Chemical shift assignment of the transmembrane helices
of DsbB, a 20-kDa integral membrane enzyme, by 3D
magic-angle spinning NMR spectroscopy
YING LI,1DEBORAH A. BERTHOLD,2,3ROBERT B. GENNIS,1–3
AND CHAD M. RIENSTRA1–3
1Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana,
Illinois 61801, USA
2Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA
3Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA
(RECEIVED September 5, 2007; FINAL REVISION November 6, 2007; ACCEPTED November 10, 2007)
The Escherichia coli inner membrane enzyme DsbB catalyzes disulfide bond formation in periplasmic
proteins, by transferring electrons to ubiquinone from DsbA, which in turn directly oxidizes cysteines in
substrate proteins. We have previously shown that DsbB can be prepared in a state that gives highly
resolved magic-angle spinning (MAS) NMR spectra. Here we report sequential13C and15N chemical
shift assignments for the majority of the residues in the transmembrane helices, achieved by three-
dimensional (3D) correlation experiments on a uniformly
frequency. We also present a four-dimensional (4D) correlation spectrum, which confirms assignments
in some highly congested regions of the 3D spectra. Overall, our results show the potential to assign
larger membrane proteins using 3D and 4D correlation experiments and form the basis of further
structural and dynamical studies of DsbB by MAS NMR.
15N-labeled sample at 750-MHz
Keywords: solid-state NMR; membrane protein; chemical shift assignment; magic-angle spinning;
disulfide bond formation
Supplemental material: see www.proteinscience.org
Membrane proteins are estimated to constitute 30%–40%
of the proteins encoded by the human genome (Liu et al.
2002), and the majority of the drugs on the market are
Despite their high abundance and importance, they are
underrepresented in current protein structure databases.
In recent years, structural biology tools including both
X-ray crystallography (Byrne and Iwata 2002) and solution
NMR (Pervushin et al. 1997; Fernandez and Wider 2003;
Tamm and Liang 2006) have been increasingly applied to
membrane proteins (Torres et al. 2003), resulting in more
than 100 unique high-resolution structures. Both structure
and dynamics can be examined by NMR, and recent
advances in solid-state NMR methods (McDermott 2004)
are especially beneficial for membrane proteins since
samples can be prepared in either native-like lipid envi-
ronments or chemically defined lipid bilayers without using
detergents. Another advantage of SSNMR is that line widths
are not correlated to the size of the protein.
Reprint requests to: Chad M. Rienstra, Department of Chemistry,
University of Illinois at Urbana-Champaign, Urbana, Illinois 61801,
USA; e-mail: firstname.lastname@example.org; fax: (217) 244-3186.
Abbreviations: BMRB, biological magnetic resonance data bank;
CP, cross-polarization; DARR, dipolar assisted rotational resonance;
SPECIFIC CP, spectrally induced filtering in combination with cross-
polarization; TPPM, two pulse phase modulation.
Article and publication are at http://www.proteinscience.org/cgi/doi/
Protein Science (2008), 17:199–204. Published by Cold Spring Harbor Laboratory Press. Copyright ? 2008 The Protein Society
SSNMR techniques capable of determining the relative
orientation of transmembrane helices from macroscopi-
cally aligned samples have been well established (Opella
and Marassi 2004). However, the spectral quality depends
on the degree of alignment. On the other hand, magic-
angle spinning (MAS) experiments do not require sample
alignment but require signals to be assigned in a site-
specific fashion that is analogous to solution NMR. Due
to the smaller chemical shift range within transmembrane
helices (relative to b-strand secondary structures), this
task can be time-consuming and technically challenging.
Moreover, few membrane proteins have solution NMR
assignments available, and so the SSNMR assignments
must be performed de novo. So far, nearly complete or
partial assignments have been reported for only a few
membrane proteins, compared with more than a dozen
soluble proteins (with molecular weight ranging from 5 to
20 kDa) that have been completely assigned (McDermott
2004; Pintacuda et al. 2007). In these few studies,
partially selective labeling schemes have been employed
to simplify spectra (Egorova-Zachernyuk et al. 2001;
Gammeren et al. 2005; Etzkorn et al. 2007) except for a
small single transmembrane protein (Andronesi et al.
2005). Here we show that the majority of the signals
from the transmembrane helices of a 20-kDa membrane
protein, DsbB, can be assigned using one uniformly13C,
15N-labeled sample and three-dimensional (3D) correla-
DsbB is an integral membrane enzyme located on
the inner membrane of Escherichia coli. Together with
soluble protein DsbA, it catalyzes disulfide bond forma-
tion in many periplasmic proteins and facilitates their
folding (Kadokura et al. 2003). DsbB contains four
transmembrane helices and binds DsbA via one of two
periplasmic loops. There is so far one published high-
resolution structural study of DsbB (Inaba et al. 2006), in
which DsbB was co-crystallized with DsbA in a cova-
lently linked complex. Efforts to crystallize DsbB in the
absence of DsbA were not successful. In the present
study, we use MAS NMR techniques to study the C41S
mutant of DsbB, which represents a transient intermedi-
ate state in the proposed reaction pathways (Inaba et al.
