Incorporation of spin-labelled amino acids into proteins

Article abstract of DOI:10.1002/mrc.1688

The elucidation of structure and function of proteins and membrane proteins by EPR spectroscopy has become increasingly important in recent years as technological advances have been made in the design of spectrometers and in the chemistry of the nitroxide group. These new developments have increased the demand for tailor-made amino acids carrying a spin label on the one hand and for reliable methods for their incorporation into proteins on the other. Here we describe methods for site-specific spin labelling of proteins. It is shown that a combination of recombinant synthesis of proteins with chemically produced peptides (expressed protein ligation) allows the preparation of site-specifically spin-labelled proteins. Copyright

Full text of DOI:10.1002/mrc.1688

MAGNETIC RESONANCE IN CHEMISTRY
Magn. Reson. Chem. 2005; 43: S34–S39
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1688
Incorporation of spin-labelled amino acids into
proteins†
1
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Christian F. W. Becker, Kester Lausecker, Maria Balog, Tamas Kalai, Kalman Hideg,
Heinz-Ju¨ rgen Steinhoff3 and Martin Engelhard1∗
1
Max-Planck-Institut fu¨ r Molekulare Physiologie, Otto Hahn Strasse 11, 44227 Dortmund, Germany
Institute of Organic and Medicinal Chemistry, University of Pe´ cs, H-7602 Pe´ cs, Hungary
Fachbereich Physik, Universita¨ t Osnabru¨ ck, Barbarastrasse 7, 49076 Osnabru¨ ck, Germany
2
3
Received 29 April 2005; Revised 17 June 2005; Accepted 17 June 2005
The elucidation of structure and function of proteins and membrane proteins by EPR spectroscopy has
become increasingly important in recent years as technological advances have been made in the design of
spectrometers and in the chemistry of the nitroxide group. These new developments have increased the
demand for tailor-made amino acids carrying a spin label on the one hand and for reliable methods for
their incorporation into proteins on the other. Here we describe methods for site-specific spin labelling of
proteins. It is shown that a combination of recombinant synthesis of proteins with chemically produced
peptides (expressed protein ligation) allows the preparation of site-specifically spin-labelled proteins.
Copyright  2005 John Wiley & Sons, Ltd.
KEYWORDS: EPR spectroscopy; site-directed spin labelling; chemical synthesis of proteins; protein ligation
additional cysteines must be eliminated from the protein.
Recently, a method was developed that allowed site-specific
incorporation of unnatural amino acids by employing an
amber suppressor tRNA chemically aminoacylated with
the desired amino acid.3,11,12 Although this elegant method
might prove generally applicable in future (using unique
transfer RNA (tRNA)/aminoacyl-tRNA synthetase pairs13),
currently only very few laboratories are equipped to apply
the scheme successfully. The third approach goes back to
the development of solid-phase peptide synthesis (SPPS),
or Merrifield synthesis.14 Modern improvements in peptide
chemistry15 allow the synthesis of proteins containing as
many as 166 amino acids.16,17 Furthermore, combining SPPS
with recombinant techniques provides the tool to introduce
unnatural amino acids at sites of choice in large proteins and
even membrane proteins.18
INTRODUCTION
Unnatural amino acids are more and more employed in
biophysics, biochemistry and medicinal chemistry1 as they
allow the design of novel proteins,2 the introduction of
biophysical reporter groups3,4 or the modification of active
centres.5 An important group of such unnatural amino acids
carries a reporter suitable for use in magnetic resonance
techniques. Site- or fragment-specific isotope labelling, for
example, enables the structural and functional analysis of
proteins by solution or solid-state NMR spectroscopy.6,7
The introduction of a paramagnetic label in the side chain
allows determination of its static and dynamic properties
by EPR spectroscopy.8,9 There are three main approaches
to modify peptides or proteins with spin labels. A very
well established method is that of site-directed spin labelling
(SDSL), which utilizes the thiol group of a cysteine residue
for the incorporation of paramagnetic methanethiosulfonates
(Mts) yielding the spin-label side chain R1.10 This approach
requires that the target protein possesses only cysteine
residues at the desired labelling sites, which could either
be naturally occurring residues or have to be introduced
by recombinant methodologies. At the same time, all
In the present paper, the chemistry of the incorporation
of spin-labelled amino acids into peptides and proteins is
critically evaluated. In a second part, it is demonstrated that
the spin label can be introduced by using a combination of
recombinant techniques and chemical peptide synthesis.
SPIN-LABELLED AMINO ACIDS
†Presented as part of a special issue on High-field EPR in Biology,
Chemistry and Physics.
There are principally two different strategies for the intro-
duction of spin labels that can be used in peptide and
protein chemistry. The first group includes functionalized
spin-labelled building blocks that can be introduced after
the synthesis of a peptide has been completed or directly
into the native protein utilizing functional groups of nat-
urally occurring amino acids like the sulfhydryl group
of cysteines. The second group relies on special amino
ŁCorrespondence to: Martin Engelhard, Max-Planck-Institut fu¨r
Molekulare Physiologie, Otto Hahn Strasse 11, 44227
Dortmund, Germany.
E-mail: matin.engelhard@mpi-dortmund.mpg.de
Contract/grant sponsor: Deutsche Forschungsgemeinschaft;
Contract/grant number: EN87/12-1, Hi 823/1-1, STE 640/4-3.
Contract/grant sponsor: Hungarian National Research
Foundations; Contract/grant number: OTKA T48334.
Copyright  2005 John Wiley & Sons, Ltd.

