Assignment of amide proton signals by combined evaluation of HN, NN and HNCA MAS-NMR correlation spectra.
ABSTRACT In this paper, we present a strategy for the (1)H(N) resonance assignment in solid-state magic-angle spinning (MAS) NMR, using the alpha-spectrin SH3 domain as an example. A novel 3D triple resonance experiment is presented that yields intraresidue H(N)-N-C(alpha) correlations, which was essential for the proton assignment. For the observable residues, 52 out of the 54 amide proton resonances were assigned from 2D ((1)H-(15)N) and 3D ((1)H-(15)N-(13)C) heteronuclear correlation spectra. It is demonstrated that proton-driven spin diffusion (PDSD) experiments recorded with long mixing times (4 s) are helpful for confirming the assignment of the protein backbone (15)N resonances and as an aid in the amide proton assignment.
- SourceAvailable from: purdue.edu[show abstract] [hide abstract]
ABSTRACT: Many proteins have modular design with multiple globular domains connected via flexible linkers. As a simple model of such system, we study a tandem construct consisting of two identical SH3 domains and a variable-length Gly/Ser linker. When the linker is short, this construct represents a dumbbell-shaped molecule with limited amount of domain-domain mobility. Due to its elongated shape, this molecule efficiently aligns in steric alignment media. As the length of the linker increases, the two domains become effectively uncoupled and begin to behave as independent entities. Consequently, their degree of alignment drops, approaching that found in the (near-spherical) isolated SH3 domains. To model the dependence of alignment parameters on the length of the interdomain linker, we have generated in silico a series of conformational ensembles representing SH3 tandems with different linker length. These ensembles were subsequently used as input for alignment prediction software PALES. The predicted alignment tensors were compared with the results of experimental measurements using a series of tandem-SH3 samples in PEG/hexanol alignment media. This comparison broadly confirmed the expected trends. At the same time, it has been found that the isolated SH3 domain aligns much stronger than expected. This finding can be attributed to complex morphology of the PEG/hexanol media and/or to weak site-specific interactions between the protein and the media. In the latter case, there are strong indications that electrostatic interactions may play a role. The fact that PEG/hexanol does not behave as a simple steric media should serve as a caution for studies that use PALES as a quantitative prediction tool (especially for disordered proteins). Further progress in this area depends on our ability to accurately model the anisotropic media and its site-specific interactions with protein molecules. Once this ability is improved, it should be possible to use the alignment parameters as a measure of domain-domain cooperativity, thus identifying the situations where two domains transiently interact with each other or become coupled through a partially structured linker.Journal of Biomolecular NMR 09/2011; 51(1-2):131-50. · 2.85 Impact Factor
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ABSTRACT: An approach for conveniently implementing low-power CN ( n ) (ν) and RN ( n ) (ν) symmetry-based band-selective mixing sequences for generating homo- and heteronuclear chemical shift correlation NMR spectra of low γ nuclei in biological solids is demonstrated. Efficient magnetisation transfer characteristics are achieved by selecting appropriate symmetries requiring the application of basic RF elements of relatively long duration and numerically tailoring the RF field modulation profile of the basic element. The efficacy of the approach is experimentally shown by the acquisition of (15)N-(13)C dipolar and (13)C-(13)C scalar and dipolar coupling mediated chemical shift correlation spectra at representative MAS frequencies.Journal of Biomolecular NMR 06/2011; 50(3):277-84. · 2.85 Impact Factor
