Genetically Encoded Spin Labels for in Vitro and In-Cell EPR Studies of Native Proteins

Article abstract of DOI:10.1016/bs.mie.2015.05.023

Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful approach to study the structure, dynamics, and interactions of proteins. The genetic encoding of the noncanonical amino acid spin-labeled lysine 1 (SLK-1) eliminates the need for any chemical labeling steps in SDSL-EPR studies and enables the investigation of native, endogenous proteins with minimal structural perturbation, and without the need to create unique reactive sites for chemical labeling. We report detailed experimental procedures for the efficient synthesis of SLK-1, the expression and purification of SLK-1-containing proteins under conditions that ensure maximal integrity of the nitroxide radical moiety, and procedures for intramolecular EPR distance measurements in proteins by double electron-electron resonance.

Full text of DOI:10.1016/bs.mie.2015.05.023

CHAPTER EIGHTEEN
Genetically Encoded Spin Labels
for In Vitro and In-Cell EPR Studies
of Native Proteins
M.J. Schmidt, A. Fedoseev, D. Summerer, M. Drescher1
Department of Chemistry, Zukunftskolleg, and Konstanz Research School Chemical Biology,
University of Konstanz, Konstanz, Germany
1Corresponding author: e-mail address: malte.drescher@uni-konstanz.de
Contents
1. Introduction
2. Experimental Guidelines
2.1 Precautions
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490
491
491
494
496
499
499
2.2 Organic Synthesis
2.3 Expression and Purification of Spin-Labeled Proteins in E. coli
2.4 EPR Measurements
Acknowledgments
References
Abstract
Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed
spin labeling (SDSL) is a powerful approach to study the structure, dynamics, and inter-
actions of proteins. The genetic encoding of the noncanonical amino acid spin-labeled
lysine 1 (SLK-1) eliminates the need for any chemical labeling steps in SDSL–EPR studies
and enables the investigation of native, endogenous proteins with minimal structural
perturbation, and without the need to create unique reactive sites for chemical labeling.
We report detailed experimental procedures for the efficient synthesis of SLK-1, the
expression and purification of SLK-1-containing proteins under conditions that ensure
maximal integrity of the nitroxide radical moiety, and procedures for intramolecular EPR
distance measurements in proteins by double electron–electron resonance.
1. INTRODUCTION
Electron paramagnetic resonance (EPR) distance measurements of
spin-labeled proteins provide valuable insights about the structure, dynam-
ics, and interactions of proteins and protein complexes in the noncrystalline
#
Methods in Enzymology, Volume 563
ISSN 0076-6879
http://dx.doi.org/10.1016/bs.mie.2015.05.023
2015 Elsevier Inc.
All rights reserved.
483

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M.J. Schmidt et al.
state (Hubbell, Cafiso, & Altenbach, 2000; Jeschke, 2012). However, a pre-
requisite for the studying of proteins by EPR spectroscopy is the presence of
paramagnetic centers. Site-directed spin labeling (SDSL) EPR enables
studying the structure and dynamics of proteins which do not contain native
paramagnetic centers. Distance measurements between two spin labels rely
on their dipole–dipole coupling that is inversely proportional to the cube of
their distance. Pulsed EPR techniques can be used to separate the dipole–
dipole interaction from other contributions of the spin Hamiltonian and
thereby provide information on distances in the 1.5–10 nm range. The most
widely used approach for EPR distance measurements is double electron–
electron resonance (DEER, synonymously used with pulsed electron double
resonance [PELDOR]) (Jeschke, 2002; Jeschke, Abbott, Lea, Timmel, &
Banham, 2006; Jeschke, Bender, Paulsen, Zimmermann, & Godt, 2004).
A variety of approaches of SDSL have previously been developed and
established. Traditionally, unique cysteines in proteins are reacted with
thiole-reactive reagents such as the methanethiosulfonate spin label
(MTSSL) in vitro (Altenbach, Marti, Khorana, & Hubbell, 1990). However,
this requires the deletion of native cysteins and the introduction of new
cysteins at the desired sites by mutagenesis. Since cysteines carry out various
essential functions in proteins, such as serving as redox-active or nucleo-
philic catalytic centers, as stabilizers of protein folds or as metal ion ligands,
this prevents the investigation of various proteins in their natural state
(Pace & Weerapana, 2013). Spin labels can also be introduced as amino acids
by chemical protein (semi)-synthesis (Klare & Steinhoff, 2009), nonsense
codon suppression with chemically aminoacylated tRNAs (Cornish et al.,
1994; Shafer, Kalai, Liu, Hideg, & Voss, 2004) or by conjugation to genet-
ically encoded, noncanonical amino acids (ncAA) containing aldehydes and
azides (Fleissner et al., 2009; Kalai, Fleissner, Jeko, Hubbell, & Hideg, 2011).
Finally, nitroxides have been introduced into proteins by complexation with
His tags (Baldauf, Schulze, Lueders, Bordignon, & Tampe, 2013). However,
these approaches employ mandatory chemical labeling steps in vitro and thus
do not fully meet the aspiring goals of protein SDSL–EPR studies, that is,
studying proteins in their native folding state, with minimal structural mod-
ification, and directly in the unperturbed, intracellular environment of their
host cells.
We recently reported the direct biosynthesis of spin-labeled proteins in
live Escherichia coli cells, overcoming the aforementioned limitations of
in vitro spin labeling (Schmidt, Borbas, Drescher, & Summerer, 2014). This
technique relies on the genetic encoding of the spin-labeled amino acid

