Download Protein structure determination by solid-state NMR

Protein structure
determination by solid-state NMR
Birgit Habenstein
Supervised by Anja Böckmann
Solid state NMR proteinaceous targets
Structural studies and structure
determination at atomic level
Membrane proteins in
liposomes
NpSRII
Fibrillar proteins
HET-s(218-289)
Large protein assemblies
Type 3 secretion system
100 nm!
1 μm!
Poyraz et al., 2010
Etzkorn et al., 2006
Siemer et al., 2006
Wasmer et al., 2008
2006
Solid state vs liquid state for proteins
No tumbling in solids
CSA
Broad lines from anisotropic interactions Magic-Angle Spinning
Dipolar couplings
θ
MAS averages out interactions which are weaker
than the spinning frequency
13C
spectrum
+ 1H decoupling
- MAS
+ MAS
- 1H decoupling + MAS & 1H decoupling
180
160
140
© N. Giraud!
120
100
80
δ1 13C
(ppm)
60
40
20
Solid-state NMR of proteins - progress
Bottleneck: spectral resolution and sensitivity
Complexity
size - sample preparation - components - labeling schemes - nmr techno/methodology
1998
2003
2008
2013
Applications
On the way to a protein structure by
solid-state NMR
preparation/
sample
preparation/ treatment
*
sample
treatment
*
experiment adaptation
*
sequential assignment
*
distance restraints *
structure calculations
preparation/
*
sample
treatment
*
experiment
adaptation
*
sequential assignment
*
distance restraints *
structure calculations
IMX microcrystals
Ure2p prion fibrils
Ure2p70-354 microcrystals
Ure2p (354 aa) prion fibrils before and after heat treatment
13C-13C
850 MHz on 1H; 18 kHz MAS; 90 kHz 1H decoupling (acquisition)
correlation spectrum: Dipolar Recoupling Enhanced by Amplitude Modulation - DREAM δ2 13C
(ppm)
δ2 13C
(ppm)
Loquet et al., JMB 2009
Tools
sample preparation/
treatment
*
Conventional: Uniform 13C-, 15N- labeling Selective labeling with 1,3 13C-, and 2 13C - Glycerol
experiment
adaptation
*
sequential assignment
*
*
distance restraints dilutes spins and by this removes
dipolar truncation
SH3
structure calculations
*
Castellani et al., 2002
Specific labeling using cell-free protein synthesis Hong et al., 1999; Castellani et al., 2002
SAIL - Stereo-array isotope labeling (Kainosho et al., 2006); Combinatorial selective labeling (Wu et al., 2006);
Production for structural studies (Noirot et al., 2010)
Deuteration + 1H detection (Reif et al., 2001)
Selective labelldrawbacks: expensive, time consuming, possibly less information per spectrum Important: Optimised rotor filling (Böckmann et al., 2009)
preparation/
*
sample
treatment
*
*
experiment
adaptation
sequential assignment
*
distance restraints *
structure calculations
Protein size
Percentage of flexible residues
Heat resistance
Protein sequence
*
sample preparation/
treatment
*
*
experiment
adaptation
sequential assignment
*
distance restraints *
structure calculations
Tools
Temperature (upspinning/experiment)
Spectrometer (magnetic field)
Spinning speed
Decoupling power (⁓ 100 kHz)
2D/3D spectroscopy
preparation/
*
sample
treatment
*
experiment
adaptation
*
sequential
assignment
3D sequential assignment:
NCACO; NCOCA; CANCO *
distance restraints *
structure calculations
limitations: low dispersion in CO dimension - use of
CB dispersion 3D NCACB; N(CO)CACB; CAN(CO)CA; CCC;
NCACX
Schuetz et al.,
Chembiochem 2010
*
sample preparation/
treatment
*
experiment
adaptation
*
sequential
assignment
*
distance restraints *
structure calculations
Tools
3D sequential assignment
NCACB; NCOCA; CANCO; N(CO)CACB; CAN(CO)CA; CCC; NCACX
Some pulse sequences for 3D sequential assignments
NCACB
N(CO)CACB
CAN(CO)CA
NCACX
CCC
200 ms!
