The use of deuteration for the structural study of larger proteins

The use of deuteration for the structural
study of larger proteins
EMBO course 2005
Daniel Nietlispach
1. Larger proteins: What are the problems ?
• nuclei relax faster due to slower tumbling:
linewidth:
Δυ =
1
πT2
τc [ns] ~ 0.4 MW [kDa]
τc :
4 ns
MW : 8 kDa
€
8 ns
16 kDa
relaxation time T2
• broader lines
• lower sensitivity of NMR experiments
correlation time τc
12 ns
24 kDa
25 ns
50 kDa
• number of signals increase with higher MW:
• increased signal overlap
2D NOESY
8 kDa (Tendamistat)
• increasing amounts of protein in NMR sample lead to solubility issues:
• may have to reduce concentrations:
probably ok if oligomeric protein, but difficulties if monomeric proteins
21 kDa (Cdc42)
Improvements that facilitate larger MW studies
• isotope labeling:
• deuteration
• selective protonation
reduction of proton density:
slow down relaxation
simplify spectra
all protons
• pulse sequences:
• better sensitivity
relaxation compensation TROSY
• new approaches
• new types of restraints:
• RDC
• cross-correlation
• chemical shift calculations
remove 1H(C):
HN, NH2
• hardware development:
• higher magnetic fields
• cryoprobes
only 1H from
Ile, Leu, Val
13
1
relative transfer efficiency
HN (ppm)
sidechain deuterated sample
1.0
coherence transfer
Data 1
1
0.6
active
0.6
0.4
Relative transfer efficiency for
HNCA on a protonated sample
as correlation time increases.
0.4
0.2
0.2
0
0
0
5 10-9
1 10-8
1.5 10-8
2 10-8
5 10 15 20 25
correlation time τc [ns]
relaxation
Sensitivity α Π nsin(πJΔ) Π mcos(πJΔ) exp(-R2 ΣΔ) }
HNCA (protonated sample)
0.8
increased resolution
1
HN (ppm)
protonated sample
F
0.8
better sensitivity
13
Cα (ppm)
Cα (ppm)
Effect of deuteration on sensitivity of 3D experiments
2.5 10-8
30
3 10-8
passive
Relaxation is mediated by molecular motion
shielding
anisotropy
• Spins are sensitive to the presence of near-by magnetic
fields e.g dipole-dipole interactions with other spins or
anisotropic chemical shielding due to non-spherical
distributions of electrons around the nucleus.
dipoledipole
interaction
molecular
reorientation
• If these external magnetic fields fluctuate randomly over
time and the changes occur in the appropriate range of
frequencies, (α
β transitions) this leads to nuclear
relaxation.
• The required fluctuations of the local magnetic fields can
be caused by e.g. brownian motion (overall rotational
tumbling of a molecule) or due to e.g. internal mobility
within a molecule.
slow tumbling
intermediate tumbling
very fast tumbling
J(ω)
• The process of relaxation is more efficient the more
motional contribution is at the appropriate frequency. A
measure of how much power is available at a particular
frequency is given by the spectral density function J(ω).
0
• T1, T2 and {H,N}-NOE are sensitive to different frequencies
and this can reveal information about the time scales of
overall tumbling and internal motion and about the
amplitude of the internal motion (dynamics) without the
need to know the type of internal dynamics present.
T2
ω: ~ 0
107
108
frequency [Hz]
T1
ωN
1012
NOE
ωH–ωN
2. Relaxation in solution
Main mechanisms contributing to relaxation in solution are:
S
B0
Θ
• dipole-dipole interaction
• chemical shift-anisotropy
R1,2DD( XY ) ~ (K/r6) S(S + 1) (γxγy )2 ∑J(ωi)
~ r-3
I
i
dipole dipole interaction
€
S = 1/2 for 1H
S = 1 for 2H (D)
γH = 6.5 . γD
€
DD( HX ) / DD( DX )
= 16 x
Transverse relaxation times as a function of molecular tumbling
(600 MHz). Relaxation contributions of DD and CSA interactions
are considered.
2.1 Impact of deuteration on relaxation contributions
Dipolar relaxation
Removable contributions to transverse relaxation
(protein tumbling with τc ~ 12ns)
internal DD
contributions
H
H
H
X
X = 13C,15N
external DD
contributions
H
Deuteration allows to remove internal and external contributions to dipole-dipole relaxation.
