DOI: 10.1002/chem.201301921
In-Situ Spin Labeling of His-Tagged Proteins:
Distance Measurements under In-Cell Conditions
Christoph Baldauf,[a] Katrin Schulze,[a] Petra Lueders,[b] Enrica Bordignon,*[b] and
Robert Tamp*[a]
Dedicated to Prof. Dr. Amy Louise Davidson (1958*–2013†), Purdue University, West Lafayette, Indiana (USA)
Abstract: New spin labeling strategies
have immense potential in studying
protein structure and dynamics under
physiological conditions with electron
paramagnetic resonance (EPR) spectroscopy. Here, a new spin-labeled
chemical recognition unit for switchable and concomitantly high affinity
binding to His-tagged proteins was synthesized. In combination with an orthogonal site-directed spin label, this
novel spin probe, Proxyl-trisNTA (P-
trisNTA) allows the extraction of structural constraints within proteins and
macromolecular complexes by EPR.
By using the multisubunit maltose
import system of E. coli: 1) the topology of the substrate-binding protein,
Keywords: chelators · electron paramagnetic resonance · membrane
proteins · molecular recognition ·
spin probes
Introduction
Electron paramagnetic spectroscopy (EPR) plays an important role in studying the structure and conformational dynamics of proteins under conditions relevant to function.[1]
Since EPR detects specifically paramagnetic centers, the
majority of proteins must be modified by an EPR sensitive
reporter, called spin label. Despite recent developments,
such as nonsense suppressor and semisynthetic approaches
using solid-phase synthesis and native chemical ligation,
site-directed spin labeling (SDSL) with unique cysteines is
still the main technique to incorporate spin labels, in vitro.[2]
The main advantage of SDSL is that the probes, usually pyrroline- or piperidine-type nitroxide radicals, are comparable
in size to amino acids, structural details can thus be extracted from EPR data, especially from distance constraints between two labeled sites. Bulkier probes, such as functionalized chelators loaded with transition metal ions (e.g., GdIII),
[a] Dr. C. Baldauf, Dr. K. Schulze, Prof. Dr. R. Tamp
Institute of Biochemistry, Biocenter
Cluster of Excellence–Macromolecular Complexes (CEF-MC)
Goethe-University Frankfurt, Max-von-Laue Str. 9
60438 Frankfurt a.M. (Germany)
E-mail: [email protected]
[b] Dr. P. Lueders, Dr. E. Bordignon
ETH Zurich, Laboratory of Physical Chemistry
Wolfgang-Pauli-Str. 10, 8093 Zurich (Switzerland)
E-mail: [email protected]
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201301921.
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2) its substrate-dependent conformational change, and 3) the formation of
the membrane multiprotein complex
can be extracted. Notably, the same
distance information was retrieved
both in vitro and in situ allowing for
site-specific spin labeling in cell lysates
under in-cell conditions. This approach
will open new avenues towards in-cell
EPR.
have also been recently introduced at surface-exposed sites
in soluble proteins and showed their potentiality in providing distance constraints in combination with other GdIII-tags
or nitroxide labels.[3] The main disadvantages of SDSL are
that the protein of interest must be purified and that it must
possess one or two cysteines at desired sites while all other
intrinsic cysteines need to be eliminated.
Here, we describe a new nitroxide probe, which binds specifically to the His-tag of proteins in purified samples as
well as in situ, in cell lysates. As a proof-of-concept we used
this novel spin probe orthogonally to the conventional
MTSL ((1-oxyl-2,2,5,5-tetramethyl-D3-pyrroline-3-methyl)
methanethiosulfonate, R1) to extract intra- and intermolecular distance constraints and to analyze formation of large
membrane protein complexes.
Results and Discussion
Spin labeling of His-tagged proteins: Figure 1 A shows the
new spin label for reversible high-affinity binding to genetically encoded His-tags, which consists of a multivalent
chemical recognition unit, grafting three N-nitrilotriacetic
acid moieties (trisNTA) and a proxyl (P) nitroxide probe on
a tetraaza macrocyclic scaffold (cyclam). The eight-step synthesis of P-trisNTA is detailed in the Supporting Information. After loading with transition metal ions, such as NiII,
P-trisNTA can coordinate up to six imidazole moieties, thus
displaying a nano- to subnanomolar affinity towards His6–10tagged proteins under physiological conditions.[4] We first
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tion of imidazole (250 mm) released the P-trisNTA from
MalE, demonstrating the specificity and reversibility of the
spin labeling strategy.
