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NMR protein studies: projections

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EU NMR-Life / Extend-NMR Meeting. October 2008, Berlin. NMR ... assignment of all (aliphatic) hydrogens. with/without Hali-Cali connections. anchor on backbone ... – PowerPoint PPT presentation

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Title: NMR protein studies: projections


1
NMR protein studies projections and
decompositions
Doroteya K. Staykova Biophysics, Department of
Chemistry University of Gothenburg
2
NMR Data Flow
Sparse (non-uniform)coupled evolutions(GFT,
projections) Decomposition Reconstruction(arti
facts) Decomposition Shape analysis for
AssignmentsShape analysis for Structure
relaxation and etc.
Recording of high-dimensionalspectra Pre-proces
sing oftime-domain data Transform(t1,t2 ) ?
(w1,w2 ) Signal identification AssignmentsS
tructure, dynamics,interaction
Large data sets,long measurementscryoprobes Ze
ro filling,linear prediction Fourier
transform,ME reconstruction Peak
picking,patterns Spin systemsNOEs, RDCs
,15N-HSQC
3
Model of projections and decompositions
S
w2
w1
wHN
High-dimensional spectrum not recorded
4
Goals
Interpret spectra with linear combinations of
chemical shifts along the indirect
dimension assignment (bb, sc), structure
w1w2-w3-w4
w1w2w3w4
wHN
wHN
w1-w2w3-w4
w1-w2-w3-w4
wHN
wHN
5
Rapid NMR Data Backbone Projections
6
Shape analysis for backbone assignment
Val 17
Glu 18
HN
HN
8.75 ppm
8.64
8.98 ppm
8.9
For each HN component with shapes forN, C,
Ca/bi-1, Ha/bi-1, Ca, Cb, Ha, Hb ? unambiguous
shifts within fragment
N
N
136.0
ppm
95.1
C
C
183.6
ppm
167.2
Ca/bi-1
Ca/bi-1
86.5
ppm
4.1
Ha/bi-1
Ha/bi-1
8.85
ppm
0.55
Ca
Ca
86.5
ppm
4.1
Cb
Cb
86.5
ppm
4.1
Ha
Ha
8.85
ppm
0.55
Hb
Hb
8.85
ppm
0.55
7
Backbone assignment
Very high dimensional data- overlap for
sequential connections- anchor for side
chains Sensitivity- S/N1 chances for false
component 10-7 SHABBA (SHape Analysis for
BackBone Assignment)- reliable- automated
8
Sco1 protein 5D Backbone projections
Copper chaperone, 172 residues (500 MHz, SNC)
(Angelo Gallo, Ivano Bertini, Florence)
9
Rapid NMR Data TOCSY Projections
10
Side-chain assignments
  • assignment of all (aliphatic) hydrogens
  • with/without Hali-Cali connections
  • anchor on backbone
  • projections from 4-5 D data(a) Hali N C
    HN(b) Hali N Ca HN(c) Hali Cali N C
    HN(d) Hali Cali N Ca HN
  • combine data sets (a)(b), (a)(c)
  • automation
  • develop, test ubiquitin

For more details, please visit our poster!
11
Rapid NMR Data NOESY Projections
12
Structure information
  • observation of short distances (via NOEs)
  • with/without H-C connections
  • projections from 4-5 D data(a) Hali N C
    HN(b) Hali N Ca HN(c) Hali Cali N C
    HN(d) Hali Cali N Ca HN
  • combine data sets (a)(b), (a)(c)
  • anchor HN on backbone
  • tested on azurin
  • Azurin
  • 128 residues
  • lt1mM
  • 600 Mhz

13
NOESY Projections
wN
4D
wNwCwHali
wNwCali
4D
5D
9.0 8.5 8.0 wHN(ppm) 7.0
9.0 8.5 8.0 wHN(ppm) 7.0
9.0 8.5 8.0 wHN(ppm) 7.0
Cross section along w at wHN9.27 ppm
lt 1 mM 30C 600 MHz
14
Components and shapes
HN
N
C
9.15 9.13
L33, M109, M121
Ca
  • 5D components from combination of two 4D data
    sets
  • Hali, N, C, HN
  • Hali, N, Ca, HN
  • (some projections repeated)

Hali
15
Distance measurements
16
Structure
RMSD 10 structures to meanbb 2-53,82-128 1.4 Å
54 side chains 1.7 Å mean to X-ray (4azu)bb
2-53,82-128 1.3 Å 54 side chains 1.5 Å
only NOEs from HNs available!
17
http//www.lundberg.gu.se/nmr/ Malmodin
Billeter J. Amer. Chem. Soc. 127 (2005) Malmodin
Billeter Magn. Res. Chem. 44 (2006) Staykova
et al. J. Biomol. NMR 42 (2008) Staykova et al.
Bioinformatics 24 (2008)
18
PRODECOMP/Shape Analysis Methods Summary
  • Robust and reliable analysis of projections
  • (Based on mathematical model consistent with NMR
    theory)
  • Automatic handling of spectra folding
  • Simultaneous decomposition of combined data sets
  • Sensitivity Optimal treatment of data with low
    S/N
  • Very high dimensional (gt5D) output overlap for
    sequential connections
  • and high-dimensional peaks for unambigous
    assignments
  • Adapted post-decomposition analysis, automation
  • Fast approach for protein 3D structure
    determination

19
Acknowledgements
Martin Billeter Jonas Fredriksson Daniel
Malmodin Wolfgang Bermel Angelo Gallo
20
Decomposition
wN
  • pick peaks in 15N-HSQC projection
  • (fixed width) interval for each wHN
  • of components overlapping intervals!
  • analyze wHali shape

3 components
wHN
Asn 47
HNN47,F114
123 components 1-11 components /
decomposition 535 components calculated 0.9 24
min. / decomposition
H46Ha
N47Hb
C112Ha
H46Hb
W48Ha
L33Hd
21
Summary of NOEs
Peaks 877ambiguous 41weak 93match? 142 Used 6
01long range 99 Other 135H-bonds (3) 129Cu,
Cys-Cys 6 Constraints 736CYANA removed 85upper
limits 651
22
Secondary structures
C3
43 H-bonds
G9
L27
H35
V99
H-bonds long-range NOEs
D93
X-ray structure
b
b
b
b
b
a
b
b
b
b
b
b
b
b
b
a
b
b
b
PREDITOR (D. Wishart)
23
Long range constraints
sequence
1 20 40 60 80 100 120
1 20 40 60 80 100 120
sequence
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