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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, 357-406 for a general review)
6.57.07.58.08.59.09.510.0
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6.57.07.58.08.59.09.510.0
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Tendamistat 8 kDa
Cdc42 21 kDa
6.57.07.58.08.59.09.510.0
108
110
112
114
116
118
120
122
124
126
128
6.57.07.58.08.59.09.510.0
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HR1b9 kDa
ττττc = 6nsec
Cdc42/ACK26 kDa
ττττc=18nsec
Effects of IncreasingMolecular Size
•number of resonances- crowded spectra
•faster relaxation- broad lines- low intensity- overlap- long experiment time
Transverse relaxation times as a function of protein size
0
50
100
150
200
250
300
4 8 12 16 20 24 28
tran
sver
se re
laxa
tion
time
T2 [m
s]
correlation time [ns]
Cαααα(D)N(H)
Cαααα(H)
Hn
Hαααα
Hββββ
Cx CyNz ∆∆∆∆= 20-24 msecCx Cy ∆∆∆∆=20-27 msec
but T2 often less than 30 msec
Sensitivity α Πα Πα Πα Πnsin(ππππJ∆∆∆∆) ΠΠΠΠmcos(ππππJ∆∆∆∆) exp(-R2 Σ∆Σ∆Σ∆Σ∆) }}}}
Linewidth ∆ν∆ν∆ν∆ν1/2 = 1/ππππT2
789 ppm 789 ppm 789 ppm 789 ppm
ττττc 5nsMW 10kDa
10ns20kDa
15ns30 kDa
25ns50kDa
Linewidth vs. Correlation Time
coherence transfer relaxation
active passive
Relaxation Mechanisms
•Dipole-Dipole interactions (DD)•Chemical shift anisotropy
HX=13C,15N
X
HH
H
internal DD contributions
external DDcontributions
R1,2 (X) ~ D.I(I+1).(γγγγXγγγγY)2 . ΣΣΣΣ J(ωωωωi)dip
i
γγγγD/γγγγH = 1/6.5
dCD/dCH = 1/16
Impact of deuteration on relaxation rates
Cαααα N Hαααα HN
rela
xatio
n ra
te c
onst
ant/H
z
0
20
40
60
80
100
removed by sidechain deuteration
fixed
ττττc =12 nsec
Impact of deuteration on relaxation rates
T2
T1
ττττc = 18 nsec
2
4
6
8
10
12
14
16
0 5 10 15 20
overall correlation time [ns]
Rat
io
T2(CD) / T
2(CH)T
2(CD) / T
2(CH)T
2(CD) / T
2(CH)
T1(CD) / T
1(CH)
Correlation time (nsec)
Rat
io
T2(CD)/T2(CH)
T1(CD)/T1(CH)
Effects of deuteration on Cαααα relaxation times
Isolated C-H pair
J(0) dominate
J(ωωωωC-ωωωωD) effects
Approaches to the Structure Determination of Larger
Proteins
• For ττττc up to ~12 ns (20 kDa) -13C/15N-labelling
• For ττττc up to ~18 ns (35 kDa) -fractional deuteration and 13C/15N-labelling
• For ττττc above ~18 ns - selective protonation and 13C/15N-labelling
Triple Resonance NMR and Random Fractional Deuteration
• Backbone assignments• Side-chain assignments• Measurement of NOE contacts
Backbone Assignments
• Deuteration reduces relaxation• Maximum sensitivity with 100%
deuteration- Grzesiek et al., (1993) J. Am. Chem. Soc.
115, 4369-4370.- Yamazaki et al., (1994) J. Am. Chem. Soc.
116, 6464-6465.- Yamazaki et al., (1994) J. Am. Chem. Soc.
116, 11655-11666.