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ABSTRACT: We present a systematic study of the effect of the level of exchangeable protons on the observed amide proton linewidth obtained in perdeuterated proteins. Decreasing the amount of D2O employed in the crystallization buffer from 90 to 0%, we observe a fourfold increase in linewidth for both 1H and 15N resonances. At the same time, we find a gradual increase in the signal-to-noise ratio (SNR) for 1H–15N correlations in dipolar coupling based experiments for H2O concentrations of up to 40%. Beyond 40%, a significant reduction in SNR is observed. Scalar-coupling based 1H–15N correlation experiments yield a nearly constant SNR for samples prepared with ≤30% H2O. Samples in which more H2O is employed for crystallization show a significantly reduced NMR intensity. Calculation of the SNR by taking into account the reduction in 1H T 1 in samples containing more protons (SNR per unit time), yields a maximum SNR for samples crystallized using 30 and 40% H2O for scalar and dipolar coupling based experiments, respectively. A sensitivity gain of 3.8 is obtained by increasing the H2O concentration from 10 to 40% in the CP based experiment, whereas the linewidth only becomes 1.5 times broader. In general, we find that CP is more favorable compared to INEPT based transfer when the number of possible 1H,1H interactions increases. At low levels of deuteration (≥60% H2O in the crystallization buffer), resonances from rigid residues are broadened beyond detection. All experiments are carried out at MAS frequency of 24kHz employing perdeuterated samples of the chicken α-spectrin SH3 domain.Journal of Biomolecular NMR 04/2012; 46(1):67-73. · 2.85 Impact Factor
Journal of Biomolecular NMR, 25: 217–223, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
Assignment of amide proton signals by combined evaluation of HN, NN
and HNCA MAS-NMR correlation spectra
Barth-Jan van Rossuma, Federica Castellania, Jutta Paulia,∗, Kristina Rehbeina, J. Hollanderb,
Huub J.M. de Grootb& Hartmut Oschkinata,∗∗
aForschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle Str. 10, D-13125, Berlin, Germany
bGorlaeus Laboratories, University of Leiden, P.O. Box 9502, 2300 RA, Leiden, The Netherlands
Received 16 September 2002; Accepted 20 December 2002
Key words: assignment, dipolar correlation spectroscopy, magic-angle-spinning, SH3 domain, solid-state MAS
In this paper, we present a strategy for the1HNresonance assignment in solid-state magic-angle spinning (MAS)
NMR, using the α-spectrin SH3 domain as an example. A novel 3D triple resonance experiment is presented
that yields intraresidue HN-N-Cαcorrelations, which was essential for the proton assignment. For the observable
residues, 52 out of the 54 amide proton resonances were assigned from 2D (1H-15N) and 3D (1H-15N-13C) het-
eronuclear correlation spectra. It is demonstrated that proton-driven spin diffusion (PDSD) experiments recorded
with long mixing times (4 s) are helpful for confirmingthe assignment of the protein backbone15N resonances and
as an aid in the amide proton assignment.
MAS solid-state NMR is rapidly developing into a
versatile tool for the structural investigation of bio-
logical systems that cannot be studied with solution
NMR and which do not easily form 3D crystals, such
brane proteins (Castellani et al., 2002; Griffin, 1998).
Prior to the detection of structural restraints that form
the input of structure calculations, assignment of the
proteinresonancesis mandatory.In the past few years,
several groups reported on solid-state NMR assign-
ment strategies for multiply-enriched, small proteins
(Straus et al., 1998; Hong, 1999; Pauli et al., 2000,
2001; McDermott et al., 2000; van Rossum et al.,
2001). The15N chemical shifts play there a key-role,
since sequence-specific assignment procedures often
rely on heteronuclear correlations between the amide
15N and the Cαof the same amino acid or the CO of
∗Present address: BAM, Richard-Willstätter-Str. 11, D-12489,
∗∗To whom correspondence should be addressed.
the previous one in the sequence. Using triple reso-
nance techniques, almost complete assignments of the
13C and15N resonances of the α-spectrin SH3 domain
were achieved (Pauli et al., 2001). The resonances of
non-exchangeable protons were assigned by 3D1H-
13C-13C correlation spectroscopy (van Rossum et al.,
2001). In this paper, we focus on strategies for the
assignment of amide proton signals. This is the third
paper in a series and, with the assignment of the amide
protons, it completes the solid-state MAS NMR as-
signment of the α-spectrin SH3 domain (van Rossum
et al., 2001; Pauli et al., 2001), that is used as an
NH groupsare importantstructuralmonitors, since
they are often involved in the formation of hydrogen-
bonds that stabilise the folding of a protein. In ad-
dition, the NH chemical shifts are sensitive to the
protein backbone conformations, therefore providing
secondary structure information. In static NMR ex-
periments on oriented membranes, the NH chemical
shifts and dipolar interaction vectors form the corner
stone of the PISEMA experiment (Wu et al., 1994).