• Perdeuteration
what is it:
• removal of all sidechain protons H → D ( > 95%)
deuteration level is as high as possible
• identical labeling pattern for all molecules
• remaining protons:
HN
sidechain H(N) of Asn, Gln, Arg
labile –OH
γ
C
C
β
H
C
benefits:
• strong reduction of external relaxation contributions
• removal of internal contributions for 13CHn.
• increase in T2 and T1 of 13CHn and 1HN
• removal of J(H,H) → sharper lines
α
N
N
C
H
O
=D
16
€
14
T1(CD) / T1(CH)
12
J(0) dominates
ratio
10
coherence transfer
J(ωC – ωD)
8
n
sensitivity
T1(CD) / T1(CH)
6
2
5
10
15
rotational correlation time [ns]
∏ sin(π JΔ)∏ cos(π JΔ)exp(–R j ∑ Δ i )
m
4
0
relaxation
20
n
i =1
• Random fractional deuteration
what is it:
• statistical removal of sidechain protons to a certain
percentage in a random fashion. H → D (0 - 80%)
• mixture of different isotopomers (varying local environment)
benefits:
• increase in T2 and T1 of HN and to lesser extent Hα (and
also sidechain H’s)
C
CβH2–CαH
increasing level of fractional deuteration
CβHD–CαH
γ
CβH2–CαD
C
CβHD–CαD
β
C
CβD2–CαH
H
CβD2–CαD
0
α
N
C
N
C
H
O
=
1H
or D
5
10
15
20
25
30
isotopomer population
correlation time tc = 18 ns. DD and CSA are taken into account. (600
MHz). Relaxation rates with increasing levels of random statistical
sidechain deuteration. Increasing deuteration level removes external
relaxation contributions.
3. Experimental considerations
• 2H decoupling during 13C transverse periods
• remove scalar coupling
• reduce effects from scalar relaxation 2nd kind and
dynamic frequency shifts due to quadrupolar
relaxation of deuterium
• Editing methods for incomplete deuteration
• selection for C-D or e.g. CH2D
τd = 1/4JCH
1
suppress all CH
isotopomers
H
τd
TC–τd
TC
13
C
y
2
WALTZ-16x
H
1/2JCH
1
H
13
C
–y
φ = x , –x
rec. = x , –x
suppress CHD1,2 and CH3
isotopomers
CT-HN(CO)CA 2D 1HN/13Cα planes with CT=28 ms.
above: with 2H decoupling; below: no decoupling
4. Approaches to structure determination of larger proteins
tentative classification of available labeling strategies for different protein sizes:
of course this will vary from protein to protein and depend on the NMR techniques used, e.g. conventional
vs. TROSY etc.
• τC < 12 ns ( ~20 kDa protein at 298K):
13
• τC < 18 ns ( ~35 kDa protein at RT):
fractional deuteration with 13C, 15N
• τC > 18 ns ( > 35 kDa protein):
perdeuteration (> 95%) with 13C, 15N
selective protonation and background deuteration with 13C, 15N
selective protonation/reverse labeling (12C); background deuteration 13C, 15N
• Backbone assignment
• Sidechain assignment
• NOE distance information
• Dipolar coupling information
C, 15N labeling should be enough
4.1 Backbone assignment strategies
• Perdeuteration
• Maximizes sensitivity thanks to very high level of background deuteration (the higher the more sensitive)
• Strong reduction of R2(Cα) and R2(HN):
→ increased sensitivity, sharper lines
• Up to 45 kDa, constant time 13C-evolution periods (CT=1/JCaCb):
→ high resolution
• HN back exchange required: sometimes difficult, unfold/refold protocol: → will loose some HN
• R1(HN) are reduced:
→ slower pulse repetition
• Out-and-back triple-resonance experiments:
in pairs: 3D HNCA/ HN(CO)CA
3D HN(CA)CB/ HN(COCA)CB e.g. HN → N → CO → CA → CB (t1) → CA → CO → N → HN
3D HN(CA)CO/ HNCO
3D intra-HN(CA)CO/ HNCO
3D intra-HNCA/ DQ-HNCA
further: 4D HN(COCA)NH
3D HN(CACB)CG
• Increased resolution using 4D approach: HNCOCA/ HNCACO (e.g. MSG 723 AA)
• Combine experiments with H/N TROSY transfer/detection scheme. For > 50 kDa probably rather 4D than 3D
Example: backbone assignment strategy for MSG
Malate synthase G from E. coli (MSG):
723 Amino acids, 81 kDa, correlation time 37 ns @ 37˚ C
• 4D TROSY HNCOCA, HNCACO and HNCOi–1CAi (to
help resolve ambiguities) shift matching via 13CO and 13Cα
• 4D HN,HN NOESY start with Ala-HNCACB to get starting
points
• start with Ala-selective 2D HN( CACB) to find starting
points (Alai and some Alai–1) Ala ~ 10% of residues in MSG
• β-sheets and loops: sequential HN–HN NOE cannot be
detected. In cases of chemical shift degeneracy, use 13Cβ shifts
from 3D TROSY experiments
• Correct 13C shifts for deuterium isotope shifts before
predicting 2nd structure e.g using CSI
Tugarinov, V.; Muhandiram, R.; Ayed, A.; Kay, L. E. J. Am. Chem. Soc. 2002, 124,
10025-10035.