HNCA HN(CO)CA
N
H
Cα
C
O
H
N
DCβ
HβCγ
Hγ
N
H
Cα
C
O
H
N
DCβ
HβCγ
Hγ
12
3
4
123
4 5
6
Backbone Experiments with Deuterated Proteins
•Out and back
•CT for 13C evolution (1/Jcc)
•1H T1s:longer recycle delaypreserve water
•Deuterium decoupling
•Removal of residual CααααH
•Sensitivity enhancement (20 nsec limit?)Shan et al (1996) JACS 118 6570-79
y -yWALTZ16x
1H15N
13Cαααα
2H
t1/2
ττττd
Tc Tc-t1/2
2Tc = 26.6 ms = 1/Jccττττd = 1.7ms = 1/4JCH
•2H restored to z-axis (lock stability)•CααααH evolves for 1/2JCH - removed by 1H decoupling
Backbone Assignments
Nietlispach et al., (1996) J. Am. Chem. Soc. 118, 407-415.
Hββββ
Hββββ
CααααC
O
H
N
Cββββ
H/D
Cαααα
Cββββ H/D
H/D
H/D
CααααC
O
H
N
Cββββ
H/D
Cαααα
Cββββ H/D
H/D
H/D
HBCB/HACANNH HBCB/HACA(CO)NNH
Isotopomer Fraction Contributionfor Cββββ peaks
CββββH2-CααααH 12.5% 4%
CββββHD-CααααH 25% 16%
CββββH2-CααααD 12.5% 17%
CββββHD-CααααD 25% 63%
CββββD2-CααααH 12.5% 0%
CββββD2-CααααD 12.5% 0%
Isotopomers in 50% 2H Proteins
Effect of Deuteration on HBCB/HACA(CO)NHfor a protein with ττττc~18ns (30kDa)
0% 2H 50% 2H 75% 2H
7.58.08.59.0
20
30
40
50
60
70
ppm7.58.08.59.0
0% 2H 50% 2H
Effect of Deuteration on HBCB/HACANNH for a protein with
18 nsec correlation time
• Backbone assignments• Side-chain assignments• Measurement of NOE contacts
Triple Resonance NMR and Random Fractional Deuteration
Side-chain Assignments
• Deuteration reduces relaxation• Maximum sensitivity with 50%
deuteration– Nietlispach et al., (1996) J. Am. Chem.
Soc. 118, 407-415.
HCC(CO)NNH
N
H
Cαααα
C
O
H
N
HααααCββββ
HββββCγγγγ
Hγγγγ
1
2
3 4
5
1
2
ppm7.58.08.59.0 7.58.08.59.0
Effect of Deuteration on HCC(CO)NNH for a Protein with a
12 nsec Correlation Time
0% 50%
Side-chain Assignments
• Deuteration reduces relaxation• Deuteration removes both
protons
C
HC
HC D
C
HC
HC
D
HCCH-COSY HCCH-TOCSY
D
D
D
D
H
Effect of Deuteration on HCCH-TOCSY for a Protein with 18 nsec
Correlation Time
0.51.01.52.02.53.03.54.04.5
4.2
4.3
4.4
4.5
4.6
0.51.01.52.02.53.03.54.04.5
4.2
4.3
4.4
4.5
4.6
0.60.81.01.21.41.61.82.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0.60.81.01.21.41.61.82.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0%-2H
50%-2H
0%-2H
50%-2H
Valα α
α β α γ1
α γ2
Ile
δ δδ γ2δ γ1
δ β
Isotope Shifts
Isotope effects:
1∆∆∆∆ 13C ~ -0.25 ppm2∆∆∆∆ 13C ~ -0.1 ppm3∆∆∆∆ 13C ~ -0.07 ppm1∆∆∆∆ 15N ~ -0.3 ppm2∆∆∆∆ 15N ~ -0.05 to -0.1 ppm
e.g 13Cαααα in 100% D protein -0.5 ppm isotope shift
weak secondary structure dependency.
Isotopomers not resolved in most experiments
• Backbone assignments• Side-chain assignments• Measurement of NOE contacts
Triple Resonance NMR and Random Fractional Deuteration
Measurement of NOE Contacts
• Deuteration affects the measurement of HN - HN, HN - HC and HC - HC cross peaks in different ways
– Nietlispach et al., (1996) J. Am. Chem. Soc. 118, 407-415.