The room temperature cw EPR spectrum of P-trisNTA
shows three narrow lines, typical for nitroxide in the fast
motional regime (Figure 1 B, grey). Spectral integration revealed a > 90 % radical content in the P-trisNTA label,
proving the integrity of the radical after synthesis and purification. After nickel loading of P-trisNTA, we observed
broadening of the nitroxide spectrum (visible as a decrease
in the intensity of the black spin-normalized spectrum in
Figure 1 B) due to interaction of the paramagnetic nickel(II)
species with the nitroxide moiety through exchange and dipolar mechanisms.
We next incubated MalE with substoichiometric ratios of
Ni-P-trisNTA to reveal the spectral features of the bound
spin label. The spectrum showed narrower lines than the
free Ni-P-trisNTA probe, visible as an increase in signal amplitude (Figure 1 B, red line). This is due to the higher rigidity of the bound probe that minimizes the intramolecular interactions between the nickel(II) and the proxyl group, as
well as to the decreased intermolecular collisions between
Ni-P-trisNTAs. Adding an excess of imidazole to the spin labeled protein reverted the spectrum back to that of the free
label in solution (Figure 1 B, cyan lines), confirming the reversibility of the Ni-P-trisNTA labeling.
Figure 1. Principle of the P-trisNTA spin labeling strategy. A) Structure
of the NiII-loaded P-trisNTA, which can coordinate up to six imidazole
moieties (X). B) Spin-normalized continuous wave (cw) X-band spectra
detected at room temperature. P-trisNTA before (grey) and after loading
with NiII (black). Ni-P-trisNTA bound to the N-terminal His6-tag of
MalE (red). Bottom, spectrum obtained after release of Ni-P-trisNTA
from MalE by addition of imidazole (250 mm, cyan), which is almost indistinguishable from that of Ni-P-trisNTA free in solution (black).
demonstrated the reversible binding of the Ni-P-trisNTA to
N-terminally His6-tagged maltose binding protein (MalE)
from Escherichia coli. After incubation with stoichiometric
amounts of P-trisNTA, complex formation was analyzed by
size exclusion chromatography (SEC) and continuous wave
(cw) EPR. In SEC, a shift of the free Ni-P-trisNTA peak
(2.0 mL) to the protein peak with slightly higher apparent
molecular weight (1.65 mL) indicated the formation of a
stable, stoichiometric complex (Supporting Information, Figure S1). Importantly, binding of Ni-P-trisNTA did not
induce dimerization or aggregation of MalE. No protein labeling was observed with the metal-free P-trisNTA. Addi-
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Determination of interspin distances and protein topology:
Determination of interspin distances by EPR provides
unique insights into the structure of proteins and multiprotein complexes.[5] The accuracy and range of interspin distances determined by pulsed EPR techniques (e.g., double
electron–electron resonance, DEER), strongly depends on
the transverse (Tm) and longitudinal (T1) relaxation times of
the nitroxide radicals.[6] Usually, DEER experiments with nitroxide probes are carried out at 50 K. At this temperature,
a maximized phase memory time (Tm) for optimal distance
sensitivity and signal-to-noise ratio is combined with a T1 in
the millisecond range, allowing for relatively fast signal accumulation (shot repetition time of a few milliseconds). To
examine its applicability for distance measurements, we first
determined T1 and Tm for the nitroxide in the Ni-P-trisNTA
at 50 K (Supporting Information, Figure S2). The T1 longitudinal relaxation of P-trisNTA moieties before and after
loading with NiII is about 1 ms, which is similar to the value
found for conventional MTSL nitroxide labels in water solutions. The phase memory time Tm of P-trisNTA is about
2 ms,. After loading with NiII, it decreases to about 0.4 ms.
Binding to MalE does not further affect the Tm (Supporting
Information, Figure S3). The time length of a DEER sequence is determined by the dipolar frequency to be detected, but in general exceeds two microseconds. Thus, the fast
Tm of the Ni-P-trisNTA species does not allow detection of
this type of nitroxides as observer spins in a DEER sequence. For the same reason, determination of distances by
DEER between two Ni-P-trisNTA species is disfavored.
However, the relatively long T1 allows the use of this new
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E. Bordignon, R. Tamp et al.
