Labelling strategies in the NMR structure determination of larger proteins

Labelling strategies in the NMR structure determination of larger proteins - Difficulties of studying larger proteins - The effect of deuteration on spectral complexity and relaxation rates - NMR expts used with different deuteration schemes - global fold and high res. structure determination - Expression of deuterated proteins Das Bild kann nicht angezeigt werden. Dieser Computer verfügt möglicherweise über zu wenig Arbeitsspeicher, um das Bild zu öffnen, oder das Bild ist beschädigt. Starten Sie den Computer neu, und öffnen Sie dann erneut die Datei. Wenn

Obstacles with regards to labelling: Selective 15 N labelling is difficult due to transamination - This shows up also when amino acid-type selective 13C, 15N labelling is desired - Use transaminase knockouts Spreading of labels due to similar pathways, eg F -> Y - Provide sufficient amounts of undesired amino acids, but be prepared to observe dilution of the desired one

Obstacles to NMR studies of larger proteins Increased Number of Resonances - Degeneracy à complex spectra (i.e. overlap) Increased Relaxation Rates - reduced sensitivity - broad linewidths

Production of randomly and specifically labelled proteins LB plate (H 2 O) (1) Minimal media for 13 C, 15 N labelling (M9): Small scale medium Salts, 2-4 g/l 13 C-glucose, 0.5-1g/l 15 N-labelled NH4Cl, in difficult cases add 13 C, 15 N- labelled algal hydrolysate, or yeast extract Large scale medium (2) Amino acid type labelling: As above but add ~100mg/l of each desired labelled amino acid (50 mg of Trp) to the otherwise fully deuterated (and/or 15 N/ 13 C labelled) medium. Add also 100mg/l of each amino acid that should not be labelled (3) Selective methyl labelling: As above but add ~50-100mg/l correctly labelled ILV precursors to the otherwise fully deuterated (and/or 15 N/ 13 C labelled) medium, provide 100 mg of all other amino acids: i.e. [100% 3,3-2 H, 100% 13 C]-α-ketobutyrate to obtain ( 1 H-δ1 methyl)-ile, and [100% 3-2 H, 100% 13 C/ 15 N]- α-ketoisovalerate to obrain ( 1 H-γ methyl)-val and ( 1 H-δ methyl)-leu

Obstacles to NMR studies of larger proteins Increased Number of Resonances - Degeneracy à complex spectra (i.e. overlap) Increased Relaxation Rates - reduced sensitivity - broad linewidths

Production of randomly and specifically labelled 2 H proteins LB plate (H 2 O) (1) Random fractional or full deuteration: Small scale medium Minimal media with desired % D 2 O supplemented with either 4g/l 100% 2 H glycerol (cheaper than 2 H glucose!) if no carbon labelling desired, otherwise 2 H, 13 C glucose or 10-20% (v/v) appropriately labelled 2 H, 13 C, 15 N-labelled algal hydrolysate Large scale medium (2) Amino acid type selective protonation ( reverse labelling ): As above but add ~100mg/l of each desired unlabelled amino acid to the otherwise fully deuterated (and/or 15 N/ 13 C labelled) medium (3) Selective methyl protonation: As above but add ~50-100mg/l correctly labelled ILV precursors to the otherwise fully deuterated (and/or 15 N/ 13 C labelled) medium: i.e. [100% 3,3-2 H, 100% 13 C]-α-ketobutyrate to get: ( 1 H-δ1 methyl)-ile, and [100% 3-2 H, 100% 13 C/ 15 N]- α-ketoisovalerate to get: ( 1 H-γ methyl)-val and ( 1 H-δ methyl)-leu (4) Others: segmental isotope labelling: self-catalytic splicing proteins using inteins alternating labels ( 12 C- 13 C- 12 C) from (2-13 C)-glycerol or (1,3-13 C 2 )-glycerol

Relaxation mechanisms: - Dipole-dipole interactions - Chemical shift anisotropy γd/γh = 1/6.5 d CD /d CH = 1/16 Result: 2 D relaxes far less efficiently than 1 H A Solution: Protein Deuteration Protonated Deuterated External rlxn. pathways compete with the desired pathway that we want to measure. Solution: remove them!

