Deuteration: Structural Studies of Larger Proteins

Transcription

1 Deuteration: Structural Studies of Larger Proteins Problems with larger proteins Impact of deuteration on relaxation rates Approaches to structure determination Practical aspects of producing deuterated proteins (See Gardner and Kay (1998) Ann. Rev. Biophys. and Biomol. Str. 27, for a general review)

2 Tendamistat 8 kda Cdc42 21 kda

3 R1b 9 kda τc = 6nsec Cdc42/ACK 26 kda τc=18nsec

4 Effects of Increasing Molecular Size number of resonances - crowded spectra faster relaxation - broad lines - low intensity - overlap - long experiment time

5 Transverse relaxation times as a function of protein size N() C α (D) transverse relaxation time T2 [ms] n C α () α 0 β correlation time [ns] Cx CyNz = msec Cx Cy =20-27 msec but T2 often less than 30 msec

6 Linewidth vs. Correlation Time coherence transfer Sensitivity α Π n sin(πj ) Π m cos(πj ) active passive relaxation exp(-r 2 Σ ) } Linewidth ν 1/2 = 1/πT 2 τ c MW 5ns 10kDa 10ns 20kDa 15ns 30 kda 25ns 50kDa ppm ppm ppm ppm

7 Relaxation Mechanisms Dipole-Dipole interactions (DD) Chemical shift anisotropy dip R 1,2 (X) ~ D. I(I+1).(γ X γ Y ) 2. Σ J(ω i ) i internal DD contributions X X= 13 C, 15 N external DD contributions γd/γ = 1/6.5 d CD /d C = 1/16

8 Impact of deuteration on relaxation rates relaxation rate constant/z τc =12 nsec Cα N α N removed by sidechain deuteration fixed

9 Impact of deuteration on relaxation rates T2 τc = 18 nsec T1

10 Effects of deuteration on Cα relaxation times Isolated C- pair T 2 (CD)/T 2 (C) T 2 (CD) / T 2 (C) J(0) dominate 12 Ratio Ratio 10 8 J(ω C -ω D ) effects 6 4 T 1 (CD)/T 1 (C) T 1 (CD) / T 1 (C) overall correlation time [ns] Correlation time (nsec)

11 Approaches to the Structure Determination of Larger Proteins For τ c up to ~12 ns (20 kda) - 13 C/ 15 N-labelling For τ c up to ~18 ns (35 kda) - fractional deuteration and 13 C/ 15 N- labelling For τ c above ~18 ns - selective protonation and 13 C/ 15 N-labelling

12 Triple Resonance NMR and Random Fractional Deuteration Backbone assignments Side-chain assignments Measurement of NOE contacts

13 Backbone Assignments NCA N(CO)CA O O 4 N Cα Cβ D C N N 4 Cα Cβ C 3 D N 6 β Cγ β Cγ γ γ Deuteration reduces relaxation Maximum sensitivity with 100% deuteration - Grzesiek et al., (1993) J. Am. Chem. Soc. 115, Yamazaki et al., (1994) J. Am. Chem. Soc. 116, Yamazaki et al., (1994) J. Am. Chem. Soc. 116,

14 Backbone Experiments with Deuterated Proteins Out and back CT for 13 C evolution (1/J cc ) 1 T1s: longer recycle delay preserve water Deuterium decoupling Removal of residual Cα Sensitivity enhancement (20 nsec limit?) Shan et al (1996) JACS

15 1 τ d 15 N 13 Cα 2 t1/2 Tc Tc-t1/2 y -y WALTZ16 x 2Tc = 26.6 ms = 1/J cc τ d = 1.7ms = 1/4J C 2 restored to z-axis (lock stability) Cα evolves for 1/2J C - removed by 1 decoupling

16 Backbone Assignments /D BCB/ACANN /D BCB/ACA(CO)NN Cβ /D Cβ /D Cα C N Cα Cα C N Cα O /D Cβ /D O /D Cβ /D β β Nietlispach et al., (1996) J. Am. Chem. Soc. 118,

17 Isotopomers in 50% 2 Proteins Isotopomer Fraction Contribution for Cβ peaks Cβ 2 -Cα 12.5% 4% CβD-Cα 25% 16% Cβ 2 -CαD 12.5% 17% CβD-CαD 25% 63% CβD 2 -Cα 12.5% 0% CβD 2 -CαD 12.5% 0%