Figure 2. Extracting protein topology using Ni-P-trisNTA. A) Crystal structure of maltose-free MalE (grey, PDB ID: 1OMP),[7] displaying the Ca atoms
of the four positions spin-labeled with the conventional nitroxide label MTSL (called R1). Dotted lines highlight distances towards residue #1, used as
reference position for the Ni-P-trisNTA label attached to the N-terminal His6-tag. B) Upper panel: normalized experimental X-band (position 13) and
Q-band primary DEER traces (positions 36, 80, 352) and 3D decaying background (dotted lines), as fitted by DeerAnalysis2011.[8] Bottom panel: form
factors F(t) obtained after background correction and fit by the Tikhonov regularization with a = 100 (dotted lines). MalE was shock frozen in Tris
buffer (50 mm, pH 7.5) containing deuterated glycerol (20 % v/v) as cryoprotectant. For positions 13 and 352 a 0.9:1 stoichiometric ratio between Ni-PtrisNTA label and protein gave rise to a modulation depth in the 0.25–0.4 range. The smaller modulation depths for positions 36 and 80 arise from a
0.75:1 stoichiometric ratio. C) Experimental distance distributions obtained by DEER. Superimposed are the Ca Ca distances of each MTSL-labeled
position to residue #1 increased by 2 nm to estimate the length of the new label. The trend of distances obtained with the new label follows that expected
from the crystal structure.
label as a “pump spin” in DEER experiments in combination with a “slower relaxing” observer spin (e.g., a conventional MTSL).
We performed X- and Q-band DEER experiments at
50 K to verify if it is possible to measure the distance between the spin-labeled His-tag and a conventional MTSL attached at four different engineered cysteines (G13C, T36C,
T80C, S352C) of MalE (Figure 2). The distances between
the MTSL labels and Ni-P-trisNTA showed the same trend
as the Ca Ca distances calculated in the crystal structure
using residue #1 as reference for the His-tag position when
considering a 2 nm offset as an estimate for the length of
the Ni-P-trisNTA label. Due to the intrinsic flexibility of the
P-trisNTA label, the experimental distance distributions between the R1 side chains and the spin-labeled His6-tag in
MalE are rather broad. Despite the high degree of disorder
of the Ni-P-trisNTA label in frozen samples, we could retrieve the topology of MalE using the novel label in combination with an MTSL site scan. This proves the specificity of
the Ni-P-trisNTA for the N-terminal His-tag of MalE and
the applicability of the orthogonal spin labeling strategy to
proteins.
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Venus flytrap motion of MalE detected with orthogonal labeling: The position 352 in the C-lobe of MalE opposes the
His6-tag and is therefore an optimal combination with the PtrisNTA spin label to monitor Venus flytrap dynamics of
MalE from the open to the closed conformation upon substrate binding (Figure 3). In the absence of maltose, the two
spin probes show a 6 nm mean interspin distance as expected from their location in the opposite lobes of MalE in the
open conformation.[7] Despite the flexibility of Ni-P-trisNTA
attached to the His6-tag of MalE and the relatively small
maltose-induced hinge motion, the new spin probe monitors
the maltose-induced protein conformational change as visible directly in the primary DEER data (Figure 3 A, grey and
blue traces). Maltose binding induces an overall shift towards smaller distances (Figure 3 B). The large distribution
observed upon maltose addition can be the fingerprint of
the flexibility of the His6-tag in the closed conformation
(Supporting Information, Figure S4), but we cannot exclude
that an equilibrium between open and closed forms exists,
analogous to a dynamic equilibrium observed by NMR spectroscopy in the absence of maltose.[9] Upon addition of imidazole to the sample in the absence of maltose, no dipolar
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In-Situ Spin Labeling of His-Tagged Proteins
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Figure 3. Conformational changes of MalE detected with Ni-P-trisNTA. A) Q-band DEER traces obtained by incubating MalE-352R1 (57 mm) stoichiometrically labeled with Ni-P-trisNTA in the absence (grey) and presence (blue) of maltose (5 mm) in Tris buffer (50 mm, pH 7.5) containing deuterated
glycerol (20 % v/v). Labeling is reversed by addition of imidazole (250 mm, red trace). Left panel; normalized experimental data V(t) and 3D decaying
background (dotted red lines) as fitted by DeerAnalysis2011.[8] Right panel: modulation-depth-normalized form factors F(t) and fit by the Tikhonov regularization with a = 100 (dotted red lines). B) Left panel: superimposed crystal structures of open (grey, PDB ID: 1OMP)[7] and closed MalE (blue, PDB
ID: 1ANF)[10] showing the Venus flytrap motion induced by maltose binding. The grey and blue clouds represent the location of the simulated R1 rotamers attached at position S352. The cones represent the flexible His6-tag to which P-trisNTA is attached. Right panel: normalized interspin distance distributions obtained by the fitting of the data in panel A. The arrow indicates the shift towards smaller distances upon substrate binding.
oscillations are visible in the DEER trace confirming the efficient release of the Ni-P-trisNTA from the His-tag and the
specificity of reversible spin labeling (Figure 3 A, red trace).