Genetically Encoded Spin Labels
485
SLK-1 (Fig. 1A) in response to the amber stop codon (TAG) (Liu & Schultz,
2010) by an evolved, orthogonal tRNAPyl/pyrrolysyl-tRNA-synthetase
(PylRS) pair (Blight et al., 2004; Chen et al., 2009; Fekner, Li, Lee, &
Chan, 2009; Gautier et al., 2010; Hoppmann et al., 2014; Li et al., 2013;
Luo et al., 2014; Plass et al., 2012; Polycarpo et al., 2006; Pott,
Schmidt, & Summerer, 2014; Schmidt & Summerer, 2013; Wan et al.,
2010; Wan, Tharp, & Liu, 2014; Yanagisawa et al., 2008). This enables
the cotranslational incorporation of SLK-1 at single and multiple user-
defined sites of proteins with high efficiency and fidelity. Possible applica-
tions of SLK-1 can best be described by a brief survey of our recent
experiments.
Figure 1 Cotranslational incorporation of spin-labeled amino acid SLK-1 into proteins in
E. coli. (A) Chemical structure of SLK-1. (B) Incorporation of SLK-1 in E. coli thioredoxin at
user-defined sites. TRX mutants with amber codons at indicated positions and with
C-terminal His6 tag were coexpressed with tRNAPyl/PylRS-SL1 in E. coli in the presence
of SLK-1. Yields of protein expressions in the presence of SLK-1 obtained from BCA
assays are shown as gray bars. SDS-PAGE analysis of proteins purified by Ni-NTA chro-
matography is shown at the bottom of the figure. (C) EPR spectrum of SLK-1 in buffer.
(D) EPR spectrum of purified TRX-D14!SLK-1 in buffer. X-band spectra at room tem-
perature and spectral simulations for panels (C and D) are shown in black and red (gray
in the print version), respectively.

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M.J. Schmidt et al.
We investigated the incorporation of SLK-1 into E. coli thioredoxin
(TRX) and performed structural studies by DEER, since this enzyme serves
as important oxidoreductase in various redox processes including dithiole–
disulfide exchange reactions. Furthermore, X-ray structural information for
this protein is available for data comparison (Katti, LeMaster, & Eklund,
1990), and the catalytic center of TRX is constituted by two redox-active
cysteines. This prohibits structural studies of the native protein by traditional
MTSSL labeling and DEER (Katti et al., 1990). Finally, TRX is a homol-
ogous protein in E. coli, which could be studied with minimal perturbation
of its natural environment and in its natural host cells, where it is bio-
synthesized and processed.
By coexpression of tRNAPyl/PylRS-SL1 in the presence of SLK-1 with
genes encoding TRX bearing a C-terminal His6 tag and containing amber
codons at different positions, spin-labeled TRX proteins are obtained with
good yields and purities after Ni-NTA purification, as indicated by SDS-
PAGE analysis and a bicinchoninic acid (BCA) assay (Fig. 1B). Yields of
SLK-1-containing TRX mutants are in the range of 2–3 mg/l expression
culture for singly labeled proteins; doubly labeled TRX proteins are typically
obtained with yields of >1 mg/l. Studies with other target proteins typically
afforded yields of >1 mg/l, being in a reasonable range for EPR studies (e.g.,
green fluorescent protein and T4 lysozyme, data not shown). Typically,
expression yields of SLK-1-containing proteins are reduced compared to
the expression of the wild-type proteins due to competition of tRNAPyl
with release factor 1 (RF1) (Liu & Schultz, 2010). A number of strategies
have been developed to increase the efficiency of amber suppression with
ncAA that may be employed, if higher yields are required. Besides optimiz-
ing expression levels of tRNA and aminoacyl-tRNA-synthetase (aaRS)
(Cellitti et al., 2008; Chatterjee, Sun, Furman, Xiao, & Schultz, 2013;
Ryu & Schultz, 2006; Young, Ahmad, Yin, & Schultz, 2010), these include
the optimization of tRNAs (Chatterjee et al., 2013; Guo, Melancon, Lee,
Groff, & Schultz, 2009), reduced amber codon recognition by RF1 by its
genomic knockout (Johnson et al., 2012, 2011; Lajoie et al., 2013;
Mukai et al., 2010), by engineering RF1 itself (Ryden & Isaksson, 1984;
Wu et al., 2013; Zhang, Ryden-Aulin, Kirsebom, & Isaksson, 1994), or
its binding site at the ribosome (Huang et al., 2010; Wang, Neumann,
Peak-Chew, & Chin, 2007), and optimization of the sequence context of
the amber codon (Pott et al., 2014).