18 Effect of Deuteration on BCB/ACA(CO)N for a protein with τ c ~18ns (30kDa) 0% 2 50% 2 75% 2

19 Effect of Deuteration on BCB/ACANN for a protein with 18 nsec correlation time ppm % 2 50% 2

20 Triple Resonance NMR and Random Fractional Deuteration Backbone assignments Side-chain assignments Measurement of NOE contacts

21 Side-chain Assignments CC(CO)NN O 1 N Cβ 3 Cα 2 C α 4 N 5 β 1 Cγ 2 γ Deuteration reduces relaxation Maximum sensitivity with 50% deuteration Nietlispach et al., (1996) J. Am. Chem. Soc. 118,

22 Effect of Deuteration on CC(CO)NN for a Protein with a 12 nsec Correlation Time ppm 0% 50%

23 Side-chain Assignments CC-COSY CC-TOCSY D C D C D C D C C D C D Deuteration reduces relaxation Deuteration removes both protons

24 Effect of Deuteration on CC- TOCSY for a Protein with 18 nsec Correlation Time α α α β Val α γ1 0% α γ % δ β δ γ1 δ γ2 δ Ile δ 0% %

25 Isotope Shifts Isotope effects: 1 13 C ~ ppm 2 13 C ~ -0.1 ppm 3 13 C ~ ppm 1 15 N ~ -0.3 ppm 2 15 N ~ to -0.1 ppm e.g 13 Cα in 100% D protein -0.5 ppm isotope shift weak secondary structure dependency. Isotopomers not resolved in most experiments

26 Triple Resonance NMR and Random Fractional Deuteration Backbone assignments Side-chain assignments Measurement of NOE contacts

27 Measurement of NOE Contacts Deuteration affects the measurement of N - N, N - C and C - C cross peaks in different ways Nietlispach et al., (1996) J. Am. Chem. Soc. 118,

28 Distance Restraints and Fractional Deuteration τ c = 12 nsec N/sidechain N/N 0% 2 50% 2 75% 2

29 Measurement of NOE Contacts J-correlated CD NOESY CD C 2 C 2 δ (ppm) δ (ppm) Similar contributions from different isotopomers lead to comparable chemical shifts

30 Structure Calculations Use of ARIA for structure determination Nilges (1995) J. Mol. Biol. 245, Global fold from selectively protonated data Use Talos to estimate backbone torsion angles from chemical shifts

31 Fractional Deuteration at 50% Improved sensitivity and linewidth No complications from different isotopomers Backbone and sidechain assignments possible 1 / 1 NOEs can be observed Applicable to proteins up to 30 kda

32 Approaches to the Structure Determination of larger Proteins For proteins of up to τ c ~ 12 ns, use 13 C/ 15 N-labelling For proteins of up to τ c ~ 18 ns, use fractional deuteration and 13 C/ 15 N-labelling For proteins τ c ~ 18 ns and above, use selective protonation and 13 C/ 15 N-labelling

33 Approaches for Proteins Larger than 30 kda Complete deuteration maximum sensitivity for backbone experiments but limited NOE information (N <-> N) Selective protonation of residues e.g AILV in deuterated background Selective protonation of Methyl-groups (Gardner J. Am. Chem. Soc. 119, 7599)

34 Triple Resonance NMR and Selective Protonation Label proteins with ILV+FY Methyl and aromatic 13 C and 1 nuclei relax more slowly Allows measurement of N-N, N-methyl and methyl-methyl NOE contacts These residues are typically found in the protein core or interfaces Biosynthetic pathways allow straightforward labelling Can adjust the number of residue types labelled

35 Triple Resonance NMR and Selective Protonation Backbone assignments Side-chain assignments Measurement of NOE contacts

36 Backbone Assignments NCA N(CO)CA O O 4 N Cα Cβ D C N N 4 Cα Cβ D C N 6 β Cγ β Cγ γ γ Deuteration reduces relaxation Maximum sensitivity with 100% deuteration ILV - Cα mainly deuterated Me-selective - Cα 100% deuterated

37 Triple Resonance NMR and Selective Protonation Backbone assignments Side-chain assignments Measurement of NOE contacts

38 Side-chain Assignments NCACB CC(CO)NN O O 6 N 5 4 Cβ 1 2 Cα 3 D C N N Cβ 2 Cα 1 D C 3 N 4 D Cγ D Cγ 1 D D Deuteration reduces relaxation Maximum sensitivity with 100% deuteration Yamazaki et al., (1994) J. Am. Chem. Soc. 116, Farmer et al., (1995) J. Am. Chem. Soc. 117,