Tugarinov, V.; Kay, L. E. J. Mol. Biol. 2003, 327, 1121-1133.
Tugarinov, V.; Kay, L. E. J. Am. Chem. Soc. 2003, 125, 13868-13878.
Example: Backbone resonance assignment of a 502 residue protein (56 kDa) 3D CT-13C H/C/N experiments
Perdeuteration ( > 98%)
• 3D TROSY CT-13C experiments
TROSY-CT-HNCA
11H-15
15N TROSY-HSQC
Department of Biochemistry
Away Day 2003
Example: Sequential assignment using 3D intra-HNCA and DQ-HNCA
H
O
N
H
C
C
C
O
N
C
C
C
C
C
O
intra-HNCA
D154
F156
A155
Y157
DQHNCA
intraHNCA
DQHNCA
intraHNCA
DQHNCA
intraHNCA
DQHNCA
intraHNCA
116.8
116.8
124.3
124.3
112.8
112.8
119.7
119.7
54
13Cα(i-1)+13Cα(i)
Ω(15N)
[ppm]
56
intra-HNCA
+
DQ-HNCA
58
60
K104
62
increased resolution
and sensitivity
64
8.14
8.14
8.50
D154
HN(CO)CA
52
116.8
8.50
7.13
1H(F3) [ppm]
A155
HNCA
116.8
HN(CO)CA
124.3
7.13
9.55
F156
HNCA
HN(CO)CA
112.8
124.3
9.55
HNCA
112.8
HN(CO)CA
119.7
HNCA
119.7
Ω(15N)
[ppm]
i-1
58
?
62
13Cα(i)
intra-HNCA
HNCA
+
HN(CO)CA
56
60
DQ-HNCA
Cross peak is
shifted “on-the-fly”
to its correct
position.
Y157
?
54
Assignment
setup for intra-
HNCA / DQ-HNCA
O
DQ-HNCA
52
Ras (1-171).GDP @ 4˚ C
~ 75 kDa . 800 MHz
K104
13Cα(i-1)
64
8.14
8.14
8.50
7.13
8.50
1H(F3) [ppm]
7.13
9.55
9.55
Nietlispach et al., J. Am. Chem. Soc. 2002, 124, 11199.
13Cα(i)
Transfer efficiency for some triple-resonance TROSY experiments
• fast 13CO relaxation with increasing τc
and at high magnetic fields
• better sensitivity for experiments that
achieve inter-residue correlation without
transffer via 13CO
Field dependence of transfer efficiency
Transfer efficiency at 800 MHz
intra-HNCA TROSY
NxCα(i)zCα(i-1)zC′z
8NzCα(i)zCα(i-1)zC′’(i-1)y
→ 4NzCα(i)zC′’(i-1)x
selective refocusing of
intra contribution
DQ-HNCA TROSY
DQ–Cαx(i)Cαx(i-1)
Peak intensity comparison for residues 4-120 of H-Ras(1-171) at 4˚C (τc ~
28ns): intra-HNCA, DQ-HNCA, HN(CO)CA, sequential-HNCA and
conventional HNCA. For large proteins and at high magnetic fields DQHNCA becomes more sensitive than the HN(CO)CA.