In MAS NMR, amide1H and15N nuclei may be used
forthe detectionofN-H···X bondlengths, forthemea-
surementof torsionangles or of HH distance restraints
(Hong et al., 1997; Schnell et al., 1998; Reif et al.,
2000; Hohwy et al., 2000; Brown et al., 2001; Zhao
et al., 2001; Song and McDermott, 2001). In particu-
lar, for the detection of long-range H-H correlations,
the amide protons are potentially useful due to their
high γ, once samples that are perdeutarated at the
non-exchangeable sites are provided. Perdeuteration
removes all strong1H-1H dipolar couplings and leads
ing mild1H-homonuclear decoupling. This makes a
peak intensities feasible, as demonstrated in a recent
communication (Reif et al., submitted).
Materials and methods
Samples of the α-spectrin SH3 domain were prepared
as described previously (Pauli et al., 2000). For the
solid-state CP/MAS NMR correlation experiments,
preparations containing typically ∼1.4 µmol (10 mg)
of (U-15N) or (U-13C,15N) α-spectrin SH3 domain
were used. The samples were confined to the cen-
tre of the rotor by use of spacers to optimise RF
All solid-state spectra were recorded with a MAS
frequencyωR/2π= 8.0kHz. The2D1H-15N and15N-
298 K at a field of 17.6 T using a wide-bore DMX-
750 spectrometer (Bruker, Karlsruhe, Germany). The
3D1H-15N-13C dataset was recorded at 280 K, with a
DMX-400 spectrometer operating at a field of 9.4 T
(Bruker, Karlsruhe, Germany). Both spectrometers
were equipped with 4 mm triple-resonance CP/MAS
probes (Bruker, Karlsruhe, Germany). The heteronu-
clear correlation experiment was obtained with the
pulse program depicted in Figure 1A, which em-
ploys phase-modulated Lee-Goldburg (PMLG) irradi-
ation during proton evolution to suppress strong1H-
homonulear dipolar interactions (Vinogradov et al.,
1999). For the15N-homonuclear correlation experi-
ment, a standard PDSD sequence was used, with a
mixing time of 4.0 s (Szeverenyi et al., 1982). The
3D1H-15N-13C experiment is shown in Figure 1B and
applies specific-CP (Baldus et al., 1998) to transfer
magnetisation selectively between the amide15N and
the13Cαof the same residue.
For PMLG decoupling a shaped-pulse was used
that mimics each frequency offset with a phase trajec-
Figure 1. Pulse programs used for the 2D1H-15N (A) and
PMLG-decoupling (Vinogradov et al.,
decoupling (1H-15N or1H-13C) was achieved with TPPM during
evolution and acquisition (Bennett et al., 1995), while continuous
wave (CW) decoupling was applied during the specific-CP (Baldus
et al., 1998).
1H-15N-13C (B) dipolar correlationexperiments.
tory that contains three phase steps (PMLG-3) (Vino-
gradov et al., 1999). The shaped pulse contains 2048
complete PMLG cycles and has a total duration τtot.
Prior to the experiments, the efficiency of the PMLG
13C signals of adamantane. This was done by observ-
ing the JCH-couplings in 1D13C spectra collected
with PMLG irradiation during data acquisition, and
by fine-tuning the pulse length τtotto yield optimally
resolved doubletand triplet line shapes for the CH and
CH2 moieties, respectively. The proton evolution in
t1was sampled at intervals τinccorresponding to two
complete PMLG cycles (typically 40 µs). The incre-
ment τincwas first calculated according to τtot/1024,
rounded off to the nearest integral multiple of 100 ns.