2005). Besides reporting the signal assignment of trans-
membrane helices, we demonstrate that four-dimensional
(4D) correlation spectra can be acquired on the same
sample with good sensitivity, illustrating the potential to
assign membrane proteins of larger size.
Results and Discussion
Four 3D chemical shift correlation experiments (NCACX,
NCOCX, CAN(CO)CX, and CON(CA)CX) were ac-
quired at 750 MHz1H frequency for signal assignment.
Three-dimensional NCACX and NCOCX experiments
form the basis of most previous chemical shift assignment
work on solid proteins by MAS NMR (McDermott 2004).
However, these two experiments are not sufficient for
uniformly labeled polytopic membrane proteins, which
typically contain a large number of hydrophobic residues
in helical conformation. Within these amino acid types,
both13C and15N chemical shifts are highly degenerate,
though to less extent for15N. In a previous study, we have
reported the method to prepare DsbB samples that give
narrow line widths (;0.5 ppm for13C and ;0.7 ppm for
15N at 750 MHz1H frequency) and partial assignment of
signals from NCOCX, NCACX, and CANCO spectra (Li
et al. 2007). The same method was first employed to
prepare a 144-kDa membrane protein, cytochrome bo3
oxidase, for MAS NMR studies (Frericks et al. 2006). In
this study, we have employed a revised sample prepara-
tion to obtain protein pellets with less bulk lipids, which
enabled us to pack a larger quantity of protein into the
rotor. The enhanced sample sensitivity allows for two
additional experiments, CAN(CO)CX and CON(CA)CX,
to be performed, which provide unambiguous connectiv-
For all 3D experiments, relatively short13C-13C mixing
times were chosen to give only intraresidue correlations.
Figure 1 shows representative two-dimensional (2D)
planes from these 3D spectra. Among these experiments,
CAN(CO)CX and CON(CA)CX 3D are more technically
challenging due to the fact that three cross-polarization
steps, two of which involve15N-13C transfers, must be
performed with high stability and transfer efficiency
(Franks et al. 2007). However, they provide a few advan-
tages over NCOCX and NCACX 3D with long13C-13C
mixing times, which can also provide a similar type of
connectivity information. First, the correlation pattern is
more predictable since long mixing times often result in
cross-peaks between spins that are not close in the pri-
mary sequence. Second, the number of cross-peaks is
approximately the same as in NCOCX and NCACX
with short mixing times rather than being significantly
increased. This presents a significant advantage to the
time-consuming stages of data analysis, which are not
yet sufficiently automated for SSNMR. Third, the CAN
(CO)CX and CON(CA)CX experiments provide unam-
eliminate the need to infer the types (intra- or interresi-
due) of correlations from peak intensity, which can be
significantly affected not only by the internuclear dis-
tances but also by molecular dynamics.
Similar to solution NMR protocols, the sequential
assignment process involves spin system identification,
the linking of spin systems, amino acid type identifica-
tion, and mapping to the amino acid sequence. Because
amide proton chemical shifts are generally not observed
in SSNMR (with notable exceptions) (Paulson et al. 2003;
13C-13C correlations and therefore
Li et al.
Protein Science, vol. 17
Hologne et al. 2006; Zhou et al. 2007a,b), more accurate
identification of amino acid types is required to effi-
ciently link spin systems and map them back to the
sequence. The iterative assignment process proceeds in
stages. In the first stage, CON(CA)CX and NCACX
spectra are combined to identify all spins from the i resi-
due and C9[i ? 1]. Similarly, NCOCX and CAN(CO)CX
spectra can be combined to identify all spins from the
i ? 1 residue, Ca[i] and N[i], forming another group of
spin systems. Subsequently, these two groups of spin
systems are compared to link i and i ? 1 residues. The
results from this linking step are highly reliable since
three backbone chemical shifts (N[i], C9[i ? 1], Ca[i]) are
matched and the overall degeneracy for this particular set
of chemical shifts is quite low. The second stage of the
linking process involves taking two residue pairs to form
a triplet by matching the chemical shifts of all13C spins
from the same residue. False matching can occur at this
stage due to the high
within residues of the same type and secondary structure.