J. Am. Chem. Soc. 2002, 124, 11199.
intra- and inter-residue connectivity using Cα shift information
HNCA
HN(CO)CA
intra-HNCA
interresidue
correlation
intraresidue
correlation
W31/
A32
F47/
L48
F178/
E179
S76/
N5/ Y77
R6
G152/
Q153
A89/ N219/
C90 E220
15N:
123ppm
N114/ Q136/
F115 F137
15N:
123ppm
15N:
123ppm
Example:
3D intra-HNCACB
reduced overlap for Cβ resonances
HN(CA)CB
E173
N174
HN(COCA)CB
intra-HN(CA)CB
Example:
Sequential assignment using 3D intra-HN(CA)CO and HNCO
H
O
J (CO,Cα)
α
C
C
J (N,Cα)
α
N
C
N
C
J (N,Cα)
J (N,CO)
intra-HN(CA)CO
J (CO,Cα)
H
O
HNCACO
intra + inter
assignment based on matching 13CO shifts
• reduced overlap in intra-HN(CA)CO
• MQ-HN(CA)CO increases signal intensity for
Ser, Thr, Gly
HNCO
inter
HN(CA)CO
2D 1HN/13CO projections of the selective intra(red)
and the conventional 3D TROSYHNCACO (blue) experiments recorded on a 80
kDa protein
sequential
sequential
Nietlispach, J. Biomol. NMR 2004, 28, 131-136
• Random fractional deuteration
• Sidechain HC resonances can be observed
• Reduction of R2(Cα,β,γ...) and R2(HN); smaller reduction for R2(Hα)
• Statistical reduction of 1H population
→ Hside, Cside assignment
→ improved sensitivity
• Sensitivity improvement is smaller than with perdeuteration
• One sample for backbone, sidechain and NOE
• Useful up to ~35 kDa (τc ~ 18ns)
• Much less problems with HN back exchange
• Mixture of various H/D isotopomers:
→ 13C isotope shift effects
• limits available resolution in 3D experiments
→ instead use 4D experiments. Keep lower resolution in each dimension
• Out-to-stay triple-resonance experiments: HC → → → HN
• 4D HBCB/HACANH and HBCB/HACA(CO)NH
• Out-and-back triple-resonance experiments e.g HNCA work too:
• requires suppression of CH isotopomer
• sensitivity reduction by a statistical factor ~ % H2O level in growth condition
50 – 60% random fractional deuteration gives increased sensitivity
HBCB/HACA(CO)NH
HBCB/HACANH
HBCB/HACA(CO)NH
0%
50%
75%
2D 1H/13C projection plane of the 4D HBCB(CACO)NH for the
deuteration levels 0%, 50% and 75%.
–CβD2–CαD–
–CβD2–CαH–
–CβΗD–CαD–
–CβH2–CαD–
–CβΗD–CαH–
–CβH2–CαH–
Magnetization transfer pathway and relative transfer efficiencies for out-to-stay
experiments as a function of the fractional deuteration level. The best sensitivity
is obtained at 50 – 60% (depending on the correlation time).
Nietlispach et al. J. Am. Chem. Soc. 1996, 118, 407-415.
0
10
20
30
40
50
60
70
Signal contribution of different isotopomers in %
4.2 Sidechain assignment
• Perdeuteration
• Assignment of 13C resonances using 3D C(CCO)NH:
→ increase in T1(13C(D)), γH = 0.25 . γH ; still, it’s quite sensitive !
• Low proton density:
→ Assign sidechain HN of Gln, Asn, Arg to increase number of protons
• Correction for 13C isotope shifts for e.g.:
• 2nd structure prediction
• to match 13C with protonated samples e.g. for HCCH-TOCSY
Farmer and Venters J. Am. Chem. Soc. 1995, 117, 4187-4188.
Secondary deuterium isotope shifts
Isotope shifts are additive with major contributions from 1-bond to ca. 3-bond:
13
C:
1
Δ
2
Δ
3
Δ
1
H 2H –0.29 ± 0.05 ppm
1
H 2H –0.13 ± 0.02 ppm
1
H 2H –0.07 ± 0.02 ppm
15
N:
1
Δ
2
Δ
1
H 2H –0.3 ppm
1
H 2H –0.05 to 0.1 ppm
Weak dependence on secondary structure: 13Cα : –0.5 ± 0.08 α-helical; -0.44 ± 0.08 β-strands
No significant shifts for 1HN and 13CO
(Δ values are based on HCA II (Venters, R. A.; Farmer, B. T.; Fierke, C. A.; Spicer, L. D. J. Mol. Biol. 1996, 264, 1101-1116.))