In-situ labeling and distance measurements under in-cell
conditions: We next explored one of the major advantages
of the new P-trisNTA spin-labeling techniques, namely the
specific and reversible in-situ labeling of His-tagged proteins
in cell extracts under in-cell conditions. E. coli cell lysates
containing MalE with the R1 spin label at position 352 were
incubated with substoichiometric amounts of Ni-P-trisNTA
in the presence of 5 mm maltose (Figure 4 A). Notably, the
DEER trace and the extracted distance distribution in the
cell lysate perfectly agree with those detected with the purified protein in the presence of maltose (Figure 4 B), thus
highlighting the specificity of the new spin labeling strategy
for in-situ EPR distance measurements.
Chem. Eur. J. 2013, 19, 13714 – 13719
Probing formation of macromolecular complexes: To probe
the assembly of macromolecular complexes, MalE labeled
with Ni-P-trisNTA was added to the maltose ABC transporter (MalFGK2) carrying a reporter nitroxide at position 205
in the periplasmic loop of the membrane subunit MalF.[11]
The two additional N-terminal His-tags in the MalK subunits are located on the opposite side of the transporter; consequently they do not interfere with the measurements.
Despite the low affinity of MalE (in the 100 mm range) to
the transporter, which generally generates serious signal-tonoise limitations in DEER experiments,[12] it was possible to
follow with Q-band DEER the complex formation with the
novel spin label on MalE. The broad distance detected between the spin-labeled His-tag of MalE and the periplasmic
loop in MalF is a clear indication for the formation of the
215 kDa macromolecular membrane multiprotein complex
(Figure 5). These results confirm that labeling by P-trisNTA
does not affect the formation of the target protein complex
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E. Bordignon, R. Tamp et al.
and demonstrate the possibility to retrieve intra- as well as
intermolecular constraints with the orthogonal spin probe.
Conclusion
Figure 4. In-situ distance measurements under in-cell conditions.
A) Analysis of the total E. coli lysate by SDS-PAGE (4–12 %, Coomassie
stained) for DEER experiments containing MalE-C352R1. MalEC352R1 (3 mg mL 1) was incubated with E. coli lysate (5 mg mL 1 of
total protein) for 5 min at room temperature and then loaded with Ni-PtrisNTA in substoichiometric ratio. B) Modulation-depth normalized
form factors in cell lysate (cyan, fit in dotted red) and purified protein
(blue, taken from Figure 3, fit in dotted red) samples. Inset: the corresponding distance distributions. The form factors and the distance distributions obtained in the in-situ experiment (cyan) are indistinguishable
from those obtained with purified MalE (blue).
The in-situ spin labeling strategy employing the Ni-P-trisNTA label enables the reversible, site-specific spin labeling of
the proteins His-tags to be used as orthogonal spin units in
combination with conventional site-directed spin labeling
approaches. The use of Ni-P-trisNTA labeling directly in cell
lysate has proven to be successful in the case of MalE. PtrisNTA tags loaded with diamagnetic metals may allow
monitoring distances within a protein complex in cell lysates,
cell membranes, or even on living cells without the need of
orthogonal nitroxide labels, and work is in progress on this
line.
Figure 5. Probing the formation of the macromolecular complex MalFGK2-E with P-trisNTA. A) X-ray structure of the multisubunit maltose transport complex MalFGK2-E (PDB ID: 3PV0)[11] with the simulated R1 rotamers at position 205 in MalF (magenta ball represents the center of the NO group of the most populated rotamers). The blue cone illustrates the position of Ni-P-trisNTA attached to the N-terminal His-tag of MalE.
B) Q-band DEER traces (T = 50 K) and analysis with DeerAnalysis2011.[8] The sample was prepared by incubating the substrate-bound MalE (52 mm), prelabeled at the His-tag with Ni-P-trisNTA (52 mm), with the detergent-solubilized MalFGK2 (58 mm) carrying the R1 side chain at position 205 in the periplasmic loop of MalF.
The His-tags at the N terminus of each MalK subunit are positioned on the opposite side of the complex (at a
distance of about 10 nm from residue 205 in MalE); thus, they do not interfere with the measurements. The
buffer used was Tris (50 mm, pH 7.5), DDM (0.02 %, w/v) with deuterated glycerol (20 % v/v). Upper panel:
Q-band primary DEER trace (black) and 3D decaying background (dashed lines). In red, the DEER trace obtained after addition of imidazole (the dipolar oscillation is strongly suppressed). Central panel: form factor
F(t) obtained after background correction and fit by the Tikhonov regularization with a = 100 (dotted line).