Incorporation of spin-labelled amino acids into proteins S35
acids like 4-amino-1-oxyl-2,2,6,6-tetramethyl-piperidine-4-
carboxylic acid (TOAC) I or pyrroline nitroxide-containing
amino acids II and III, respectively (Scheme 1), which are
directly incorporated into the peptide during chemical syn-
thesis.
In spite of the large number of very successful applica-
tions, several serious shortcomings of the method of cysteine
exchange mutagenesis and SH-specific spin labelling have
to be considered, which may include distortions of the pro-
tein conformation or unspecific labelling in the presence
of more than one SH group. Furthermore, the extended
length of spin labels like the commonly used 1-oxyl-2,2,5,5-
tetramethylpyrroline-3-methyl-sulfonate (MTSSL) as com-
pared to naturally occurring amino acid side chains and
the conformational flexibility (Fig. 1) does not unequivocally
allow relating of the data obtained directly to the proper-
ties of the side chain in its native state. For example, the
label could fold in an environment different from that of the
original side chain, thereby experiencing conditions that can
falsely be attributed to the native state.
However, these disadvantages can be overcome by
the recently developed techniques for the incorporation of
unnatural amino acids into proteins and by taking advan-
tage of tailor-made amino acids. New, N-Boc–protected
paramagnetic amino acids with various side chains (e. g.
differences in polarity, orientation and conformationally
constrained structures) obtained by O’Donnell synthesis
have been reported very recently.20–22 The synthesis of a
paramagnetic amino acid containing the simplest and small-
est 3-methylene-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-
1-yloxyl radical moiety in the side chain (III in Scheme 1)
is reported in the next section. Spin-label amino acids with
reduced residual side chain motion provide defined ori-
entations with respect to the backbone. This will simplify
discrimination between protein backbone dynamics and its
modulation upon conformational changes and residual side
chain dynamics. Furthermore, the reduced residual motion
and the defined orientation of the nitroxide side chain
with respect to the backbone will provide reliable data of
inter-spin distances and relative orientations of the nitroxide
side chains (molecular axes orientations). Using sets of dou-
bly spin-labelled engineered proteins, the determination of
Figure 1. Conformational space of two spin-labelled amino
acids (MTSSL (a) and II (b)). The double bond character of the
peptide bond in conjugation with the double bond in the
five-membered ring is indicated by dots.
structural details and conformational changes with atomic
resolution will be achievable.
Figure 1(a) presents all flexible bonds within the R1
side chains bound to a cysteine in comparison with
those of the unnatural amino acid II. For the R1 side
chain, molecular dynamics (MD) simulations have been
performed (Beier and Steinhoff, unpublished results). For
rotation around the C˛–Cˇ bond, MD simulations reveal a
potential according to the familiar three-peaked behaviour
showing the gauche/anti states of pure sp3 hybridizations.
Both –C–S– potentials show almost ideally staggered
conformations. The torsion angle distributions of the –S–S–
°
bond reveal two characteristic states around 90 and 270 .
In these cases, the lone-pair electrons in the 3pꢀ atomic
orbitals of both sulfur atoms are orthogonal to each
other and their repulsion is minimized. Rotation around
the last bond, –Cε–C1–, shows the highest flexibility.
Particularly, apart from the trans conformation between
the –Sυ–Cε– and the double bond, all orientations are
possible. If the entire R1 side chain is located in a more
restrictive environment, the rotation about –Cε–C1– is
dominant. Since this bond is nearly parallel to the nitroxide
bond, it leads to an anisotropic reorientational motion
of the spin label approximately around the molecular
x-axis.
A low flexibility is expected for the side chain of the
unnatural amino acids II and III (Fig. 1(b)). The partial double
bond character of the N–C bond keeps the four atoms HNCO
in a plane and also restricts the reorientation about the single
bond connecting the ring to this plane. For side chain III, the
Scheme 1. Chemical structures of nitroxide-containing amino
acids.
Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2005; 43: S34–S39