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487
In EPR measurements, the spectral shape of free SLK-1 corresponds to a
fast tumbling small molecule, as expected (Fig. 1C). Spectral simulations
(Stoll & Schweiger, 2006) revealed a rotational correlation time τc ¼40 ps.
The EPR spectrum for SLK-1 incorporated into E. coli TRX shows a com-
parably low mobility of the nitroxide moiety (Fig. 1D, τc ¼250 ps). For
structural investigations of E. coli TRX by DEER, purified, doubly labeled
protein (e.g., TRX-D14/R74!SLK-1) is dialyzed into aqueous buffer.
Before shock freezing in liquid nitrogen in order to trap the macromolecular
conformation, 20% (v/v) glycerol was added to obtain a glassy matrix. The
DEER measurements were performed at T¼50 K to optimize relaxation
rates of the spin system and to avoid reorienting of the spin–spin inter-
connecting vector due to molecular tumbling. The latter would average
the dipole–dipole interaction which is required for distance determination.
Depending on the labeling strategy adopted, both intermolecular and intra-
molecular distances can be measured. Distances between molecules can, in
fact, be obtained from proteins with a single SLK-1 incorporated. Double
incorporation of SLK-1 can instead be used to measure distances within pro-
teins. When measuring intramolecular distances, the contribution of dis-
tances to neighboring proteins should be suppressed. Therefore, a singly
labeled control protein (e.g., TRX-D14!SLK-1) is required for control
measurement. For TRX-D14!SLK-1, obtained DEER data are in full
agreement with a homogeneous 3D distribution of spin labels as expected
for a singly labeled protein in solution. For TRX-D14/R74!SLK-1
(Fig. 2A), the background corrected DEER data result in the corresponding
distance distributions by model-free analyses using DEERAnalysis2013
(Fig. 2B; Jeschke et al., 2006).
An important aspect of SDSL–EPR is the influence of the linker flexi-
bility of a spin label on the distance distribution. This factor can be predicted
via rotamer approaches (Polyhach, Bordignon, & Jeschke, 2011).
In case of SLK-1, the experimental distance distribution for TRX-D14/
R74!SLK-1 is in agreement with the theoretically predicted distance dis-
tribution between traditional MTSSL moieties at the same incorporation
sites (Fig. 2B). More importantly, a direct experimental comparison
between TRX-D14/R74!SLK-1 and the corresponding MTSSL-labeled
mutant TRX-C33S/C36S/D14C/R74C exhibits a similar distance distri-
bution (Fig. 2C). For the latter protein, additional maxima are observed that
were identified as artifacts caused by oligomerization via disulphide bridges

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M.J. Schmidt et al.
Figure 2 DEER distance measurements in E. coli thioredoxin (TRX). (A) Cartoon repre-
sentation of the crystal structure of TRX (pdb entry 2TRX) (Katti et al., 1990). Amino acids
at both incorporation sites of SLK-1 (D14 and R74) are shown as sticks, the correspond-
ing Cα–Cα distance is indicated as dotted red line. Positions of catalytic cysteines
C33 and C36 are indicated. (B) Distance distribution for TRX-D14/R74!SLK-1 (red)
compared to the theoretically predicted distance distribution between two MTSSL
moieties at corresponding positions (blue). (C) Distance distribution for TRX-D14/
R74!SLK-1 (red) compared to the distance distribution of MTSSL-labeled TRX-C33S/
C36S/D14C/R74C (black). (D) Distance distribution for TRX-D14/R74!SLK-1 (red)
compared to the theoretically predicted distance distribution between two SLK-1 at
the same positions (blue).
during the labeling protocol. This effect was not observed for TRX-D14/
R74!SLK-1, where an introduction of surface-exposed cysteines is not
required. These data indicate that SLK-1 is a useful structural probe with
similar spectral characteristics as MTSSL and that SLK-1-based DEER mea-
surements can deliver insights without artifacts from chemical labeling steps
associated with MTSSL. To further analyze the flexibility of the SLK-1 side
chain (Polyhach et al., 2011), we established rotamer libraries for the predic-
tion of SLK-1 distance distributions on the basis of a model of the protein
structure (Fig. 2D).
Recent studies have shown that singly SLK-1-labeled proteins can selec-
tively be detected in the host E. coli cells where they are biosynthesized and

