STUDIA UNIVERSITATIS BABES-BOLYAI, PHYSICA, SPECIAL ISSUE, 2003
SOLID-STATE NMR TECHNIQUES FOR STRUCTURAL INVESTIGATIONS
OF PROTEINS
Claudiu Filip
National Institute for Research and Development of Isotopic and Molecular
Technology, Cluj-Napoca, Romania
Abstract: Applications of Solid-State NMR spectroscopy to structural
investigation of large proteins requires extending its techniques from
selectively to uniformly labeled compunds. Here we present two novel
MAS NMR pulse sequences for simultaneous measurement of carbonnitrogen distances in uniformly 13C,15N-labeled solids.
Introduction
Solid-state nuclear magnetic resonance (SS-NMR) spectroscopy
currently undergoes a remarkable evolution from being mainly physicsoriented method for studying small molecules in inorganic or organic
materials toward biology-oriented applications handling complex clusters of
atoms for structure analysis of large bio-molecules. Recent advances in
magic-angle-spinning (MAS) SS-NMR instrumentation and methodology
[1] have greatly contributed to extending its applicability from selectively to
multiply 13C,15N labeled peptides and proteins. In a first step of obtaining
full 3D structures for large proteins, site-specific resonance assignments are
a source of valuable structural information. Whereas NMR experiments for
sequential resonance assignments generally exploit the strongest spin-spin
interactions and are relatively straightforward to implement in uniformly
13 15
C, N labeled systems, long-range distance measurements in these systems
are more complicated. The nuclei form a tightly coupled network via
through-space (dipolar coupling) and through-bond (J-coupling) spin-spin
interactions; consequently, the strongest couplings can interfere with the
accurate determination of the weaker ones. In the following we present new
MAS NMR pulse sequences for simultaneous measurement of multiple
carbon-nitrogen distances in uniformly 13C,15N labeled solids. The
experiments are demonstrated in model U-13C,15N labeled tri-peptides N-formyl-LMet-L-Leu-L-Phe (MLF), where 26 13C-15N distances up to ~5-6 Å were
determined.
CLAUDIU FILIP
Long-range distance constraints in uniformly 13C,15N-labeled polypeptides
The pulse sequences employ transferred echo double resonance (TEDOR) for 13C15
N coherence transfer and 15N and 13C frequency labeling for site-specific
resolution, and as such build on several previously described 3D TEDOR
experiments. The novel feature of the 3D TEDOR pulse sequences presented here
is that they are specifically designed to circumvent the detrimental effects of
homonuclear 13C-13C J-couplings on the measurement of weak 13C-15N dipolar
couplings. Homonuclear J-couplings: (i) generate undesirable anti-phase and
multiple-quantum (MQ) spin coherences, which lead to spurious cross-peaks and
phase-twisted lines in the 2D 15N-13C correlation spectra, and thus degrade the
spectral resolution and prohibit the extraction of reliable cross-peak intensities and
(ii) significantly reduce cross-peak intensities for strongly J-coupled 13C sites (e.g.,
CO and C). The first pulse sequence (Fig. 1a) employs z-filter periods to suppress
the anti-phase and MQ coherences and generates 2D spectra with pure absorption
mode peaks for all TEDOR mixing times. The second sequence (Fig. 1b) uses
band-selective 13C pulses to refocus J-couplings between 13C spins within the
selective pulse bandwidth and 13C spins outside the bandwidth.