• Random fractional deuteration
• Assignment of H/C using 50–60% D sample: 4D HC(CCO)NH → same sample as for backbone assignment
• HCCH TOCSY:
• lower sensitivity due to less protons; sharper lines
• longrange correlations benefit and are more sensitive
correlation time τc = 18 ns
2D 1H/13C projection of the 4D HC(CCO)NH for
different levels of sidechain deuteration. Best
sensitivity is achieved around 50%.
4.3 NOE distance information
• Perdeuteration
• HN–HN NOE: 4D HNNH NOESY (HMQC, HSQC, TROSY etc.)
(Grzesiek et al. J. Am. Chem. Soc. 1995, 117, 9594-9595; Farmer et al. J. Biomol. NMR. 1996, 7, 59-71.)
• typically up to 5Å but further possible (slower spin diffusion and longer selective T1 (diagonal signal)
• very long mixing times ( < 1.2 s) → up to 8Å inter-proton distances. (Mal et al., J. Biomol. NMR 1998, 12, 259-276)
• Not enough restraints to calculate accurate global folds (RMSD > 8Å). Particularly poor if large content of α-helices.
• Additional NOE restraints are required:
• sidechain HN: R, N, Q, W are often in interior of protein. However, many are solvent exposed, exchanging rapidly.
• sidechain HC: selective protonation approaches
HN/NH2
H-Ras
all protons
• Fractional deuteration
• 15N separated NOESY benefit from 50% deuteration.
• 13C separated NOESY loose in sensitivity
• deuteration level of 50–60% → one sample for backbone and sidechain assignment
• 50–60% D is also a reasonable compromise to get NOE information
• various isotopomers contribute similarly to diffferent experiments → less problems with isotope shifts
• clearly not good enough for proteins > 35 kDa → instead: perdeuteration, selective protonation
1
Hali/1HN planes from a 3D NOESY 15N HSQC recorded at
0% and 50% fractional deuteration showing the often more
intense and better resolved peaks of the deuterated sample.
NOE peak intensities as a function of deuteration
level. Relaxation and population effects are taken
into account.
5. Selective protonation approaches
• additional NOE distance information
• selective 1H labeling of individual amino acid types or site-specific protonation:
• entire A.A.: Smith et al. J. Biomol. NMR 1996, 8, 360-368. Metzler et al. J. Am. Chem. Soc. 1996, 118, 6800-6801.
• methyl groups: Gardner et al. J. Am. Chem. Soc. 1997, 119, 7599-7600
• highly deuterated background
• 13C or 12C labeling at protonated positions (reverse labeling) (Vuister et al. J. Am. Chem. Soc. 1994, 116, 9206)
e.g. attractive for aromatic residues
Advantages of selective protonation
• protonation sites are part of the protein core
• scheme adaptable for the system under study
• varying number of residues can be labeled
• labeling techniques unproblematic (add precursors: amino acids or α-ketoacids)
• aromatic residues give many structurally important NOEs
Residue-selective protonation: more NOEs but faster relaxation. Methyl protonation is most sensitive.
• Residue-selective protonation
• protonated Ile, Leu. Val, (Ala)
• backbone assignment as for perdeuterated sample
• HC(CCO)NH for protonated residue assignment
• 13C NOESY, CT-13C-NOESY
• sensitivity suffers from intra-residue interactions
• Phe, Tyr, Trp aromatic ring-selective protonation
• aromatic residues: provide additional information for global fold
Rajesh et al. J. Biomol. NMR 2003, 27, 81-86.
determination e.g. for α-helical proteins
• Cβ and Cα positions are deuterated → assignment HNCA/CB
•12C in aromatic positions (slow relaxation)
1H
amide-rejected 1D spectrum showing the selective aromatic
ring sidechain protonation that can be achieved through the use
of shikimic acid in the growth medium.
Aromatic region of YUH1 from amide-rejected homonuclear 2D
1H-TOCSY together with strips from 3D 15 N-separated NOESYHSQC with NOEs between amide protons and aromatic protons.