Genetically Encoded Spin Labels
489
processed (Schmidt et al., 2014). Thorough washing of cells with Luria
Broth (LB) media reduced background signals arising from free SLK-1 in
E. coli, as indicated by measurements of control expressions that did not
include SLK-1, an amber codon in the target TRX gene, or the plasmid
for coexpression of the tRNAPyl/PylRS-SL1 pair (Fig. 3A). These experi-
ments indicate that our approach affords sufficient expression levels of
endogenous, spin-labeled proteins for in-cell SDSL–EPR measurements
and that free SLK-1 can be selectively washed out of cells under conditions
that leave the spin-labeled protein intact and localized in the cells. This pro-
vides a starting point for future in-cell EPR studies of proteins with increased
Figure 3 Detection of SLK-1-labeled proteins in E. coli and stability of SLK-1 under con-
ditions of protein expression in E. coli. (A) Cartoon: workflow of the sample preparation
for EPR measurements of intracellular TRX-R74!SLK-1. Plasmids encoding tRNAPyl
,
PylRS-SL1, and TRX-R74!TAG are transformed into E. coli, and proteins are expressed
in the presence of SLK-1. Cells are washed with LB media, pelleted, and subjected to EPR
measurements. Diagram: EPR signal intensities for EPR measurements of washed E. coli
cells with negative controls omitting SLK-1, the amber codon at position 74 or the
tRNAPyl/PylRS-SL1 pair as shown in the figure. a.u.¼arbitrary units. (B) EPR signal decay
for free SLK-1 in E. coli culture in LB medium at temperatures as indicated. (C) EPR signal
decay for free SLK-1 in pelleted and lysed (uncleared) E. coli lysates at temperatures as
indicated.

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M.J. Schmidt et al.
biorelevance, i.e., without the need for microinjection of in vitro spin-
labeled molecules into Xenopus oocytes or transfection protocols (Azarkh
et al., 2011, 2012, 2013; Igarashi et al., 2010; Krstic et al., 2011;
Martorana et al., 2014; Qi, Groß, Jeschke, Godt, & Drescher, 2014).
A critical property of nitroxides for EPR studies in biological environ-
ments is the stability of the radical moiety. Nitroxides have been reported to
be prone to chemical conversion in all previously studied intracellular con-
texts (Belkin, Mehlhorn, Hideg, Hankovsky, & Packer, 1987; Couet,
Brasch, Sosnovsky, & Tozer, 1985; Krstic et al., 2011), resulting in signal
decay (loss of paramagnetism). This limits the sensitivity and applicable max-
imal measurement times of SDSL–EPR studies of proteins. SLK-1-
containing proteins expressed in E. coli typically exhibit integrity degrees
of ꢀ50–70% (the integrity degree is defined as the percentage of intact, para-
magnetic SLK-1 per initially incorporated SLK-1). Note that the labeling
degree with SLK-1, i.e., the incorporation without taking into account
potential conversion of the radical, is determined by the translation fidelity
and thus is >99%) (Schmidt et al., 2014). However, the overall integrity
degree for doubly labeled proteins is decreasing in a multiplicative manner
compared to singly labeled proteins and this can prevent successful DEER
measurements. It is thus very important to take any measures that prevent
SLK-1 conversion during protein expression, lysis, and purification. Signal
decay measurements with free SLK-1 in E. coli expression cultures or in
lysed, uncleared E. coli cells indicate that both temperature and incubation
time are critical parameters that affect the integrity degree (Fig. 3B and C).
For high degrees, minimal expression temperature and times are
recommended, as well as a fast lysis and purification protocol at low
temperature. Additionally, the presence of serine protease inhibitors such
as phenylmethylsulfonyl fluoride (PMSF) or 4-(2-aminoethyl)ben-
zensulfonylfluoride (AEBSF) during lysis has been observed to strongly
decrease the undesired conversion of free SLK-1 (data not shown).
2. EXPERIMENTAL GUIDELINES
Here, we provide detailed experimental guidelines on how the genet-
ically encoded spin label SLK-1 can be used to obtain long-range distance
constraints for structural studies of native, endogenous proteins. The entire
experimental design starting from the synthesis of SLK-1 via genetic
encoding, protein expression, and EPR measurements to data analysis is
described.