Subsequently, τtotwas recalculated as (τinc· 1024).
This was done to ensure synchronisation of n · τinc
with the shaped pulse for large n. For similar reasons,
be chosen arbitrarily, but should be set to 0 µs or to a
small multiple of τinc/2. The PMLG decoupling was
optimised for the SH3 preparations by adjusting the
1H RF strength to yield similar1H pulse lengths as
foundfortheadamantanesample. Forall SH3 samples
that we have studied, this results in RF powers that are
about 10% higher than for adamantane.
The protons were decoupled by use of the two-
pulse phase-modulation (TPPM) decoupling scheme
during all acquisition periods and during the indirect
15N evolution in the correlation experiments (Bennett
et al., 1995). The TPPM decoupling was optimised di-
rectly on the SH3 domain preparations, yielding pulse
lengths of typically 7.0 µs for a phase-modulation
angle of 15 degrees. For the specific CP, RF pow-
ers corresponding to nutation frequencies of ∼15 kHz
(15N) and ∼20 kHz (13C) were applied. The amide
15N were irradiated close to resonance and the Cαoff-
resonance. The13C offset was optimised for maximal
Cαsignal, using a 1D version of the pulse program
shownin Figure1B (i.e., withoutthe evolutionperiods
Results and discussion
An initial step to the assignment of the amide signals
can be taken by a combined evaluation of 2D1H-15N
and15N-15N correlation spectra. Figure 2A shows a
relationspectrum of uniformly15N labelled α-spectrin
SH3 domain. The data were obtained at a field of
17.6 T with the sequence depicted in Figure 1A, using
1H-homonuclear decoupling during proton evolution.
A cross-polarisation contact of 170 µs was applied
to build-up heteronuclear1H-15N correlations. This
short contact time ensures that the spectrum is selec-
bondedNH pairs are observed.For these stronglycou-
pled spin-pairs, coherent transfer leads to a rapid rise
in the15N signal intensity during the first ∼150 µs of
the CP and results in strong correlations that contain
the relevant information. In contrast, the information
becomes obscured by proton spin-diffusion processes
for longer mixing times (>1 ms) and the selectivity
is lost, although some additional15N signal intensity
may be obtained. In Figure 2B, a 2D15N correlation
spectrum is shown, that was recorded at a field of
et al., 1982) and is used as a ruler in the assignment
procedure.A long PDSD mixing time of 4.0 s was ap-
plied to exchange magnetisation between the weakly
coupled15N spins. Analysis of the15N-15N PDSD
experiment revealed that most of the observed cross-
peaks are related to transfers between the amide15N
spins of sequential residues. As an example, the corre-
Table 1. Solid state and solution NMR assignment of the1HNand15N
signals of the α-spectrin SH3 domain
ResidueChemical shift (ppm)
aAt T = 298 K.
bResolved from the 3D spectrum (T = 280 K).
Figure 2. (A) Contour plot of a 2D PMLG-decoupled1H-15N heteronuclear dipolar correlation spectrum of precipitated (U-15N) α-spectrin
SH3 domain, recorded at a field of 17.6 T and with a spinning frequency ωR/2π = 8.0 kHz. The data were obtained at a temperature of 298 K,
using a short ramped CP contact of 170 µs. (B) Contour plot of a 2D15N-homonuclear dipolar correlation spectrum of precipitated (U-15N)
α-spectrin SH3 domain, recorded at a field of 17.6 T, with a spinning frequency ωR/2π = 8.0 kHz and at a temperature of 298 K. The data
were obtained using a PDSD mixing time of 4.0 s. The dashed line indicates the correlation walk from P54 to K60. Note that the amides of A56
and Y57 have almost identical chemical shifts and a cross-peak can not be resolved from the diagonal.
lations in the subsequence P54 to K60 are depicted in
Figure 2B. Other cross-peaks could be identified and
assigned in a similar fashion and the chemical shifts
are listed in Table 1.
