chorismate mutase lacking a preorganized structure

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 (1H) [ppm]

1 (15N) [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

CU

H = J IzSz + x(t) Ix + y(t) Iy

Definition of the optimization problem for isolated 2 spin ½ system

S

IDD(IS), CSA(S) and CSA(I) interactions

max

(Khaneja et al, PNAS, 2003, 100, 13162)

Differential driving of the manifolds Iand I by

selective rf-pulse

Iz = Iz+ I z → Iz

I z = 2Iz Sz

Ii= Ii(1/2E +Sz)

Ii= Ii(1/2E Sz) Iz

I z

Excitation profile of polychomatic pulse

Polychomatic pulse wave-form and spin trajectory

Polarization transfer using polychromatic irradiation

2 (1H) [ppm]

1 (15N) [ppm]

CRINEPTPOLY-C

PC-SPI spectra of free CR1 and CR1/C3b complex

CR1/C3b complex

CR122 kDa

CR1/C3b complex220 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

SubstrateSubstrate

54 kDa „moonlight“ chaperone with PPIase activity

15N relaxation measurements of free FkpA at 600 MHz

15N relaxation measurements with FkpA at 600 MHz

1H-15N RDCs measurements in the presence of Pf1 phages

Histogramm of RDCs values in two media

C12E5 / hexanol/H2OLn-Alkyl-poly(ethylene glycol)/n-alkyl alcohol and glucopone/n-hexanol mixtures

Phages Pf1

RDCs values in Pf1 medium

RDCs values in Pf1 medium

A schematic model of intramolecular dynamics in FkpA

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

Chemical shifts mapping

Equilibrium binding of FkpA to substrates: (1) reduced and carboxymethylated bovine -lactalbumin, (2) RNAse AS

Kd = 540 m

Protein Quality Control in the ER

Substrates recognized by GT

RNase B RNase BS RNase BS 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 15N-1H HSQC

RNase A:complete assignmentis available

Assignment of S-Protein

6.007.008.009.0010.00

105.00

110.00

115.00

120.00

125.00

130.00

7498

99

62

94

96 41 91

124

60

61

72

70 68

112

123

77

6597

40?

76

109

124

100

75

7144?

83

120?

6395

111

7964

56

5790

21

69 28?

30?

78 67

113

58

59

`39?

46

110

1H (ppm)

15N (ppm)

RNase S Protein:• Line broadening• Resonance doubling

RNase S:an additional set of resonances is observed

RNase A:complete assignmentis available

S peptide

cleavage

conformational exchange

Chemical Shift Difference between S protein and RNase A

Fast Amide Proton Exchange

15N-Relaxation measurements

Rex by cross-correlated relaxation

0

20

40

60

80

100

120

20 30 40 50 60 70 80 90 100 110 120Residue Number

R2

, s-1

R2

R2 - Rex fr. CCR

Concentration Scan

1.06 mM

Concentration Scan

0.2 mM

Concentration Scan

0.08 mM

Ratio between peak volumes corresponding to oligomerization states of RNAse AS

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 0.2 0.4 0.6 0.8 1 1.2 1.4

R = Voligomeric/Vmonomeric

RNAse AS [mM]

Lys 60

Delution Chaperone

Gln 65

Delution Chaperone

Leu 91

Delution Chaperone

Lys 95

Delution Chaperone

Conformational dynamics in S Protein

S Protein N S Protein Uku

kf

[S Protein]n

>30ms

~80 Hz

/(2 2) 30Hzck

kc

15N relaxation measurements of FkpA/S-protein complex at 600 MHz

R1[ 1/ s]

0

0. 5

1

1. 5

2

0 50 100 150 200 250

R2[1/ s]

0102030405060708090

100

0 50 100 150 200 250

15N relaxation measurements of free FkpA 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