1 H detection of perdeuterated samples, recrystallized from H 2 O/D 2 O = 1:9: SH3 with 1 H, 2 H, 13 C and 15 N By Rasmus Linser, Vipin Agarwal, Veniamin Chevelkov, Bernd Reif Linewidths: 24 ±5 Hz Use of standard pulse solution programs R. Linser et al., Sensitivity Enhancement using paramagnetic relaxation in MAS solid state NMR of perdeuterated proteins, J. Magn. Res, subm.

Proton-detected solid state NMR of 2D crystals CP INEPT Art(MP) 15N ArtM(P) 15N 11

Transverse relaxation times of different nucleus types as a function of molecular correlation time T2 ms Evolution of 13 C- 15 N couplings: C x C y N z optimal Δ ~ 22-25 ms τ c ns But often have T2 < 30 ms so signal is largely decayed at the end of such an evolution time

Effect of deuteration on the relaxation rates of different nuclei For a protein with τ c = 12 ns 100 Relaxation rate constant (Hz) 80 60 40 20 0 Cα N Hα HN Variable (can be eliminated by replacing 1 H with 2 D) Fixed (rlxn. Due to CSA and DD intxns with H N ) From: Figure 2, Nietlispach et al., (1996) JACS, 118, p. 410

2 H isotope effects Isotopomers in 50% 2 H proteins 1 Δ 13 C(D) ~ -0.29 ppm 1 Δ 13 C gly (D) ~ -0.39 ppm 2 Δ 13 C(D) ~ -0.13 ppm 3 Δ 13 C(D) ~ -0.07 ppm 1 Δ 15 N(D) ~ -0.3 ppm 2 Δ 15 N(D) ~ -0.05 to -0.1 ppm Isotopomer % present % contribution to C β peaks C β H 2 -C α H 12.5 4 C β H 2 -C α D 12.5 17 C β D 2 -C α H 12.5 0 C β D 2 -C α D 12.5 0 C β HD-C α H 25.0 16 C β HD-C α D 25.0 63 - Where n Δ 13 C represents the n-bond isotope effect per 2 D. - Can see effects as far as 4 bonds away from a 2 D. - Small secondary structure dependence. - Distribution in total 2 D isotope effect can lead to broadened 13 C resonances (as peaks from different isotopomers are usually not individually resolved) See: Venters et al., (1996) J. Mol. Biol., 264, 1101-1116

Effect of deuteration on the Cα relaxation rate in 3D HNCA for 47 kda DHBPS protein, τ c = 20 ns Signal intensity Signal intensity

Deuteration therefore improves sensitivity and resolution Equivalent planes of 3D HNCA spectra

Backbone assignments Perdeuterated and very highly deuterated proteins - maximum sensitivity (i.e. min. relaxation rates) with 100% 2 H (perdeuteration) - CT for 13 C evolution - no isotopomer effects D/ D/ D/ D/ - need out and back expts - need 2 H decoupling HNCA HNCACB HN(CO)CA HN(CO)CACB - lose 1 H s: source of NOE restraints - lose all C α -H

Production of randomly and specifically labelled 2 H proteins LB plate (H 2 O) (1) Random fractional deuteration: Small scale medium Minimal media with desired % D 2 O supplemented with either 4g/l 100% 2 H glycerol (cheaper than 2 H glucose!) or 10-20% (v/v) appropriately labelled 2 H, 13 C, 15 N- labelled algal hydrolysate Large scale medium (2) Amino acid type selective protonation ( reverse labelling ): As above but add ~100mg/l of each desired unlabelled amino acid to the otherwise fully deuterated (and/or 15 N/ 13 C labelled) medium (3) Selective methyl protonation: As above but add ~50-100mg/l correctly labelled ILV precursors to the otherwise fully deuterated (and/or 15 N/ 13 C labelled) medium: i.e. [100% 3,3-2 H, 100% 13 C]-α-ketobutyrate to get: ( 1 H-δ1 methyl)-ile, and [100% 3-2 H, 100% 13 C/ 15 N]- α-ketoisovalerate to get: ( 1 H-γ methyl)-val and ( 1 H-δ methyl)-leu (4) Others: segmental isotope labelling: self-catalytic splicing proteins using inteins alternating labels ( 12 C- 13 C- 12 C) from (2-13 C)-glycerol or (1,3-13 C 2 )-glycerol