Therefore, at this stage, accurate determination of amino
acid types is essential to eliminate the triplets that are not
present in the amino acid sequence. For DsbB, this can be
done for a large percentage of residue pairs since the
three most abundant amino acid types (Leu, Ala, and Val)
are among those that can be unambiguously determined
from Ca, Cb, and Cg chemical shifts. This is expected to
be generally the case for the transmembrane regions of
other membrane proteins since they are always featured
13C chemical shift degeneracy
by high abundance in hydrophobic residues, among which
most of the nonaromatic types can be easily determined.
Figure 2 shows a strip plot of 3D spectra that contain
cross-peaks from the triplet A73-V75. If a single amino
acid type cannot be assigned, a generic type consisting of
several types of amino acids with similar chemical shift
ranges could be assigned and sequence information can
be used in a similar way. Linking triplets and mapping
Figure 1. Representative 2D planes from 3D (A) NCACX, (B) CAN(CO)CX, (C) NCOCX, and (D) CON(CA)CX chemical shift
correlation spectra acquired on a [U-13C,15N] DsbB C41S sample (;1 mmol) at 750-MHz1H frequency, 12.5-kHz MAS frequency,
and 223 K; 75-kHz1H TPPM decoupling (6.5 ms, 9°) was used during indirect evolution periods and acquisition period, and 90-kHz
CW decoupling was used during15N-13C and13C-15N SPECIFIC CP periods. Additional acquisition and all processing parameters for
each spectrum are included in the Electronic supplementary material.
Figure 2. A strip plot of residues A73–V75. The plot consists of strips
from four 3D spectra: (A) CON(CA)CX, (B) NCACX, (C) NCOCX, and
Chemical shift assignment of DsbB by MAS NMR
them back to the sequence is straightforward and there-
fore is not discussed here.
By following the protocol described above, we were
able to assign 90% of the signals that were detected in at
least one of the 3D spectra. The remaining 10% of signals
were not assigned due to either very high degeneracy in
the chemical shifts of many Leu residues or signals
missing in one or two 3D spectra. The assignment table
is included in the Supplementary material and has been
submitted to the BMRB as entry 15,546 (Seavey et al.
1991). Figure 3 shows the backbone torsion angles of all
assigned residues determined from assigned chemical
shifts by the program TALOS (Cornilescu et al. 1999).
All assigned residues have helical secondary structure
except for G61 and A62. These two residues are located
at the end of the second transmembrane helix and the
beginning of the short intracellular loop. Other signals in
this loop are missing, and similarly, we find no long
stretches of amino acids consistent with the periplasmic
region. Since essentially all of the signals observed in the
spectra have been assigned at least by amino acid type,
we conclude that the signals from two periplasmic
regions must be below the detection limit in the 3D
spectra. The missing signals can be explained by either
low CP efficiency caused by motion or inhomogeneous
line broadening caused by multiple conformations in slow
chemical exchange. If the former is the case, the overall
13H-13C CP enhancement factor determined from
one-dimensional (1D) experiments with and without CP
should be significantly lower than that for a completely
rigid protein since ;90 out of 176 residues are located out-
side of the transmembrane helices. However, the (1H-13C)
CP enhancement factor remains ;2.0 throughout the
temperature range from 223 K to 283 K, which is close
to the values previously detected for completely rigid
proteins (Franks et al. 2005); likewise the15N-13C transfers
proceed with high efficiency (;50% for N-C9 and ;40%
for N-CA), which is only possible if the majority of the
protein backbone is rigid. In addition, at 283 K we did not
values than 15 ms, suggesting that the periplasmic regions
are not mobile even when the actual sample temperature
is close to room temperature. Therefore, we conclude that
static disorder is the reason why periplasmic signals are too
broad to be detected in the 3D spectra yet contribute to the
overall integrated intensity of 1D CP-MAS spectra. In the
X-ray structure (Inaba et al. 2006), the second periplasmic
region contains an additional helix, which was not observed
in our spectra. This we attribute to the likelihood that these
residues are more disordered when DsbB is not in complex
with DsbA. This structural difference is consistent with
biochemical studies (Kadokura and Beckwith 2002; Inaba
et al. 2005) that suggest the intrinsic flexibility of the
second periplasmic region of DsbB is likely to be important
for its function.
To evaluate the potential to assign larger membrane
proteins, such as receptors and channels with seven or
more transmembrane helices (von Heijne 2006), we
acquired a 4D CANCOCX spectrum on the same DsbB
sample. The polarization transfer pathway is the same as
the 3D CAN(CO)CX experiment, but in addition, an
evolution period is incorporated while the polarization
resides on the CO spin (Franks et al. 2007). Figure 4
shows representative 2D planes from this 4D spectrum
with assignments labeled. Many strong cross-peaks can be
detected with chemical shifts in each dimension match-
ing very well with the 3D data. Since 4D experiments are
often associated with long sampling time, the choice of
acquisition and sampling parameters can affect spectral
quality significantly. A detailed investigation of how 4D
experiments can be efficiently implemented on membrane
protein samples is beyond the scope of this work; likely
the fast methods developed in solution NMR in the last
several years, including GFT, nonlinear sampling, and
projection reconstructions (Szyperski and Atreya 2006),
will be of benefit to acquire 4D spectra with higher digital
resolution. However, the quality of the initial spectrum
acquired at 500 MHz1H frequency leads us to believe that
the assignment of larger membrane proteins can benefit
from 4D experiments.