Bottom panel: obtained distance distribution. As a reference, the Ca Ca distance between residue #1 of
MalE and residue 205 of MalF increased by 2 nm is 4.6 nm.
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2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Experimental Section
Materials: All chemicals and reagents
were purchased from Sigma–Aldrich
unless otherwise noted. His6-MalE
(wild-type and single cysteine variants)
was purified from the cytosolic fraction of E. coli strain JM109 harboring
plasmid pCB06 or derivatives as previously described.[13] To remove tightly
bound maltose, purified MalE variants
were subjected to a denaturation/refolding procedure using 6 m guanidine
hydrochloride as described.[14] The
MalFGK2 mutant carrying the nitroxide label MTSL at position 205 in the
F subunit was prepared as previously
described.[15] For the labeling of MalE,
MalE (25 mL) carrying the R1 label at
position 352 (100 % labeling efficiency) at a concentration of 240 mm was
mixed with buffer (30 mL) containing
the Ni-P-trisNTA (200 mm) and incubated for 5 min. Before shock freezing,
deuterated glycerol (20 mL) was
added. The other MalE R1-labeled
mutants were prepared in a similar
way. A 0.9:1 stoichiometric ratio between Ni-P-trisNTA label and protein
was used for MalE carrying the nitroxide label at positions 352 and 13, and
a 0.75:1 stoichiometric ratio for MalE
carrying the nitroxide label at positions 36 and 80.
For in-situ labeling in cell lysate,
E. coli cells BL21ACHTUNGRE(DE3)pLysS (50 mg)
were grown in LB medium (20 mL),
overnight. The cell paste was resuspended in Tris 50 mm (500 mL), pH 7.5,
NaCl buffer (300 mm) and sonicated
three times for 4 min on ice to lyse the
cells. The suspension was centrifuged
for 3 min at 25 000 g to remove the
debris. The protein concentration
(17 mg mL 1) was determined with the
Chem. Eur. J. 2013, 19, 13714 – 13719
In-Situ Spin Labeling of His-Tagged Proteins
Bradford assay. MalE (25 mL) carrying the R1 label at position 352
(100 % labeling efficiency) at a concentration of 240 mm was mixed with
cell lysate (25 mL) containing maltose (5 mm) and incubated for 5 min.
Buffer (30 mL) containing the Ni-P-trisNTA (200 mm) was added to the
solution and incubated for 5 min. Before shock freezing, deuterated glycerol (20 mL) was added. The sample was analyzed by SDS-PAGE to visualize the content of MalE with respect to the other proteins.
Methods: Continuous wave (cw) X-band EPR experiments were performed with a Bruker Elexsys E500 spectrometer equipped with a
Bruker Elexsys super high sensitive probehead at room temperature.
Samples were loaded into EPR glass capillaries (0.9 mm inner diameter,
sample volume 15 mL) and recorded with 100 kHz field modulation,
2 mW microwave power, 0.1 mT modulation amplitude. For spin-normalization, the derivative EPR spectrum was divided by the double integral
of the derivative spectrum.
X- and Q-band DEER measurements were performed at 50 K. The Xband Bruker Elexsys E580 spectrometer was equipped with a Bruker
Elexsys MS3 probehead, which allows tubes with 3 mm outer diameter.
Observer pulse lengths of 32 ns for p/2 and p pulses as well as an
ELDOR p pulse of 12 ns were used, with a frequency separation of
65 MHz. The Q-band homemade spectrometer was equipped with a
homemade rectangular cavity allowing the same tubes used at X-band
with 3 mm outer diameter.[16] Observer pulse lengths of 12 ns for p/2 and
p pulses as well as an ELDOR p pulse of 12 ns were used to avoid orientation selection artifacts. A frequency separation of 100 MHz was used.
Perdeuterated glycerol was used to increase sensitivity. Deuterium nuclear modulations were averaged (eight cycles with 16 ns time increment of
the first interpulse delay). Traces were accumulated for 12–24 h. The
background of the DEER primary data [V(t)] was fitted with a 3D homogeneous distribution of distances with the software DeerAnalysis2011.[8]
Acknowledgements
We thank all the members of the Tamp laboratory for experimental
help and discussions, as well as E. Schneider (Humboldt University
Berlin) for providing the MalE and MalFGK2 mutants. E.B. thanks
S. Bçhm for spin labeling the MalE mutants and G. Jeschke (ETH
Zurich) for helpful discussions. The German Research Foundation
(SFB807-Transport and Communication across Biological Membranes)
supported this work.
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2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Received: May 18, 2013
Published online: August 26, 2013
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