• Methyl protonation
• protonated methyl groups of Ile (δ1 only), Leu, Val with highly deuterated background
• well resolved
O
• sharp lines
H15N 13CD
• interior of protein
13C
O
O
13CD 13CH
3
• V,L most common A.A. at molecular interfaces
13CH
3
H15N 13CD
13CD
2
13CD 13CH
3
13CH
3
• uniform 13C labeling
• backbone:
assignment as for perdeuterated sample (out-and-back)
• sidechain:
assignment of methyl groups → connect to backbone
13C
O
O
H15N 13CD
13C
13CD
CD3
13CD
2
13CH
3
• 3D methyl–(H)C(CCO)NH and methyl–H(CCO)NH
(Gardner, K. H.; Konrat, R.; Rosen, M. K.; Kay, L. E. J. Biomol. NMR 1996, 8, 351-356.)
• NOE:
too low resolution in methyl-region of conventional 13C NOESY
• Val, Ile selective NOESY → 3D (HM)CMCB(CMHM) NOESY
(Zwahlen et al. J. Am. Chem. Soc. 1998, 120, 4825-4831)
• methyl-selective 13C,13C NOESY
(Zwahlen et al. J. Am. Chem. Soc. 1998, 120, 7617-7625)
• methyl-selective HQQF NOESY
(Nietlispach, 1998)
O
Example: Improved resolution in 3D NOESY using CT-13C methyl selective experiment
1
H [ppm]
13 C
[ppm]
HSQC
HQQF
1H
[ppm]
left: Resolution enhancement obtained in the methyl selective HQQF experiment
compared with the 13C HSQC. right: Sensitivity improvement obtained with
HQQC compared with other methyl selective experiments.
1
H (CH3) [ppm]
left: Pulse sequence of the heteronuclear quadruple quantum
filtered CT-13C HQQF experiment. (CT-period = 6Δ = 24 ms).
above: Strip plots (F2 = methyl 13C) from a 3D NOESY 13C
HQQF recorded on 50% fractionally deuterated
Cdc42.GPPNHP.
Proton density comparison for different protonation levels
p21 H-Ras (21 kDa)
Ile, Val, Leu
methyl groups
HN/NH2
HN, methyl, aromatic
aromatic F, W, Y
all protons
Example: Long range NOEs in ILV (methyl)-FYW (aromatic)-1H YUH1
13
C-13C strips from 3D 13C-separated (t1,t2)
NOESY
I202C δ1
I36C δ1
1
I55C δ1
H-1H Strips from 3D 15N- or 13C-separated (t2) NOESY showing aromatic-HN,
HN-HN, methyl-HN, aromatic-methyl and methyl-methyl NOEs.
L231 H N
L231 HN L231 Hδ1#
Y33 Hε#
I202C δ1
I36Cδ1
L231 H δ1#
Y33 Hδ#
I55Cδ1
C NOESY
N- NOESY
13
A32 HN
V202 Hγ2#
V52 Hγ1#
C-NOESY
L48C δ2
G230 HN
I55 Hδ1
V52 Hγ2#
13
L39C δ1
G232 HN
L231 Hδ2#
N-NOESY
L45C δ2
L48C δ1
V52Cγ1
L231 Hδ1#
15
V52Cγ1
Aromatic 1H-methyl NOESY
V52C γ2
15
V207C γ1
Aromatic 1H- H N NOESY
W81 Hη2
Example: Global fold of YUH1 calculated from unambiguous NOEs measured
on ILV/FYW- protonated sample
NOEs
CH3–CH3
intra
total
149
268
aro–CH3
30
H2N–HN and HN–HN
intra
181
sequential
672
total
1616
Coordinate RMSD (Å) at initial stage:
backbone heavy atoms (all residues):
all heavy atoms (all residues):
backbone heavy atoms (β-sheet region):
all heavy atoms (β -sheet region):
*without aromatic distance restraints.
2.64 (3.20*)
3.28 (3.86*)
1.10 (1.64*)
1.84 (2.48*)
Ito et al. 2002
Example: CH3–selective protonation for global fold determination of MBP
Mueller, G. A.; Choy, W. Y.; Yang, D. W.; Forman-Kay, J. D.; Venters, R. A.; Kay, L. E. J. Mol. Biol. 2000, 300, 197-212.
