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Protein/Protein and Protein/Ligandinteractions in large and dynamicallydisordered systems studied by NMR insolution
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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