S36
C. F. W. Becker et al.
Scheme 2. The reaction scheme for the synthesis of the paramagnetic Boc-protected amino acids 5 and 6. (a) 10% aq. NaOH,
CH2Cl2, Bu4NHSO4; (b) 5% aq. H2SO4, EtOH; (c) Boc2O, THF; (d) 10% aq. NaOH, EtOH.
sterical restrictions due to the interaction of the ring with the
backbone provide a spin-label probe with even less mobility.
can be overcome by employing Fmoc-based synthesis
strategies either on the solid phase in combination with
HF-cleavages or in solution as described by Marchetto
et al. and Monaco et al., respectively.31,32 In both cases,
Fmoc-protected TOAC (I) was used to achieve successful
incorporation of the spin-labelled amino acids into short
peptides. The solution-phase approach yielded the pure,
EPR-active TOAC-containing peptide without any further
treatment, whereas the HF-cleavage requires reconstitution
of the N-oxide under mildly basic conditions to revert
the protonation of the nitroxide followed by oxidation.
Another approach to achieve site-specific incorporation of
a spin label into a peptide synthesized by Boc-chemistry
was presented by McNulty et al.19 They used N˛-Boc, Nˇ-
Fmoc-L-2,3-diaminopropionic acid [Boc-Dap(Fmoc)-OH] as
a scaffold to couple a spin label to the side chain of this
amino acid (Scheme 1, II) just prior to HF-cleavage. This
avoids exposure of the spin label to repeated cycles of TFA
treatment, which can result in irreversible destruction of
the nitroxide. Reconstitution of full spin activity was again
achieved by treatment with a weak base.
We have tried to achieve direct incorporation of a
pyrroline-based nitroxide amino acid into peptides by Boc-
chemistry relying on the fact that pyrroline nitroxides
are less sensitive towards reducing, oxidative and acidic
condition, probably due to a lower accessibility of the
nitroxides when compared with six-membered rings such
as in TOAC.33 Incorporation and deprotection of amino
acid III positioned at or close to the N-terminus using
Boc-chemistry in combination with an in situ neutralization
protocol34 could be successfully conducted. However, nine
coupling cycles after introduction of III into the peptide
chain, decomposition of the nitroxide-containing amino acid
III became obvious, and after 14 cycles, no product could
be identified anymore. Comparison of synthesis results
obtained for similar sequences where the nitroxide amino
acid was replaced by leucine or L-2,3-diaminopropionic acid
(Dap) clearly indicated that the five-membered nitroxide
amino acid is also prone to irreversible destruction under
Boc-synthesis conditions. This irreversible destruction of the
spin label can probably be avoided if the nitroxide moiety is
masked during peptide elongation, e.g. as a benzyl protected
hydroxylamine (Hideg et al., unpublished). Deprotection of
the O-benzyl-hydroxylamine should occur upon treatment
with HF and the resulting hydroxylamine can be oxidized to
produce the paramagnetic nitroxide.
CHEMICAL SYNTHESIS OF SPIN-LABELLED
AMINO ACIDS
The chemical synthesis of the novel spin-labelled amino
acid 5 (Scheme 2) was carried out by alkylating ethyl
N-diphenylmethylene glycine 1 with paramagnetic allylic
bromide23 2 under phase-transfer conditions24 in aque-
ous NaOH/CH2Cl2 in the presence of Bu4NHSO4. This
procedure afforded the monoalkylated product 3, which
could be readily hydrolysed under acidic conditions to the
corresponding amine25 4, without affecting the N-oxyl rad-
ical moiety. The treatment of DL-amino acid esters with
t-butoxycarbonyl anhydride in THF gave the correspond-
ing protected N-Boc amino acid ethyl esters 5, which can
be hydrolysed20 to amino acid 6 ready for use in SPPS
(Scheme 2).
CHEMICAL SYNTHESIS AND
SEMI-SYNTHESIS OF PROTEINS CONTAINING
SPIN LABELS
The chemical synthesis and semisynthesis of proteins
relies on the ability to produce the constituent peptides,
typically by SPPS, either based on the Boc or Fmoc
protection strategy. Chemoselective ligation methods, like
native chemical ligation (NCL), are then used to link two
ore more of the resulting peptides in a head-to-tail fashion
or to attach them to recombinantly produced proteins.26–28
The synthesis and purification of peptides consisting of
up to 60 amino acids is now routinely done and even
the incorporation of a wide variety of biophysical probes
(fluorophores, isotope labels, sterically hindered amino
acids) can be achieved.29,30 This often requires identification
of synthesis conditions that are chemically compatible with
the desired biophysical probe as well as optimization of the
coupling condition by varying activation agents, solvents
and coupling times to avoid inefficient coupling reactions.
However, the site-directed incorporation of spin-labelled
amino acids in a given protein by chemical means still
presents a challenge. This is mostly due to the delicate
nature of the nitroxide moiety that is readily protonated
under acid conditions, such as TFA treatments during
Boc-synthesis or cleavage from the solid support during
Fmoc synthesis, and can then decompose. This problem
Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2005; 43: S34–S39