• methyl protonated Ile (δ1), Leu, Val
Global fold of MBP in complex with
β-cyclodextrin
(370 residues, 42 kDa)
NOE:
H-bonds:
dihedral:
HN–HN
HN–CH3
CH3–CH3
826
769
348
48
555
5.5Å RMSD
N domain
C domain
measure 5 dipolar couplings to orient peptide plane
2.2Å RMSD
Sidechain assignment in very large proteins
Malate synthase G from E. coli (MSG): 723 Amino acids, 81 kDa, correlation time 37 ns @ 37˚ C
Tugarinov, V.; Kay, L. E. J. Am. Chem. Soc. 2003, 125, 13868-13878.
previously:
methyl protonated Ile (δ1), Leu, Val with 2H background and uniform 13C
TOCSY: HM(CCO)NH and (HM)CM(CCO)NH and [H,N]-TROSY implementations of it
beyond 60 kDa sensitivity become too low:
due to branching at Cβ (Val), Cγ (Leu): makes TOCSY problematic
Cme
CM
Cb/Cg
Ca
Approach 1: COSY type correlation Methyl → HN
Ile:
3D (HM)CM(CGCBCA)NH
problematic for Leu, Val due to similar pro-R,S CH3 shifts
Leu, Val:
reverse label one methyl group to 12CD3
= linearize CC-spin system
Leu: 3D (HM)CM(CGCBCA)NH
Val: 3D (HM)CM(CBCA)NH
HN
Approach 2: Methyl-detected out-and-back experiments
• higher sensitivity than approach 1: 5 – 10 x
• benefits from methyl reverse labeling to 12CD3 for Val, Leu
• 3D experiments: Cme/Cali/Hme and Cme/CO/Hme
• 3D HMCM[CG]CBCA
• 3D HMCM([CG]CBCA)CO
• sequence specific CH3 assignments by matching
13
Ca, 13Cb, 13CO with backbone experiments
Tugarinov, V.; Kay, L. E. J. Am. Chem. Soc. 2003, 125, 13868-13878.
High-resolution 13C CT-HMQC of methyl region (CT=28ms)
Effect of external relaxation sources on 13C HMQC
usage of linearized spin systems in MSG
Reduced concentration of methyl protons leads to
higher resolution and sensitivity
Tugarinov, V.; Kay, L. E. J. Am. Chem. Soc. 2003, 125, 13868-13878.
Methyl-TROSY
Tugarinov V. et al. J. Am. Chem. Soc. 2003, 125, 10420-10428.
Tugarinov V. et al. J. Biomol. NMR 2004, 28, 165-172.
13
1
C: 8 transitions of which 50% relax slowly
H: 10 transitions of which 50% relax slowly
TROSY: keep fast and slow relaxing components separate
HMQC transfers 50% slow → slow
HSQC transfers 19% slow → slow
Cancellation of HH and CH dipolar interactions → field independent
for ωcτm >> 1
Remove external HH dipolar interactions that degrade performance:
• Ile(δ1)-13CH3
rest: 2H and 13C for assignment
rest: 2H and 12C for NOE
(strong external HH between pro-R,S CH3 in Leu, Val:
• Leu, Val mono-methyl 13CH3,12CD3
rest: 2H and 13C for assignment
rest: 2H and 12C for NOE
ZQ-line narrowing in Methyl-TROSY:
reduces intra- and inter-methyl dipolar interactions
Tugarinov et al. J. Am. Chem. Soc. 2004, 126, 4921-4925.
HMQC > HSQC
τc ~ 118 ns
Global fold determination of the 82 kDa MSG (723 residues)
Methyl protonation approach. MSG: 22% I, L, V residues
Global fold based uniquely on NMR data.