Genetically Encoded Spin Labels
491
2.1 Precautions
For reliable results and maximal yields in the organic synthesis of SLK-1, all
described organic reactions should be performed under anhydrous condi-
tions and an inert atmosphere of argon in oven-dried glassware with mag-
netic stirring. During the expression and purification of SLK-1-containing
proteins, the exposure of SLK-1 to air and cell media should be kept to a
minimum to obtain maximal integrity degrees. SLK-1 should be added only
shortly before induction of protein expression, and expression time should
be minimal and at the minimal possible temperature. Most importantly, the
lysis and purification have to be performed as fast as possible, at 4 °C, and
PMSF should be included during lysis. Once purified, SLK-1 is stable in pro-
teins at room temperature, but storage temperature should be as low as pos-
sible as a precaution. Note that the use of any reductants such as
dithiothreitol (DTT) or β-mercaptoethanol will reduce SLK-1 to the
EPR-inactive hydroxylamine. As for all labeling techniques, control exper-
iments aimed at excluding distortions of the original protein behavior due to
incorporation of SLK-1 are essential.
2.1.1 Experimental Strategy
Most promising labeling positions are those that result in distance distribu-
tions that fall in the optimum distance range (1.5–6 nm). For their identifi-
cation, analyzing high-resolution structural models, for instance, those
obtained from the pdb database (http://www.rcsb.org/pdb/) may be a good
starting point.
2.2 Organic Synthesis
Nitroxide amino acid SLK-1 can be synthesized from commercially avail-
able starting materials by N,N0-disuccinimidyl carbonate-(DSC)-mediated
carbamate formation between 3-hydroxymethyl-1-oxy-2,2,5,5-tetramethyl-
pyrroline and Nα-Boc-protected L-lysine, followed by deprotection with
trifluoroacetic acid (Fig. 4).
This protocol affords SLK-1 with high purity and with an overall yield of
84%. The reaction outcome of the carbamate coupling step can be quickly
assessed by TLC and high-resolution mass spectrometry (HRMS). The final
product can additionally be analyzed by 1H and 13C NMR after a reduction
to the hydroxylamine with formic acid to confirm the identity and ensure
high purity of SLK-1 for subsequent molecular biology experiments.

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Figure 4 Scheme of synthesis of nitroxide amino acid SLK-1 from commercially avail-
able starting materials.
2.2.1 Materials
Reagents were purchased from the following suppliers: 2,2,5,5-
tetramethyl-3-pyrrolin-1-oxyl-3-carboxylic acid was purchased from Acros
Organics; Red-Al® sodium bis(2-methoxyethoxy)aluminum hydride solu-
tion (ꢁ60 wt.% in toluene) and trifluoroacetic acid were purchased from
Sigma-Aldrich; N,N0-disuccinimidyl carbonate, 98%, and N,N-
diisopropylethylamine, 99%, were purchased from Abcr; and triethylamine
was purchased from Merck. Flash chromatography: Roth 60 F254 (230–400
mesh) silica gel. Solvents used for chromatography were ethyl acetate,
DCM, and methanol, technical grade. Reaction solvents were purchased
in anhydrous quality and stored over molecular sieve. Acetonitrile, toluene,
and DMF were purchased from Sigma-Aldrich. 1H and 13C NMR spectra
were recorded at ambient temperature on a Bruker Avance III 400 instru-
ment. Signal positions are reported in δ/ppm with their characteristics den-
oted as s (singlet), d (doublet), dd (double doublet), t (triplet), m (multiplet),
and br (broad). HRMS measurements were performed on a Bruker
Daltonics micrOTOF II. DCM, dichloromethane; TLC, thin-layer chro-
matography; MeOH, methanol; and TFA, trifluoroacetic acid.
2.2.2 Synthesis of Nitroxide Amino Acid SLK-1
(a) Carbamate coupling step
1. Dissolve 3-hydroxymethyl-1-oxy-2,2,5,5-tetramethyl-pyrroline
(3.6 g, 21.14 mmol, 1 equiv.) in acetonitrile (40 ml).
2. Add triethylamine (11.73 ml, 84.6 mmol, 4 equiv.) and cool the
solution in an ice bath under argon atmosphere.
3. Add N,N0-disuccinimidyl carbonate (8.13 g, 31.72 mmol,
1.5 equiv.) in one portion and stir the solution at 0 °C, then at room
temperature overnight (13 h).

Genetically Encoded Spin Labels
493
4. Concentrate the reaction mixture under reduced pressure, dry in
vacuo, and filter the residue through a pad of silica gel using ethyl
acetate as solvent.
5. Concentrate the filtrate under reduced pressure, dissolve it in
anhydrous DMF (20 ml), and add it dropwise to a suspension of
Boc-Lys-OH (7.81 g, 31.72 mmol, 1.5 equiv.) and N,N-
diisopropylethylamine (10.8 ml, 63.44 mmol, 3 equiv.) in anhy-
drous DMF (40 ml) at 0 °C.
6. Stir the reaction mixture for 20 h at room temperature.
7. Quench the reaction with water (300 ml) and extract it with ethyl
acetate (6ꢂ100 ml). Wash the organic phase with water
(4ꢂ200 ml) and dry over MgSO4. Evaporate the solvent and purify
the obtained yellow oil by flash column chromatography (DCM,
DCM/1% MeOH!DCM/2% MeOH) to yield (S)-2-((tert-
butoxycarbonyl)amino)-6-((((1-oxy-2,2,5,5-tetramethylpyrroline-3-
yl)methoxy)carbonyl) amino)hexanoic acid as a yellow foam
(7.94 g, 17.94 mmol, 85%). TLC (DCM/MeOH 95:5)
Rf¼0.28. HR-ESI MS (m/z): [M+H]+ calculated for
[C21H37N3O7]+: 443.2626, found: 443.2607. [M+Na]+ calculated
for [C21H36N3O7 ꢃNa]+: 465.2446, found: 465.2432.
(b) Deprotection step
1. Dissolve (S)-2-((tert-butoxycarbonyl)amino)-6-((((1-oxy-2,2,5,5-
tetramethylpyrroline-3-yl)methoxy) carbonyl)-amino)hexanoic acid
(3.2 g, 7.23 mmol) in DCM (20 ml).
2. Add trifluoroacetic acid (6 ml) dropwise at room temperature and
stir the reaction mixture for 40 min. Avoid longer reaction times
and monitor the reaction progress by TLC to exclude undesired
cleavage of the Nε-carbamate bond.
3. Remove the solvent and coevaporate the residual oil once with
methanol (100 ml).
4. Add diethyl ether (5ꢂ100 ml) to the residual oil, decant the sol-
vent, and dry the remaining oil in vacuo to obtain a white powder
under reduced pressure. This yields the TFA salt of SLK-1 ((S)-2-
amino-6-((((1-oxy-2,2,5,5-tetramethylpyrroline-3-yl)methoxy)
carbonyl)amino)hexanoic acid) as a white foam (3.27 g, 7.16 mmol,
99%).HR-ESI MS (m/z): [M+H]+ calculated for [C16H29N3O5]+:
343.2102, found: 343.2091.
(c) Reduction of SLK-1 to corresponding hydroxylamine for NMR
characterization