Incorporation of spin-labelled amino acids into proteins S37
Scheme 3. Coupling of spin label to the ε-amino group of
lysine.19
In order to demonstrate incorporation of a spin label
into semi-synthetic proteins using peptides synthesized
by Boc-chemistry, we took refuge in an approach simi-
lar to that described by McNulty et al.19 and introduced
an orthogonally protected lysine residue (Boc-Lys(Fmoc)-
OH) into the peptide sequence (CGK(SL)GHHHHHH,
the His6 tag was introduced for purification purposes).
Coupling of the spin label was achieved after com-
plete assembly of the peptide and selective deprotec-
tion of the ε-amino group of lysine with 20% piperi-
dine in N,N-dimethylformamide (DMF) by using 2-
(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflu-
orophosphate (HBTU) activation in the presence of DIEA
as a base (Scheme 3).
Scheme 4. Mechanism of native and expressed protein ligation.
Ligation’) by using an expressed protein ligation (EPL)
scheme.
EPL and the closely related NCL strategies rely on the
reaction of the sulfhydryl group of an N-terminal cysteine
with a C-terminal thioester (Scheme 4). After rearrangement
through an S ! N acyl shift, a native peptide bond is formed.
The reaction can be performed even in the presence of other
unprotected cysteine residues because of a first reversible
reaction and a second irreversible step. The combination
of recombinant techniques and peptide chemistry allows
introduction of the spin labels at every position of the protein.
The only requirements are N-terminal cysteine residues
that can be generated by protein splicing, proteolysis of
recombinant proteins and chemical synthesis of peptides and
C-terminal thioesters that are accessible by protein splicing
and chemical synthesis.
The spin-labelled peptide was then cleaved from the
°
solid support using liquid HF at 0 C in the presence of
5% p-cresol as a scavenger and yielded a paramagnetic
compound (data not shown). The applicability of the method
was demonstrated by fusing CGK(SL)GHHHHHH to the
Ras-binding domain of c-Raf1 (RBD) at its C-terminus (Fig. 2
and section ‘Spin Labelling of RBD By Expressed Protein
It should be noted that applying the EPL technique to a
spin-labelled peptide is inherently problematic because of the
use of a large excess of thiols to mediate the transesterification
step and to keep the cysteine residues in a reduced state.35
The reducing properties of frequently used thiols like
thiophenol in NCL or sodium 2-mercaptoethanesulfonate
(Mesna) in EPL can lead to reduction of the nitroxide to the
corresponding hydroxylamine and therefore render the label
inactive,36 which makes it necessary to reoxidize the sample
after the ligation step. However, reoxidation of the spin label
might be accomplished by treatment with a weak base in
the presence of atmospheric oxygen with or without added
PbO2.
SPIN LABELLING OF RBD BY EXPRESSED
PROTEIN LIGATION
Using the strategy outlined above, a semi-synthetic protein
that carries a site-specifically incorporated spin label within
its C-terminus was generated. As a test system for this
approach, the RBD was used. This protein is a downstream
Figure 2. Strategy for the C-terminal spin labelling of RBD.
Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2005; 43: S34–S39

S38
C. F. W. Becker et al.
thioesters by recombinant means. The combination of chem-
ically synthesized peptides with recombinant polypeptides
greatly increases the versatility and applicability of chemical
synthesis of labelled proteins. It has now become possible to
introduce spin labels via these methods not only in the C-
or N-terminal part of the protein but also in its centre, thus
allowing EPR studies with a new quality of labelled proteins.
Acknowledgements
This work was supported by grants from Hungarian National
Research Foundations (OTKA T48334) and Deutsche Forschungs-
gemeinschaft (EN87/12-1 for M. E., Hi 823/1-1 for K. H. and STE
640/4-3 for H-J. S).
Figure 3. EPR spectra of the spin-labelled peptide
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