Distance Restraints and Fractional Deuteration
0% 2H 50% 2H 75% 2H
NH/NH
NH/sidechain
ττττc = 12 nsec
Measurement of NOE Contacts
• Similar contributions from different isotopomers lead to comparable chemical shifts
CHD
CH2
δ (ppm)
J-correlated NOESY
CHD
CH2
δ (ppm)
Structure Calculations
• Use of ARIA for structure determination
– Nilges (1995) J. Mol. Biol. 245, 645-660
• Global fold from selectively protonated data
• Use Talos to estimate backbone torsion angles from chemical shifts
Fractional Deuteration at 50%
•Improved sensitivity and linewidth
•No complications from different isotopomers
•Backbone and sidechain assignments possible
•1H/1H NOEs can be observed
•Applicable to proteins up to 30 kDa
Approaches to the Structure Determination of larger
Proteins
• For proteins of up to ττττc ~ 12 ns, use 13C/15N-labelling
• For proteins of up to ττττc ~ 18 ns, use fractional deuteration and 13C/15N-labelling
• For proteins ττττc ~ 18 ns and above, use selective protonation and 13C/15N-labelling
Approaches for Proteins Larger than 30 kDa
Complete deuterationmaximum sensitivity for
backbone experiments but limited NOE information (HN <-> HN)
Selective protonation of residues e.g AILV in deuterated background
Selective protonation of Methyl-groups(Gardner J. Am. Chem. Soc. 119, 7599)
Triple Resonance NMR and Selective Protonation
• Label proteins with ILV+FY• Methyl and aromatic 13C and 1H
nuclei relax more slowly– Allows measurement of NH-NH,
NH-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
Triple Resonance NMR and Selective Protonation
• Backbone assignments• Side-chain assignments• Measurement of NOE contacts
Backbone Assignments
• Deuteration reduces relaxation• Maximum sensitivity with 100%
deuteration• ILV - Cαααα mainly deuterated• Me-selective - Cαααα 100% deuterated
HNCA HN(CO)CA
N
H
Cαααα
C
O
H
N
DCββββ
HββββCγγγγ
Hγγγγ
N
H
Cαααα
C
O
H
N
DCββββ
HββββCγγγγ
Hγγγγ
12
3
4
123
4 5
6
Triple Resonance NMR and Selective Protonation
• Backbone assignments• Side-chain assignments• Measurement of NOE contacts
Side-chain Assignments
• Deuteration reduces relaxation• Maximum sensitivity with 100%
deuteration– Yamazaki et al., (1994) J. Am. Chem. Soc.
116, 11655-11666.– Farmer et al., (1995) J. Am. Chem. Soc.
117, 4187-4188.
N
H
Cαααα
C
O
H
N
DCββββ
DCγγγγ
D
1
2 3
4
1
CC(CO)NNH
N
H
Cαααα
C
O
H
N
DCββββ
DCγγγγ
D
12
34
5
6
HNCACB
Side-chain Assignments
(H)CC(CO)NNH
N
H
Cαααα
C
O
H
N
DCββββ
DCγγγγ
Hγγγγ
2
3 4
5
1
2
Gardner et al (1996) J. Biomol. NMR 8, 351-356.
Triple Resonance NMR and Selective Protonation
• Backbone assignments• Side-chain assignments• Measurement of NOE contacts
Measurement of NOE Contacts
• 3D 15N- and 13C-NOESY
• 3D Val/Ile (HM)CMCB(CMHM) NOESY
– Zwahlen et al., (1998) J. Am. Chem. Soc. 120, 4825-4831.
• 3D 13C/13C-NOESY– Zwahlen et al., (1998) J. Am. Chem.
Soc. 120, 7617-7625.