Sidechain assignments Effect of deuteration level on signal intensity HBCB/HACANH HBCB/HACA(CO)NH Relative transfer efficiency Relative transfer efficiency 2.5 2.0 1.5 1.0 0.5 0.0 % random fractional deuteration % random fractional deuteration From: Figure 4. Nietlispach et al., (1996) JACS, 118, p. 412

HBCB/HACANH Relative transfer efficiency Relative transfer efficiency % random fractional deuteration 2.5 2.0 1.5 1.0 0.5 0.0 HBCB/HACA(CO)NH 0% 2 H 50% 2 H 75% 2 H Protein with τ c =18 ns (30 kda) Optimum transfer efficiency ~ 50% 2H % random fractional deuteration From: Figures 6 and 7, Nietlispach et al., (1996) JACS, 118, p. 413

Sidechain assignments HCC(CO)NH Protein with τ c =12 ns (20 kda) Optimum transfer efficiency ~ 50% 2H From: Figure 7. Nietlispach et al., (1996) JACS, 118, p. 413

Sidechain assignments Similarly for: HCCH-TOCSY and HCCH-COSY 0% 2H Protein with τ c =12 ns (20 kda) Optimum transfer efficiency at ~ 50% 2H 50% 2H

Effect of deuteration on NOE intensities Relative intensity fractional deuteration Deuteration affects NOEs between different pairs of nuclei, differently: H N -H N : large improvement with increasing 2 H H C -H N : intensity reduction after 40% H C -H C : immediate fall-off in intensity From: Figure 8. Nietlispach et al., (1996) JACS, 118, p. 414

1 H population in 47 kda DHBPS (all 1 H) H C H N

1 H N population in DHBPS (non-exchangable sidechain 1 H C replaced by 2 H C ) H N

Effect of deuteration on resolution and sensitivity 2D 1 H NOESY spectra: H N -H N region Protonated DHBPS 100% Deuterated DHBPS Large improvement with 100% 2 H

Effect of deuteration on different NOE-types H C -H N NOEs strong loss of intensity after 50% 2 H H N -H N NOEs get more intense as % 2 H increases 0% 2 H 50% 2 H 75% 2 H From: Figure 10. Nietlispach et al., (1996) JACS, 118, p. 414

Proteins with τ c >18 ns (>35 kda): (1) Amino-acid-type specific protonation in a deuterated background reverse-labelling F,Y: 14 N, 1 H F I L V T,I,V,L: 14 N, 1 H Others: 15 N, 2 H T Y - FYTIVL share similar biosynthetic pathways (minimize label scrambling) - Can choose residue types to be labelled from within related groups. Used for DHBPS: 47 kda dimer τ c = 20 ns - Typically found in the protein core or interface regions

1 H population in 47 kda DHBPS (all 1 H) H arom H others

Sidechain 1 H C population in DHBPS: 1 H-FYTIV ( 1 H N also present, not shown) H arom H T,I,V,L

NOEs in 100% 15 N, 2 H { 14 N, 1 H FYTIVL} sample Possible NOEs : 15 1 H N (all) à 1 H N (all) 14 15 But only: 1 H C (FYTIVL) à 1H N (all) in aliphatic region 15 15 14 From: Figure 1, Kelly et al., (1999) J. Biomol. NMR, 14, p. 80

Specific protonation of FYTIVL dramatically improves sensitivity in 47 kda DHBPS: 2D 1 H NOESY spectra DHBPS DHBPS