Due to the selectivity and the high resolution in
the15N dimension, a large number of NH signals
can be assigned unambiguously from the 2D exper-
iment of Figure 2A (Table 1). There is, however,
for a small number of NH pairs overlap of the15N
chemical shifts, which prohibits the complete proton
assignmentonthebasis ofthe 2D1H-15N datasetonly.
exploiting the relatively well-resolved correlations in
a NCA experiment (Pauli et al., 2001). This can be
Figure 3. Plot of a 3D PMLG-specific CP HNCA correlation experiment, displayed with a single contour (blue). The 3D dataset was recorded
from precipitated (U-15N,13C) α-spectrin SH3 domain, at a field of 9.4 T and at a spinning frequency ωR/2π = 8.0 kHz. The spectrum was
obtained at a temperature of 280 K. The ω1-ω2(1H-15N) and ω2-ω3(15N-13C) projections of the 3D experiment are shown in red.
Figure 4. Assignment of the amides of T24, G28 and Q50. (A) shows a section of the 2D1H-15N experiment of Figure 2A, centred around
the15N chemical shift of the three residues (∼116.6 ppm). In (B), a plane from the 3D dataset is shown, extracted at the same15N chemical
shift. Finally, (C) shows a strip from a 2D NCA experiment, recorded from (U-15N,13C) α-spectrin SH3 domains at a field of 9.4 T and using
a spinning frequency ωR/2π = 8.0 kHz.
done by correlating the1H-15N signal with the Cαof
the same residue in a 3D (1H-15N-13C) heteronuclear
correlation experiment (Figure 3), using the pulse se-
quenceshown in Figure 1B. The methodcombinesthe
PMLG-decoupled1H-15N experiment in Figure 1A
with specific CP following the nitrogen evolution in
t2(Baldus et al., 1998), to transfer magnetisation se-
lectively from the backbone15N to the Cα. In this
way, each residue gives rise to a single intra-residue
1HN-15N-13Cαcorrelation in the 3D spectrum. The
resolution enhancement obtained in the 3D HNCA
correlation experiment allows unambiguous assign-
ments of the amide protons. This is illustrated in
with the15N centred around 116.5 ppm, close to the
amide15N chemicalshift forthethree residues. Dueto
overlap in the nitrogen dimension, it is not possible to
assign the amide protons of T24, G28 and Q50 unam-
biguouslyfrom the 2D experiment.On the other hand,
the Cαresonate with different chemical shifts for T24,
Q50 and G28, at 61.9 ppm, 53.4 ppm and 45.1 ppm,
respectively. Hence the signals from the three residues
are fully resolved in the NCA dimension of the ex-
periment (cf. Figures 4B and C) and the three amide
protons can be assigned unambiguously from the ω1-
ω3slice extracted from the 3D dataset with an ω215N
shift near 116.6 ppm (Figure 4B). The assignment of
theamide protonsis listed in Table1, togetherwiththe
shifts found in solution NMR, for pH 3.5 and pH 7.5.
The1HNthat we could not detect are from the first
seven residues on the N-terminus (M1-E7), and from
the residues N47 and D48.
The15N-15N correlation network along the protein
backbone observed in the15N-15N correlation spec-
trum was useful for cross-checking our previously re-
ported15N assignment (Pauli et al., 2001). Two more
15N signals were identified that were not previously
assigned from the NCA-type experiments (Pauli et al.,
2001). D62 at the C-terminus was tentatively assigned
signal of V46 was identified from correlations with
E45 (Figure 2B). Consistently, a weak correlation was
sample recorded at 9.4 T, that we now can assign to
V46 (data not shown). This correlation has a chemical
shift of 125.7 ppm for the15N and of 60.0 ppm for the
13Cα, in line the previously reported Cαassignment
(Pauliet al., 2001).Combiningthese assignmentswith
the 2D1H-15N and 3D1H-15N-13C experiments, the
amide proton signals of V46 and D62 can be assigned
and are included in Table 1.
