Protein structure determination by solid-state NMR

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1 Protein structure determination by solid-state NMR Birgit Habenstein Supervised by Anja Böckmann

2 Solid state NMR proteinaceous targets Structural studies and structure determination at atomic level Membrane proteins in liposomes NpSRII Fibrillar proteins HET-s( ) 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.,

3 Solid state vs liquid state for proteins No tumbling in solids Broad lines from anisotropic interactions CSA Dipolar couplings Magic-Angle Spinning θ MAS averages out interactions which are weaker than the spinning frequency 13 C spectrum + 1 H decoupling - MAS + MAS - 1 H decoupling + MAS & 1 H decoupling N. Giraud! δ1 13 C (ppm)

4 Solid-state NMR of proteins - progress Bottleneck: spectral resolution and sensitivity Complexity size - sample preparation - components - labeling schemes - nmr techno/methodology Applications

5 On the way to a protein structure by solid-state NMR sequential assignment distance restraints

6 preparation/ sample sequential assignment distance restraints IMX microcrystals Ure2p prion fibrils Ure2p microcrystals Ure2p (354 aa) prion fibrils before and after heat 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

7 sequential assignment Tools Conventional: Uniform 13 C-, 15 N- labeling Selective labeling with 1,3 13 C-, and 2 13 C - Glycerol dilutes spins and by this removes dipolar truncation distance restraints SH3 Castellani et al., 2002 Hong et al., 1999; Castellani et al., 2002 Specific labeling using cell-free protein synthesis 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 + 1 H detection (Reif et al., 2001) Selective labelldrawbacks: expensive, time consuming, possibly less information per spectrum Important: Optimised rotor filling (Böckmann et al., 2009)

8 sequential assignment distance restraints Protein size Percentage of flexible residues Heat resistance Protein sequence

9 Tools sequential assignment distance restraints Temperature (upspinning/) Spectrometer (magnetic field) Spinning speed Decoupling power ( 100 khz) 2D/3D spectroscopy

10 sequential assignment distance restraints 3D sequential assignment: NCACO; NCOCA; CANCO 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

11 sequential assignment distance restraints Tools 3D sequential assignment NCACB; NCOCA; CANCO; N(CO)CACB; CAN(CO)CA; CCC; NCACX Some pulse sequences for 3D sequential assignments NCACB NCACX N(CO)CACB CCC 200 ms! CAN(CO)CA Schuetz et al., Chembiochem 2010

12 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... Ni(CO)CAi-1CBi-1! x x CAiNi(CO)CXi-1! NiCAiCBi! x CAiNi(CO)CXi-1! x

13 sequential assignment distance restraints Tools 3 complementary approaches 1. Proton - mediated s: CHHC, NHHC (Zhang et al. 1992, Lange et al. 2002) semi-quantitative measurement of 1 H- 1 H distance; < 5Å 2. Proton - assisted s: PAR, PAIN (DePaepe et al. 2008, Lewandowski et al. 2006) 3. Proton - driven s: DARR, PDSD (Takegoshi 2001 et al.)

14 sequential assignment distance restraints Tools 3 different approaches 1. Proton - mediated s: CHHC, NHHC 2. Proton - assisted s: PAR, PAIN 3. Proton - driven s: DARR Crh (85 aa) CHHC PAR DARR 200 µs, 500 MHz 9 ms, 900 MHz 200 ms, 500 MHz Loquet et al., JACS 2008 De Paepe et al., 2008

15 Ambiguities sequential assignment Δ = 0.1 ppm Not realistic with al lineshape Number of cross-peaks after distance restraints Δ = 0.25 ppm peak-picking = 1231 Average number of ambiguities per crosspeaks n av =16.3 Number of cross-peaks which encode <25 ambiguities = 998 Δ = 0.5 ppm -> potentially ambiguities Number of cross-peaks < 25 ambiguities is insufficient Δ = 0.75 ppm Residue number Loquet et al., JACS 2008 Resolve ambiguities. selectively labeling reduces ambiguities - and information content ARIA

16 sequential assignment distance restraints structure calculations HET-s( ) prion 2KJ / -- N/CHHC, PDSD, PAR Melckebeke et al.(in press) H. Oschkinat!

17 Ure2 prion - structural investigations Saccharomyces cerevisiae! 354 amino acids kda - active in dimeric soluble form! Ure2p1-354 fibrils Ure2p1-93 fibrils Ure2p crystals

18 Ure2 prion - structural investigations Ure2p1-354 Full-length fibrils Ure2p1-354 N-terminal fibrils Ure2p1-93 C-terminal microcrystals Ure2p Ure2p1-93 δ1 13 C (ppm)! Ure2p DARR (20 ms mixing); 850 MHz on 1 H; 18 khz MAS; 90 khz 1 H decoupling (acquisition) δ2 13 C (ppm)!

19 Ure2 prion - structural investigations sequential assignment distance restraints?! challenge (354 aa): structure determination: 4D, selective labeling, cell-free synthesis(noirot, 2010)

20 Anja Böckmann! François Penin! Simon Megy! Antoine Loquet (MPI Göttingen)! Claire Noirot! Carole Gardiennet...and the rest of the group!