Schuetz et al.,
Chembiochem 2010
*
sample preparation/
treatment
*
experiment
adaptation
*
sequential
assignment
*
distance restraints *
Tools
Ccpnmr Analysis
marking + scrolling through planes
overlay in different dimensions
use of all resonances
strip in vertical and horizontal directions
...
structure calculations
Ni(CO)CAi-1CBi-1!
x
NiCAiCBi!
x
CAiNi(CO)CXi-1!
x
x
CAiNi(CO)CXi-1!
preparation/
*
sample
treatment
*
experiment
adaptation
*
sequential assignment
*
distance
restraints *
structure calculations
Tools
3 complementary approaches
1. Proton - mediated experiments: CHHC, NHHC
(Zhang et al. 1992, Lange et al. 2002)
semi-quantitative measurement of 1H-1H distance; < 5Å
2. Proton - assisted experiments: PAR, PAIN (DePaepe et al. 2008, Lewandowski et al. 2006)
3. Proton - driven experiments: DARR, PDSD (Takegoshi 2001 et al.) Tools
preparation/
*
sample
treatment
3 different approaches
*
experiment
adaptation
*
sequential assignment
*
distance
restraints *
1. Proton - mediated experiments: CHHC, NHHC 2. Proton - assisted experiments: PAR, PAIN
3. Proton - driven experiments: DARR structure calculations
Crh (85 aa)
CHHC
200 µs, 500 MHz
Loquet et al., JACS 2008
PAR
9 ms, 900 MHz
De Paepe et al., 2008
DARR
200 ms, 500 MHz
sample preparation/
treatment
*
*
Ambiguities
experiment
adaptation
Δ = 0.1 ppm
*
Not realistic with experimental lineshape
sequential assignment
Number of cross-peaks after *
distance
restraints *
structure calculations
peak-picking = 1231
Δ = 0.25 ppm
Average number of ambiguities per crosspeaks nav=16.3
Number of cross-peaks which encode
<25 ambiguities = 998
Δ = 0.5 ppm
-> potentially 12700 ambiguities
Number of cross-peaks < 25
ambiguities is insufficient
Δ = 0.75 ppm
Residue number
Resolve ambiguities.
Loquet et al., JACS 2008
selectively labeling reduces ambiguities - and information content
ARIA
preparation/
*
sample
treatment
*
experiment
adaptation
*
sequential assignment
*
distance
restraints *
structure
calculations
HET-s(218-289) prion
© H. Oschkinat!
2KJ3
3
71
7085
0.64/ --
N/CHHC, PDSD, PAR
Melckebeke et al.(in press)
Ure2 prion - structural investigations
Saccharomyces cerevisiae!
354 amino acids - 40.5 kDa - active in dimeric soluble form!
Ure2p1-354 fibrils Ure2p1-93 fibrils
Ure2p70-354 crystals
Ure2 prion - structural investigations
Ure2p1-354 Full-length fibrils Ure2p1-354
N-terminal fibrils Ure2p1-93 C-terminal microcrystals
Ure2p70-354 δ1 13C (ppm)!
Ure2p1-93
Ure2p70-354
DARR (20 ms mixing); 850 MHz on
1H; 18 kHz MAS; 90 kHz 1H decoupling (acquisition)
δ2 13C (ppm)!
Ure2 prion - structural investigations
preparation/
sample
preparation/ treatment
*
sample
treatment
*
experiment adaptation
*
sequential assignment
*
distance restraints *
structure calculations
?!
challenge (354 aa):
structure determination: 4D, selective labeling, cell-free synthesis(Noirot, 2010)
Anja Böckmann!
François Penin!
Simon Megy!
Antoine Loquet (MPI Göttingen)!
Claire Noirot !
Carole Gardiennet
...and the rest of the group!