Residues that are difficult to assign from the15N-
15N PDSD experiment are prolines because most of
the correlations involving the back-bone nitrogens of
these residues are very weak and below the limit set
by the contours in Figure 2B. Proline is the only type
of residue that has a non-protonated amide nitrogen,
and coherence transfer mediated by15N signal broad-
ening induced by NH dipolar couplings during the
PDSD mixing will be less effective. Since prolines
resonate downfield of the amide response, the PDSD
sequence can be expected to be less efficient for15N
magnetisation transfer between prolines and residues
that resonate more upfield. Indeed, transfer between
the amides of P54 and A55 is observed, that have rel-
atively closely spaced chemical shifts of 136.8 ppm
and 129.2 ppm, respectively, but not between P54 and
V53, the latter resonating around 110 ppm. Likewise,
sequential correlations between P20 (137.7 ppm) and
S19 (111.4ppm)orR21 (112.3ppm), that have a large
difference in chemical shift, are not detected.
Some correlations were detected in the 2D15N-
15N spectrum that could not be assigned to transfers
between amides of sequential residues. Such corre-
lations involve long-range transfers and provide re-
straints for the calculation of the fold of the SH3 do-
main from the solid-state NMR data (Castellani et al.,
2002). For instance, a correlation is observed that can
be identified as V23-F52 and/or V23-Y15. According
to the solution NMR structure of the α-spectrin SH3
domain (Blanco et al., 1997), the distance between
the amides is 4.1 Å for V23 and F52, and 9.0 Å for
V23 and Y15. The observed correlation most likely
involves transfer over the shortest distance, between
V23 and F52. Likewise, it was found that S19 cor-
relates with E17 (5.8 Å) and/or E22 (4.9 Å). Since
the long-range correlations can not be assigned un-
ambiguously from the current solid-state data, they
were included as ‘ambiguous’ restraints in the struc-
ture calculations presented recently (Castellani et al.,
It has been shown that amide proton signals can be
assigned unambiguously from the 2D and 3D dataset
in Figures 2–4, providing that13C and15N assign-
ments exist. Together with the assignment of the non-
exchanging protons reported previously (van Rossum
et al., 2001), nearly all1H of the α-spectrin SH3
domain have been assigned. This is the largest sys-
tem for which a nearly full1H solid-state MAS NMR
assignment has been obtained till date. Most of the
cross-peaks in the triple-resonance experiment are
fully resolved, even at a moderately low field strength
of 9.4 T. The 3D spectrum may also serve as a poten-
tially important building block for obtaining structural
restraints, if combined with suitable homonuclear or
heteronuclear transfer schemes. In addition, the 3D
HNCA experiment is considerably more resolved as
compared to the 2D NCA experiment recorded at the
same magnetic field strength (Pauli et al., 2001). The
resolution enhancement achieved by adding the1H
dimension to the NCA experiment may be instrumen-
tal for the sequential assignment, if combined with
HNCO experiments performed in parallel.
The15N assignment obtained from the15N-15N
correlation experiment is fully consistent with the as-
signment reported previously (Pauli et al., 2001). In
this respect, the15N chemical shift information con-
tained in the15N homonuclear correlation experiment
is basically the same as the one obtained from the
NCA/NCO-type triple resonance experiments. The
experiment ‘tells’ which pairs of15N correlate and
provides information about which amides are con-
nected via sequential residues. Hence, the15N-15N
experiment provides an independent check of the15N
assignments, since it relies on direct transfer between
the sequential15N of the protein backbone and not on
a two-step transfer mechanism via the Cαand/or CO.
It shouldthereforebeconsideredas an experimentthat
can be performed in parallel with the NCA(CX) and
the assignment, and to reduce ambiguity in an early
stage in the assignment procedure.
Support from the DFG (grant no.: SFB 449) and from
the EU (grant no.: BIO4-CT97-2101) is gratefully ac-
knowledged.The authors thank Bernd Reif for helpful
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