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M.J. Schmidt et al.
1. Dissolve SLK-1 (100 mg, 0.22 mmol) in 6 ml H2O/formic acid
(1:1, v/v) and stir for 24 h at room temperature.
2. Remove the solvent under reduced pressure and coevaporated 3ꢂ
with 50 mM HCl (15 ml) to afford the HCl salt of (S)-2-
amino-6-((((1-hydroxy-2,2,5,5-tetramethylpyrroline-3-yl)methoxy)
carbonyl)amino)hexanoic acid (81 mg, quant.). 1H NMR
(400 MHz, D2O), δ: 5.90 (s, 1H, H–C]C–), 4.66 (m, 2H,
–CH2–), 3.71 (t, J¼6.1 Hz, 1H, Hα), 3.12 (t, J¼6.7 Hz, 2H,
–CH2–), 1.84 (m, 2H, –CH2–), 1.63–1.25 (m, 16H, 2ꢂ –CH2–,
4ꢂ –CH3). 13C NMR (101 MHz, D2O), δ: 171.99, 157.67,
138.40, 129.27, 77.78, 76.05, 59.89, 52.73, 39.94, 29.34, 28.35,
23.98, 22.79, 21.60, 21.43, 21.39.
2.3 Expression and Purification of Spin-Labeled Proteins
in E. coli
2.3.1 Materials
2.3.1.1 E. coli Strains and Expression Plasmids
E. coli strains GH371 and Top10 afford the described expression yields and
integrity degrees of SLK-1-containing proteins. Other E. coli expression
strains compatible with the used expression plasmids may be used, but yields
and integrity degrees may differ, the latter owing to the unknown origin of
nitroxide conversion in E. coli.
Expression of SLK-1-containing proteins requires the coexpression of
tRNAPyl and PylRS-SL1, a PylRS mutant previously evolved for the
genetic encoding of SLK-1. Plasmid pEVOL_PylRS-SL1 (Schmidt et al.,
2014) provides high-expression levels for this purpose, though other plas-
mids may be used. pEVOL-PylRS-SL1 (p15A origin of replication, chlor-
amphenicol resistance) encodes tRNAPyl under control of the proK
promoter and two copies of PylRS-SL1 under control of a glnS0 promoter
and an araBAD promoter, respectively (Young et al., 2010). In principle, the
target protein can be expressed under control of various promoters from
plasmids with compatible origin of replication. The described experiments
made use of pBAD plasmids with ampicillin resistance and colE1 origin of
replication (pBAD-TRX_D14/R74TAG, pBAD-TRX_D14TAG, or
pBAD-TRX_R74TAG).
2.3.1.2 Culture Media, Reagents, and Lysis/Purification Buffers
Media and reagents were purchased from the following suppliers: LB media,
carbenicillin, chloramphenicol, PMSF (phenylmethanesulfonylfluoride),