39 Side-chain Assignments ()CC(CO)NN O N Cβ 3 Cα 2 D C 4 N 5 D 1 Cγ 2 γ Gardner et al (1996) J. Biomol. NMR 8,

40 Triple Resonance NMR and Selective Protonation Backbone assignments Side-chain assignments Measurement of NOE contacts

41 Measurement of NOE Contacts 3D 15 N- and 13 C-NOESY 3D Val/Ile (M)CMCB(CMM) NOESY Zwahlen et al., (1998) J. Am. Chem. Soc. 120, D 13 C/ 13 C-NOESY Zwahlen et al., (1998) J. Am. Chem. Soc. 120, D QQF-NOESY

42 Methyl-Selective Correlation Experiments all 1 transverse (16 msec) φ 3 φ 1 DEPT- QQC 1 13C (Kessler, 1989) ξ 2 CT t 1 /2 CT t 1 /2 φ rec BB a φ 2 QQF φ 5 φ 1 φ 3 t 1 /4 /2 φ rec QQF 1 13C ξ 4 φ 4 t 1 /4 + b 3 t 1 /2 2 CT c t 1 /4 + BB G 1 G 2 G 2 G 3 G 3 G 4 G 4 G z a y b 2 1x C y c 8 1x C y 2z 3z (5 evolution of J C ) 6 1/Jcc = 24 msec only 1 proton transverse between b and c

43 Sensitivity Improvement in QQF (a) QQF (b) QQC (c) DEPT-QQC (c) QQF-SQC (ppm)

44 Resolution Improvement in QQF Cdc42 methyl region I126 γ2 I117 γ2 I46 γ2 A142 β C (ppm) 8 0 V77 γ1 V85 γ2 I4 γ L165 δ L112 δ2 1.4 L δ1 L53 δ (ppm) L112 δ

45 Structure Calculations Use ARIA for structure determination Nilges (1995) J. Mol. Biol. 245, Restrain φ and ϕ angles in early stages and slacken off later

46 Limited Restraints in Structure Determination Ras: Mixed α and β structure Reasonable size (21kDa) Single domain Numbers of Methyls and Aromatics: 11 Ile 11 Leu 15 Val 5 Phe 9 Tyr 0 Trp

47

48 N/N 2 Ns only - 13Å RMSD

49 Ile, Val, Leu

50 Aromatic

51 Structures from Limited Restraints Ras NMR Structure 0.6Å RMSD 101 N-N 1171 N-S/C 1862 SC/SC 13 C/ 15 N ILVFY 2.0Å RMSD 101 N-N (ass-ass) 390 N-S/C (ass-amb) 431 S/C-S/C (ass-ass) 13 C/ 15 N ILV 15 N FY 5.0Å RMSD 101 N-N (ass-ass) 390 N-S/C (ass-amb) 231 ILV-ILV (ass-ass) 174 ILV-FY (ass-amb) 26 FY-FY (amb-amb) 13 C/ 15 N ILV 15 N FY 1.7Å N NOEs to 5Å As above with 330 N-N NOEs

52 Structures from Limited Restraints: Effect of Dipolar Couplings Clore et al (1999) JACS BAF - 89 residues all α CVN residues 2 domains - all β BAF CVN N-N N-Me N-arom Me-Me Me-arom arom-arom 5 5 Total bonds RDC bonds + N + Me ~4Å accuracy Adding aromatics Å (BAF) 1.53Å (CVN) Adding RDC Å (BAF) 1.10Å (CVN) RDC: N,N-C,N-C,Cα-,Cα-C

53 Conclusions Selective Protonation optimises the sensitivity of experiments used to correlate side-chain and backbone resonances allows one to obtain limited NOE data and structural models use of orientational restraints should improve structures

54 Practical Aspects of Producing Deuterated Proteins Random fractional deuteration LB/ 2 O LB/50% D 2 O OD 600 = 0.4 M9 in 50% D 2 O with either [50% 2, 100% 13 C/ 15 N] algal hydrolysate or [100% 2 / 13 C] glucose

55 Practical Aspects of Producing Deuterated Proteins Selective protonation Use 2 / 13 C glucose, amino acids and: C 3 -CD 2 -CO-COO- [3,3-2 ]- 13 C-2-ketobutyrate C 3 -CD 2 -CD(CD 3 )-CD(ND 3+ )-COO- isoleucine Gardner and Kay (1997) J. Am. Chem. Soc. 119, LB/ 2 O LB/95-99% D 2 O OD 600 = 0.4 M9 in 95-99% D 2 O [100% 2 / 13 C] glucose [100% 3,3 2 2, 100% 13 C] 2-ketobutyrate [100% α/β- 2, 100% 13 C/ 15 N] Val