In summary, we have employed a robust protocol to
assign the majority of the signals from the transmembrane
helices of DsbB C41S mutant by MAS NMR. Each 3D
spectrum can be acquired on a DsbB sample containing
;1 mmol protein within a few days. With the improved
sensitivity and resolution of the data sets presented here,
13C signals with significantly longer T2
Figure 3. Backbone torsion angles of assigned residues in DsbB C41S
determined by TALOS analysis.
Li et al.
Protein Science, vol. 17
tentatively assigned signals from our previous study were
in most cases confirmed; a subset of the previous assign-
ments were determined to be in error, and in all cases the
present study should be considered definitive. Our results
demonstrate the feasibility of assigning the transmem-
brane helices with relatively high chemical shift degen-
eracy using uniformly labeled samples. The approach
presented here is generally applicable to membrane pro-
teins that can be efficiently expressed in isotope-enriched
Materials and Methods
DsbB was expressed as previously described (Li et al. 2007)
from E. coli C43 (DE3) containing a plasmid encoding DsbB
C41S with a 6-His tag and mutation of two nonessential
cysteines, C8A and C49V (Inaba et al. 2004). DsbB was solu-
bilized from isolated membranes using 1% dodecylmaltoside
and purified using metal chelate chromatography on Talon cobalt
resin, by elution with 120 mM imidazole (pH 6.0). Fractions
containing DsbB were pooled and concentrated, followed by
dialysis against 25 mM Tris (pH 7.5) to remove dodecylmaltoside
and imidazole. Centrifugation of the dialyzed suspension in a
fixed-angle rotor (1 h, 100,000g) produced a hard pellet consisting
of contaminating protein and lipid. Further centrifugation in a
swinging bucket rotor (20 h, 100,000g) separated the resulting
magenta-colored supernatant into a viscous deep red pellet (DsbB
C41S) and a colorless solution. The pellet was then packed into a
3.2-mm thin wall rotor with an active volume of ;36 mL using
several low speed centrifugations (5–10 min, 3000g). The13C and
15N signals detected in 1D CP-MAS experiments correspond to
20 mg (;1.0 mmol) of packed protein, which was determined by
comparing to the total integrated signals of a standard protein of
known quantity (GB1) (Franks et al. 2005).
SSNMR experiments were performed either on a 750 MHz (1H
frequency) three-channel Varian Inova spectrometer or a 500 MHz
(1H frequency) four-channel Varian InfinityPlus spectrometer.
Both spectrometers were equipped with 3.2 mm
Balun MAS probes. The typical p/2 pulse widths were 2.5 ms
on1H, 2.9 ms on13C, and 5.6 ms on15N for the 750 MHz probe
and were 2.4 ms on1H, 2.4 ms on13C, and 7.3 ms on15N for the
500 MHz probe. The flow rate of sample cooling gas was main-
tained at ;90 scfh, with the gas temperature measured at the
output of the delivery stack. For 3D15N-13C-13C and13C-15N-13C
correlation experiments, band-selective SPECIFIC CP (Baldus
et al. 1998) was used for polarization transfer between
for13C-13C mixing. All pulse sequences were implemented with
tangent ramped CP (Hediger et al. 1994) and TPPM1H decou-
pling (Bennett et al. 1995) at ;75 kHz during indirect evolution
and acquisition periods.
Data were processed with nmrPipe (Delaglio et al. 1995),
employing zero filling and Lorentzian-to-Gaussian apodization
for each dimension before Fourier transformation. Back linear
prediction and polynomial baseline correction (frequency domain)
were applied to the direct dimension. Additional acquisition and
processing parameters for each spectrum are included in either the
figure captions or Supplementary material. Chemical shifts were
referenced externally with adamantane, with the downfield13C
resonance referenced to 40.48 ppm (Morcombe and Zilm 2003).
Peak picking and assignment were performed with Sparky (http://
13C, and DARR (Takegoshi et al. 2001) was employed
This work was supported by the National Institutes of Health
(NIGMS and Roadmap Initiative R01GM075937). We thank
Dr. Kenji Inaba and Professor Koreaki Ito for providing the
plasmid encoding DsbB and the University of Illinois School of
Chemical Sciences NMR Facility for technical support.
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