Genetically Encoded Spin Labels
495
and Coomassie Brilliant Blue were purchased from Carl Roth; L-arabinose
was purchased from Sigma-Alrich; B-Per lysis reagent was purchased from
Thermo Scientific; imidazole was purchased from Abcr; Ni-NTA agarose
slurry was purchased from Qiagen; and paper filter spin column and BCA
assay were purchased from Pierce. Amicon Ultra centrifugal columns with
a molecular weight cutoff of 3 kDa were purchased from Millipore.
(a) 4ꢂ PBS: 548 mM NaCl, 10.8 mM KCl, 40 mM Na2HPO4, 7.2 mM
KH2PO4, pH 7.4.
(b) Wash buffer: 50 mM NaH2PO4, 300 mM NaCl, pH 8.0, containing
20 mM imidazole.
(c) Elution buffer: 50 mM NaH2PO4, 300 mM NaCl, pH 8.0, containing
500 mM imidazole.
2.3.2 Procedure
2.3.2.1 Expression and Purification of SLK-1-Containing Proteins
(a) Protein expression
1. Cotransform E. coli GH371 with plasmids pEVOL-PylRS_SL1 and
a pBAD plasmid encoding the target gene with amber codons at the
chosen position of SLK-1 incorporation under control of an
araBAD promoter (e.g., pBAD-TRX_D14/R74TAG, pBAD-
TRX_D14TAG, or pBAD-TRX_R74TAG) and select on LB
agar plate supplemented with 34 μg/ml chloramphenicol and
50 μg/ml carbenicillin.
2. Inoculate a single clone in LB media supplemented with 34 μg/ml
chloramphenicol and 50 μg/ml carbenicillin and grow overnight at
37 °C and 200 rpm shaking.
3. Dilute this culture 50-fold into fresh LB media supplemented with
34 μg/ml chloramphenicol and 50 μg/ml carbenicillin. Grow the
culture at 37 °C and 200 rpm shaking and at an OD600 of
0.5–0.6, add 3 mM SLK-1 and induce with 0.2% (w/v) L-arabinose.
4. After 4 h growth under the same incubation conditions, harvest the
culture by centrifugation (10 min, 3320ꢂg, room temperature,
4 °C). The pellet can be stored at ꢄ20 °C.
(b) Protein purification
1. Resuspend the pellet obtained from protein expression. For a 5 ml
expression culture, use 500 μl B-Per lysis reagent containing 1 mM
PMSF for resuspension. Incubate at 4 °C for 10 min with shaking.
2. Pellet the mixture by centrifugation (2 min, 20,817ꢂg, room tem-
perature, 4 °C), recover the supernatant and add 10 μl 500 mM

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M.J. Schmidt et al.
imidazole and 50 μl Ni-NTA agarose slurry. Shake the suspension
at 1000 rpm for 10 min at 4 °C and transfer to a paper filter spin
column.
3. Centrifuge (106ꢂg, 10 s, room temperature) and wash resin 2ꢂ
with 500 μl, 4ꢂ with PBS, and 2ꢂ with 500 μl wash buffer by
resuspension of the resin and centrifugation (106ꢂg, 10 s at room
temperature).
4. Elute protein 3ꢂ with 50 μl elution buffer by addition and centri-
fugation (1000 rpm, 10 s at room temperature).
5. Confirm the identity and analyze the purity of the obtained protein
by SDS-PAGE and staining with Coomassie Brilliant Blue. Quan-
tify using a BCA assay.
6. For EPR measurements, dialyze proteins 3ꢂ against 10 mM Tris,
pH 7.4, using a Slide-A-Lyzer dialysis column with 10 kDa MW
cutoff.
2.4 EPR Measurements
2.4.1 EPR Spectrometers
For monitoring the integrity degree, a simple benchtop CW-EPR X-band
spectrometer is sufficient. We usually use an X-band MiniScope spectrom-
eter (MS200, magnettech GmbH) equipped with a variable temperature
unit (Temperature Controller TC-H02, magnettech GmbH).
For increased sensitivity, it is recommended to perform the DEER
experiments in Q-band. Cooling with liquid helium is useful because it opti-
mizes sensitivity, but not a prerequisite for these measurements. Alterna-
tively, liquid nitrogen can be used. We performed all EPR DEER
experiments in Q-band at T¼50 K using an Elexsys E580 spectrometer
(Bruker Biospin) with EN 5107D2 probehead, equipped with a helium
gas flow system (CF935, Oxford Instruments).
2.4.2 Procedure
2.4.2.1 Monitoring Integrity Degrees by CW-EPR Measurements
1. Load samples into glass capillaries (Bluebrand, outer diameter 1 mm)
with typical sample volumes of 20 μl.
2. Obtain CW-EPR spectra in liquid solution. Typical experimental
parameters are a modulation amplitude of 800 mG and microwave
attenuation of 15 dB of the microwave source (P0 ¼100 mW). The
signal-to-noise ratio can be improved by accumulation of several spectra.