• 3D HQQF-NOESY
Gz
13C
1H
CT
∆∆ ∆ ∆φrec
1φ
ξ 2
φ3
BB
13C
1H
∆ ∆ ∆φrec
1φ
ξ 4
φ3
BB
φ2
φ4
φ5
QQF
3∆ − t 1/2
t 1/4t 1/4 ∆/2
∆t 1/4 + ∆t 1/4 +
CT − t 1/2t 1/2
G3 G3 G4 G4G2 G2G1
2 CT
DEPT-HQQC
Methyl-Selective Correlation Experiments
HQQF
(Kessler, 1989)
all 1H transverse (16 msec)
a Hyb 2H1xCyc 8H1xCyH2zH3z (5∆∆∆∆ evolution of JCH)
6∆∆∆∆ ≈ 1/Jcc = 24 msec
only 1 proton transverse between b and c
a
b
c
-0.50.00.51.01.5H (ppm)1
(a) HQQF
(b) HQQC
(c) DEPT-HQQC
(c) QQF-HSQC
Sensitivity Improvement in HQQF
0.00.20.40.60.81.01.21.41.6
10
2
4
6
8
0
2
4
6
8
C (
ppm
)13
H (ppm)1
I126γ2
I117γ2
I46γ2
A142 β
V85γ2
I4γ2V77
γ1
L165δ2
L112δ2
L165δ1
L53δ2
L112δ1
Resolution Improvement in HQQFCdc42 methyl region
Structure Calculations
• Use ARIA for structure determination
– Nilges (1995) J. Mol. Biol. 245, 645-660
• Restrain φφφφ and ϕϕϕϕ angles in early stages and slacken off later
Limited Restraints in Structure Determination
Ras:•Mixed αααα and ββββ structure•Reasonable size (21kDa)•Single domain
Numbers of Methyls and Aromatics:
11 Ile11 Leu15 Val5 Phe9 Tyr0 Trp
NHs only - 13Å RMSD
HN/NH2
Ile, Val, Leu
Aromatic
13C/15N ILVFY2.0Å RMSD101 HN-HN (ass-ass)390 HN-S/C (ass-amb)431 S/C-S/C (ass-ass)
13C/15N ILV 15N FY5.0Å RMSD101 HN-HN (ass-ass)390 HN-S/C (ass-amb)231 ILV-ILV (ass-ass)174 ILV-FY (ass-amb)26 FY-FY (amb-amb)
13C/15N ILV 15N FY1.7ÅNH NOEs to 5ÅAs above with 330 HN-HN NOEs
Structures from Limited Restraints
Ras NMR Structure0.6Å RMSD 101 HN-HN 1171 HN-S/C1862 SC/SC
Structures from Limited Restraints:Effect of Dipolar Couplings
Clore et al (1999) JACS 121 6513-14
BAF - 89 residues all ααααCVN - 101 residues 2 domains - all ββββ
BAF CVNHN-HN 106 101HN-Me 40 84HN-arom 18 18Me-Me 25 70Me-arom 51 53arom-arom 5 5Total 245 331
Hbonds 37 40
RDC 259 334
H-bonds + HN + Me ~4Å accuracyAdding aromatics - 1.37Å (BAF) 1.53Å (CVN)Adding RDC - 0.91Å (BAF) 1.10Å (CVN)
RDC: HN,N-C�,HN-C�,Cαααα-H,Cαααα-C�
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
Practical Aspects of Producing Deuterated Proteins
• Random fractional deuteration
LB/H2O LB/50% D2O
M9 in 50% D2O witheither [50% 2H, 100% 13C/15N]algal hydrolysate or[100% 2H/13C] glucose
OD600 = 0.4
Practical Aspects of Producing Deuterated Proteins
• Selective protonation• Use 2H/13C glucose, amino acids and:
CH3-CD2-CO-COO- [3,3-2H]-13C-2-ketobutyrate
CH3-CD2-CD(CD3)-CD(ND3+)-COO- isoleucine
Gardner and Kay (1997) J. Am. Chem. Soc. 119, 7599-7600.
LB/H2O LB/95-99% D2O
M9 in 95-99% D2O
[100% 2H/13C] glucose
[100% 3,3−3,3−3,3−3,3−2H2, 100% 13C] 2-ketobutyrate
[100% αααα/ββββ-2H, 100% 13C/15N] Val
OD600 = 0.4