The NMR spectra were recorded at 11.7 T (500.1 MHz 1H, 125.8 MHz 13C,
50.7 MHz 15N) using a triple-resonance MAS probe. The spinning frequency of
10.0 kHz was used in the all experiments and regulated to 5 Hz and the samples
Figure 1: Z-Filtered (ZF) and band-selective (BASE) 3D TEDOR pulse sequences. Narrow
and wide solid rectangles represent /2 and  pulses, respectively. In (a) the modifications
to the recently described 3D TEDOR experiment [] are highlighted by gray rectangles. The
modifications consist of two z-filter periods, , which eliminate undesired multiple quantum
and anti-phase spin coherences generated by 13C-13C J-evolution (see text for details). In
(b) band- selective 13C Gaussian  pulses refocus 13C-13C J-couplings to nuclei outside the
 pulse bandwidth
SOLID-STATE NMR TECHNIQUES FOR STRUCTURAL INVESTIGATIONS OF PROTEINS
were centerpacked in the rotors to minimize the effects of rf inhomogeneity. TheU13 15
C, N labeled tri-peptide N-formyl-L-Met-L-Leu-L-Phe (MLF) represents an
excellent model system for the 3D TEDOR experiments as its three-dimensional
structure (see Figure 2) has been already determined using X-ray crystallography
[2].
Using a zero order approximation [3] we proceed to describe the 3D
TEDOR experiments by neglecting all orientation dependent terms. In summary,
the main features of the simulation model are: (i) for the ith 13C nucleus coupled to
Ni 15N spins all Ni 13C-15N distances are determined by simultaneously fitting the
experimentally observed buildup of cross-peaks located at (Nk, Ci), k = 1,2,,Ni,
using eqs (3)-(5) in Ref. [3] with Ni + 2 fit parameters (Ni dipolar coupling
constants, Dik, a relaxation rate, i, and an amplitude scaling factor, i), (ii) no
Figure 2: X-ray crystal structure of N-formyl-L-Met-L-Leu-L-Phe-OMe (MLF)
knowledge of dipolar tensor orientations and no powder averaging are
required, and (iii) the cross-peak intensities at long mixing times are inversely
proportional to the number of 15N spins, Ni, simultaneously coupled to the 13C
 
N
1
nucleus, Vik (t mix )  2 i , which places a practical limit on the magnitude of the
weakest 13C-15N couplings that can be determined using 3D TEDOR methods (for
the tri-peptide investigated in this work 13C-15N dipolar couplings corresponding to
distances in the 4-6 Å regime could be determined). We note that for ZF-3D
TEDOR the buildup of cross-peaks for the ith 13C nucleus also depends on the
coupling topology of the mi J-coupled 13C nuclei. These parameters are held
constant in the simulations since 13C-13C J-couplings in peptides do not vary
significantly for different types of couplings (i.e., CO-C, C-C, etc.) [4].
Figure 3 shows representative 2D 15N-13C correlation spectra in N-formyl13 15
[U- C, N]L-Met-L-Leu-L-Phe for the ZF-3D TEDOR experiment. A large
CLAUDIU FILIP
number of dipolar correlations (~20) are observed in the aliphatic 13C spectral
region for longer mixing period (Figure 10c), and each cross-peak carries
information about 13C-15N internuclear distances. The 13C and 15N resonance
Figure 3: Representative two-dimensional slices from broadband 3D TEDOR experiments
in N-formyl-[U-13C,15N]L-Met-L-Leu-L-Phe. The 2D spectra shown in (a) and (c) (with the
1D slices corresponding to Met, Leu and Phe 15N resonances shown in (b) and (d)) were
recorded at 500 MHz 1H frequency and r/2 = 10 kHz ± 5 Hz using the pulse sequence in
Figure 1a with the z-filter delays of  = 200 s. Additional experimental parameters are
described in the text. The 3D data set consisted of 16 2D spectra acquired for TEDOR
mixing times up to 18 ms and incremented in a non-linear fashion in order to maximize the
information content and minimize the duration of the experiment. Each 2D spectrum was
acquired as (19, 1024) complex points in (t1, t2), with time increments of (800, 20) s
resulting in acquisition times of (14.4, 20.5) ms. Each FID was 128 scans, with a 3 s
recycle delay, yielding a total measurement time of ~67 h.