Genetically Encoded Spin Labels
497
3. If required, the spectra can be analyzed using Matlab R2008b (The
MatWorks, Inc.) and the toolbox EasySpin 2.6 (Stoll & Schweiger, 2006).
4. Determine signal intensities via the double integral of the first derivative
EPR spectrum and can be compared to the obtained protein
concentration.
2.4.2.2 DEER Experiments
1. The dead-time free four-pulse DEER sequence is shown in Fig. 5.
2. Estimate the required length of the measured dipolar evolution, which
is approximately given by tmax ꢅτ2 (where tmax is the maximum dipolar
evolution time and τ2 is the separation time between the primary echo
and the second π pulse of the observer sequence). The required tmax
depends on the expected value of the distance to be measured. With
tmax ¼2 μs, the shape of the distance distribution is reliable up to a dis-
tance of 3 nm (reliable distribution limit). The mean distance and the
width of its distribution are reliable up to a distance of 4 nm (reliable
width limit), whereas the mean distance, but not the distribution width,
is reliable between 4 and 5 nm (reliable mean distance limit). Beyond
5 nm no reliable mean value can be determined. All these limits scale
with the cubic root of tmax (Jeschke, Chechik, et al., 2006; Stoll &
Schweiger, 2006).
3. Cool the cryostat down to a working temperature of 50 K. Readjust
the resonant microwave frequency to the center of the resonator if nec-
essary. This is the pump frequency.
4. Apply the Hahn-echo sequence π/2–τ–π–τ–echo (π/2 and π pulses are
typically 16 and 32 ns long, respectively, and τ¼300 ns). Adjust shot
repetition time, video amplifier bandwidth, microwave attenuation,
and magnetic field, phase to maximize the echo.
Figure 5 Scheme of dead-time free, four-pulse DEER sequence.

498
M.J. Schmidt et al.
5. To adjust the pump pulse at this microwave frequency (pump fre-
quency), use the inversion recovery pulse sequence π–τ1–π/2–τ2–π–
τ2–echo. The first π pulse is applied via the ELDOR channel, its fre-
quency is set to the pump frequency. This pulse is placed 400 ns before
the Hahn-echo sequence. Optimize the ELDOR channel π-pulse
length to have maximum negative echo amplitude corresponding to
inversion.
6. Set the operating frequency 50 MHz lower than the pump frequency.
This value is defined as the observer frequency. Apply the Hahn-echo
sequence π/2–τ–π–τ–echo (τ¼300 ns) for +hxi and ꢄhxi channels.
Maximize the echo intensity by varying the pulse length at zero atten-
uation. Adjust the phases so as to observe a phase shift of 180° between
the two-pulse channels.
7. Apply the π/2–τ1–π–τ1–echo–τ2–π–τ2–refocused echo sequence at the
observer frequency (τ1 ¼300 ns, τ2 initial value¼1 μs). Set the SpecJet
scale factor to one. Adjust τ2 by inspecting the refocused echo. Set τ2 to
the longest value at which the refocused echo can still be clearly rec-
ognized. Typical values are between 1 and 3.5 μs.
8. Apply the DEER sequence (Fig. 5). The pump inversion pulse (at the
pump frequency on resonance with the microwave cavity) with a length
determined in step 5 starts with a delay time <2τ1, but large enough not
to overlap with the first observer π pulse. The pump pulse is swept within
the experiment in 8 ns steps resulting in the DEER trace.
9. Set the phase cycle for the observer sequence to be: π/2(+hxi)–π(hyi)–
π(hyi) and π/2(ꢄhxi)–π(hyi)–π(hyi).
10. For deuterated samples, apply nuclear modulation suppression by
averaging DEER traces for 10 different τ1 values incremented by 8 ns.
11. Set the integration window symmetrically over the refocused echo.
12. Set the number of averages for the SpecJet to 1. Adjust the gain so that
the signal intensity does not exceed the SpecJet window.
13. Increase the number of scans and shots per point to repeat the exper-
iment as often as required to obtain an acceptable signal-to-noise ratio.
Depending on spectrometer stability, one should not exceed accumu-
lation times of 24 h. We usually use 18 h.
2.4.3 Analysis of DEER Data
1. Install DeerAnalysis 2013.2 in your Matlab environment following the
instructions in the user manual. Perform the data analysis according to
the DeerAnalysis manual (http://www.epr.ethz.ch/software).

Genetically Encoded Spin Labels
499
2. Use an experimental background function derived from the individually
measured DEER traces of the protein with a single SLK-1 incorporated.
3. Extract the distance distribution for the doubly labeled protein with the
model-free Tikhonov regularization method (Chiang, Borbat, & Freed,
2005; Stoll & Schweiger, 2006). The modulation depth of the DEER
curve might be significantly depressed by chemical reduction of the
spin label.
4. In order to systematically analyze the influence of the different steps in
the signal treatment process (baseline correction, signal-to-noise ratio,
choice of regularization parameter, etc.) on the results of Tikhonov reg-
ularization, conduct systematic variation of parameters and monitoring
changes in the resulting distance distribution using the DeerAnalysis soft-
ware. Since the uncertainties in the parameters discussed above may be
correlated, a statistical analysis of the distance distribution obtained by
variations in a multidimensional parameter space is required.
A thorough analysis monitoring the uncertainties in different steps of
the data postprocessing (starting time of the background fitting, period
of background fitting, background density, white noise) allows for val-
idation of the presented distance distributions.
ACKNOWLEDGMENTS
M.J.S. acknowledges a Hoechst Fellowship of the Aventis Foundation. This work was
supported the DFG within SPP1601.
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