• Assignment:
backbone and CH3: U-[15N,13C,2H], Ileδ1-[13CH3], Leu,Val-[13CH3,12CD3]
Tugarinov et al., PNAS 2005, 102, 622-627
• NOE: 3D [H,N]-& methyl-TROSY, 4D CH3-CH3
HN–HN:
CH3–CH3:
HN–CH3:
U-[15N,2H] in H2O
U-[15N,2H], Ileδ1-[13CH3], Leu,Val-[13CH3,12CD3] in D2O
U-[15N,2H], Ileδ1-[13CH3], Leu,Val-[13CH3,12CD3] in H2O
long-range contacts
HN–HN:
99
CH3–CH3:
386
HN–CH3:
142
total longr.: 627
all NOEs:
1531
only few as high α-helical content
shows strength of CH3 labeling
almost 1 per residue
φ,ψ values from chemical shift (TALOS): 1066
rmsd:
5.6 Å
rmsd 5.6 Å
α-helices ok: i → i±1, i → i±3
β-sheet: show shorter than in X-ray. Due to 2H no Hα
contacts between proximal strands!
↑↑: HN(i) ⇔ HN(j) >4.0Å; ↓↑: HN(i) ⇔ HN(j) >3.3Å
Best rmsd 4.1Å
• Inclusion of orientational restraints:
RDC (1H-15N):
415
13
changes in CO shifts: 300
rmsd 4.1 Å
Conformational space of 2nd structure elements is
reduced. A few couplings per residue are enough to
achieve correct alignment.
NOE data provides translational information.
6. Practical aspects of producing deuterated proteins
Freshly transformed E.coli (e.g. BL21 (DE3)), minimal medium D2O:
growth rate ↓ , biomass ↓ , protein ↓
Approaches:
1) Quantity of protein more important than deuteration level: max. 75-80% 2H incorporation
A)
Plate onto solid H2O-based minimal or rich medium. Increase level of D2O on
plates for gradual adaptation.
B)
Growth in solution: Small scale prep. Minimal medium. Adapt bacteria to grow in
deuterated medium by culturing in increasingly higher levels of D2O. OD600 < 0.6
→ spin, remove cultures. Resuspend in fresh medium (A600 <0.1, log-phase)
=cell resuspension approach. 1H-glucose.
Fractional deuteration:
“Per”deuteration:
no adaptation, 1H-glucose, ~ % D2O
75-80% 2H using approach B).
2) High deuteration levels: Perdeuteration of sidechains (>85%)
D2O, absolutely no H2O. Adaptation procedure for cells (e.g. 10, 25, 60, 90%), 2H-glucose.
Many cells survive and express protein when transferred from H2O → D2O without adaptation.
2
H-glucose (more reliable for protein expression), 2H-acetate, 2H-glycerol, 2H-succinate, 2H-pyruvate.
D → H back exchange incomplete (particularly 2nd structure) → unfold/refold protocol.
3) Site-specific protonation, highly deuterated background
Residue protonation: 13C, 2H background
minimal medium, D2O, 2H-glucose(13C), 15NH4Cl
+ protonated amino acids or precursors (> 50mg/l)
but: D2O will replace 30-80% of Hα.
or: auxotrophic strains
+ e.g.shikimate → F,Y,W ring protonation with α,β-2H
Methyl protonation, 13C, 2H background
minimal medium, D2O, 2H-glucose(13C), 15NH4Cl
[2,3-2H] 15N,13C 2-Ketoisovalerate (> 80mg/l) → Leu, Val CH3
[3,3-2H] 15N, 13C 2-Ketobutyrate (> 50mg/l) → Ile CH3(δ1 only)
mono-Methyl protonation for “linearized” spin system approach: 13C,2H background
as above but use:
(13CH3)(12CD3)–[2,3-2H] 15N,13C 2-Ketoisovalerate
2
H-glucose(13C)
for CH3-TROSY
mono-Methyl protonation for isolated 13CH3 spin system: 12C,2H background
as above but use:
mono-methyl 13CH3–2-Ketoisovalerate
mono-methyl 13CH3–2-Ketobutyrate
2
H-glucose (12C)
Further reading:
K. H. Gardner, L. E. Kay, Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 357-406.
The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins.
R. A. Venters, R. Thompson, J. Cavanagh, J. Mol. Struct. 2002, 602, 275-292.
Current approaches for the study of large proteins by NMR.
V. Tugarinov, P. M. Hwang, L. E. Kay, Annu. Rev. Biochem. 2004, 73, 107-146.
Nuclear Magnetic Resonance Spectroscopy of high-molecular weight proteins.
L.-Y. Lian, D. A. Middleton, Prog. Nucl. Magn. Reson. Spectrosc. 2001, 39, 171-190.
Labelling approaches for protein structural studies by solution-state and solid-state NMR.