The 13C and 15N resonance assignments for MLF have been presented in previous
studies [5]. The 3D TEDOR cross-peak buildup curves for selected 13C sites
SOLID-STATE NMR TECHNIQUES FOR STRUCTURAL INVESTIGATIONS OF PROTEINS
Figure 4: 3D TEDOR cross-peak buildup curves in N-formyl-[U-13C,15N]L-Met-L-Leu-LPhe. The buildup curves were acquired using the pulse sequence in Figure 1a and
correspond to cross-peaks observed for the Met C (a) and Leu C (b) resonances in the 2D
correlation spectra in Figure 3. For each 13C the experimental buildup curves
corresponding to the Met (), Leu() and Phe () 15N sites were fit (—) using analytical
expressions based on the zeroth order Bessel function approximation. The values of 13C-13C
1
J-coupling constants used in the fits were 55 Hz for JCO C  and 34 Hz for all other Jcouplings. The cross-peak buildup curves shown in the figure correspond to carbonnitrogen distances in the 2.5-6 Å regime. Similar simulations were performed for the
remaining cross-peaks (c.f. Figure 3) and the 13C-15N internuclear distances are
summarized in the table
corresponding to distances in the 2.5-6 Å range are shown in Figure 4. Similar
experiments were performed using the BASE-3D TEDOR pulse sequence [3]
(spectra not shown) and the results are presented in the Table.
Conclusions
In conclusion, we have presented two 3D TEDOR NMR pulse sequences
for the simultaneous measurement of multiple carbon-nitrogen distances in
uniformly 13C,15N labeled in site-specific fashion. The novel feature of the
sequences is that they are compensated for the detrimental effects of 13C-13C Jcouplings on the measurements of long-range carbon-nitrogen distances. The
experiments are robust and straightforward to implement in practice. Qualitatively,
the presence of 13C-15N correlations in the 2D spectra in itself provides direct
information about three-dimensional structure. More accurate distance
measurements were performed using a simple analytical simulation model, which
accounts explicitly for multiple spin-spin couplings contributing to cross-peak
buildup.
CLAUDIU FILIP
Table: InternuclearC-N distances N-formyl-L-Met-L-Leu-L-Phe-OMe (MLF)
atoms
Met(N)
Leu(N)
Phe(N)
Met(C’)
Met(C)
Met(C
Met(C
Met(C
Leu(C2
Met(C’)
Met(C)
Met(C
Met(C
Met(C
Leu(C’)
Leu(C)
Leu(C
Leu(C2
Met(C’)
Met(C
Met(C
Met(C
Leu(C’)
Leu(C)
Leu(C
Leu(C2
Phe(C’)
Phe(C)
Phe(C
ZF-3D TEDOR
—
1.5
2.5
3.8
5.3
6.1
—
2.6
3.2
5.0
5.8
—
1.5
2.6
3.9
—
3.9
5.6
5.2
—
2.3
3.2
6.0
—
1.4
2.6
rC–N (Å)
BASE
3D TEDOR
2.5
—
2.6
—
—
—
1.3
—
3.2
—
—
2.4
—
2.6
—
3.4
3.8
—
—
1.3
—
3.1
—
2.7
—
2.6
X-ray
2.40
1.46
2.50
3.04
5.71
6.28
1.33
2.44
3.20
4.56
5.93
2.46
1.46
2.50
3.63
3.41
4.06
5.43
5.62
1.34
2.43
3.12
5.38
2.37
1.47
2.53
Acknowledgment. The author acknowledges financial support from the Romanian
Academy by the Research Grant No. 22 /2003.
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[4] J. Cavanagh, W. Fairbrother, A.G. Palmer, N. Skelton, Protein NMR spectroscopy:
Principles and Practice, Accademic Press, San Diego (1996)
SOLID-STATE NMR TECHNIQUES FOR STRUCTURAL INVESTIGATIONS OF PROTEINS
[5] C.P. Jaroniec, B. Tounge, J. Hertzfeld, R.G. Griffin, J. Am. Chem. Soc., 123, 3507
(2001)