Selecting subsets of NOEs with heteronuclear half-filters Definitions: Filter = S-H suppression Edit = S-H selection (where S = 13 C or 15 N) Edited No S-H Only S-H

Heteronuclear half-filters ω2 half-filtered NOESY 2H y S z Half-filter in t2 ω2,ω2 double half-filtered NOESY Edited No S-H Only S-H Half-filter in t1 Half-filter in t2

15 N-editing and 15 N half-filtering in 15 N, 2 H { 14 N, 1 H FYTIVL} sample 15 N-ω2-edited NOESY see only 15 N-H in F2 15 14 15 15 N-ω2-filtered NOESY see no 15 N-H in F2 15 15 14 From: Figures 1 and 2, Kelly et al., (1999) J. Biomol. NMR, 14, p. 80,81

Proteins with τ c >18 ns (>35 kda): (2) Selective methyl protonation: improves resolution and sensitivity: { 1 H(CH 3 )I δ1 LV}-sample Labelling methodology: Goto et al., (1999) J. Biomol. NMR, 13, p. 369-74

Methyl-protonation reduces ambiguity in NOESY expts 100% 13 C, 15 N, 2 H, { 1 H(CH3)I δ1 LV} 100% 13 C, 15 N, 75% 2 H

47 kda DHBPS structure ensemble and statistics Samples used in the determination of the DHBPS structure: - 100% 13 C, 15 N, 75% 2 H - 100% 13 C, 15 N, 1 H - 100% 2 H, 15 N { 14 N, 1 H FYTIVL} - 100% 2 H, 15 N { 14 N, 1 H PF} - 100% 13 C, 15 N, 2 H { 1 H(CH 3 )I δ1 VL}, RESTRAINTS Total exp restraints Total NOE restraints sequential medium range long range H-bond restraints Dihedral angle restraints 1008 674 295 139 240 67 267 RMSD ensemble mean backbone 2.28±0.51Å 1.5Å

Optimal labelling/deuteration strategies for larger proteins τ c <10-12 ns (~20 kda) uniform 13 C/ 15 N labelling usually sufficient τ c ~12-18 ns (~35 kda) uniform 13 C/ 15 N labelling combined with perdeuteration or random fractional deuteration τ c >18 ns (>35 kda) uniform or selective 13 C/ 15 N labelling combined with selective protonation in a deuterated background

Conclusions 15 N/ 13 C labelling (up to ~20 kda) 15 N/ 13 C labelling combined with random fractional deuteration and perdeuteration (~20-35 kda): improves sensitivity and resolution Selective protonation, reverse-labelling strategies (>35 kda): Amino acid type specific protonation Methyl protonation Both allow collection of specific sets of long-range NOE restraints to aid in structure and global fold determination Segmental isotope labelling: intein technology for very large proteins Alternate labelling: removal of strong 13 C- 13 C scalar and dipolar contributions during studies of sidechain dynamics

Small to medium-sized proteins: overlap resolved with 2 or 3 Dimensions 3D NMR

Medium to larger proteins: Using a Fourth Dimension, from 3D to 4D NMR 1D strip from 3D 2D 13C-1H Hyperplane from 4D Spectrum

Obstacles to NMR studies of larger proteins 19 kda cofilin (τ c =10 ns) 47 kda DHBPS (τ c =20 ns) 2D NOESYs H N à H N region

Obstacles to NMR studies of larger proteins 8 kda Tendamistat 21 kda Cdc42 2D NOESYs H C à H N region

Heteronuclear purge pulses (low pass J-filters) 2H y S y see all non S -H MQC see no S-H

Sensitivity and linewidth as a function of molecular correlation time Sensitivity α e -(ΣΔ/T2) Linewidth, Δν 1/2 = 1/πT2 As τ c increases, the rlxn time constant T2 decreases, resulting in two problems: (1) sensitivity reduction and (2) increase in linewidths MW 10 kda 20kDa 30 kda 50 kda τc 5 ns 10 ns 15 ns 25 ns