Site-specific tagging proteins via a rigid, stable and short thiolether tether for paramagnetic spectroscopic analysis

Article abstract of DOI:10.1039/c4cc08493d

Increasing the stability of protein bioconjugates and improving the resolution of protein complexes is important for spectroscopic analysis in structural biology. The reaction of phenylsulfonated pyridine derivatives and protein thiols generates a stable, rigid and short thiolether tether, which is valuable in high-resolution spectroscopic measurements. This journal is

Full text of DOI:10.1039/c4cc08493d

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Site-specific tagging proteins via a rigid, stable
and short thiolether tether for paramagnetic
spectroscopic analysis†
Cite this:DOI: 10.1039/c4cc08493d
Received 30th October 2014,
Accepted 23rd December 2014
Yin Yang,‡ Jin-Tao Wang,‡ Ying-Ying Pei and Xun-Cheng Su*
DOI: 10.1039/c4cc08493d
www.rsc.org/chemcomm
Increasing the stability of protein bioconjugates and improving the
resolution of protein complexes is important for spectroscopic
analysis in structural biology. The reaction of phenylsulfonated
pyridine derivatives and protein thiols generates a stable, rigid and
short thiolether tether, which is valuable in high-resolution spectro-
scopic measurements.
Scheme 1 Site-specific tagging proteins in a short, rigid and stable manner
via thiol–ether bond formation between the 4-(phenylsulfonyl) pyridine
moiety and the protein thiol.
Site-specific labeling proteins with paramagnetic or fluorescent
tags provide a powerful technique for determining the structures,
interactions and dynamics of proteins and protein–ligand com-
plexes by paramagnetic nuclear magnetic resonance (NMR),1 elec-
tron paramagnetic resonance (EPR),2 and fluorescent resonance
energy transfer (FRET).3 Many efforts have been made towards high-
yield selectivity in site-specific labeling of proteins with a paramag-
netic or fluorescent tag including disulfide bond4 or thiolether
bond5 formation, or via click-chemistry between non-natural amino
acids and functional tags.6 The flexibility of the functional tag in a
protein bioconjugate averages the paramagnetic effects or deloca-
lizes the paramagnetic/fluorescent center, resulting in deteriorated
resolution in the structures determined by these spectroscopic
methods. Therefore, restriction of the paramagnetic or fluorescent
tag is beneficial in improving structural resolution determined by
means of paramagnetic NMR, EPR and FRET. Several strategies
have been proposed to increase the rigidity of the tag including
coordination of the protein to the paramagnetic center, increasing
the size of the tag, and optimizing the ligation site in a protein.7
However, an efficient method in tagging a protein is of high
importance for high-resolution spectroscopic analysis especially
in situ/or in vivo in structural biology.5b,8 We herein present a new
way of site-specific tagging of proteins with a rigid, stable and short
thiolether ether via the reaction of phenylsulfonated pyridine
derivatives and protein thiols (Scheme 1). The high performance
of these new paramagnetic tags in rigidity and stability has been
evaluated by paramagnetic NMR spectroscopy.
The phenylsulfone moiety is a valuable building block in
organic synthesis, however, it has not been considered as a
good leaving group under mild reaction conditions.9 To explore
the reactivity of 4-phenylsulfonyl pyridine tags to a free thiol
in aqueous solution, we synthesized two lanthanide tags, L1
(4-(phenylsulfonyl)-pyridine-2,6-dicarboxylic acid, 4PS-DPA) and
L2 (4-phenylsulfonyl-(pyridin-2,6-diyl)bismethylenenitrilo tetrakis-
(acetic acid), 4PS-PyMTA) (Fig. 1, details of synthesis of L1 and L2
are provided in the ESI†). L1 and L2 are the phenylsulfonated
derivatives of DPA and PyMTA, respectively. We show that the
phenylsulfonyl group at the 4-position of pyridine is a thiol-
specific reaction reagent in protein bioconjugations via the
formation of a thiolether bond (Scheme 1), with a similar
mechanism to a recent report of Julia–Kocienski like reagents
with free thiols.10 The reaction of L1 and L-cysteine was first
analysed by high resolution NMR spectroscopy in D2O at pD 7.5
State-Key Laboratory of Elemento-organic Chemistry, Innovation Center of Chemical
Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, China.
E-mail: xunchengsu@nankai.edu.cn
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4cc08493d
‡ Contributed equally in this work.
Fig. 1 Lanthanide binding tags of pyridine derivatives containing a phenyl-
sulfone or vinyl group.
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with a proton frequency of 600 MHz. Significant chemical shift
perturbations were observed for the resonances of 10 mM L1
after addition of 30 mM L-cysteine. By analysis of 2D NOESY,
TOCSY, 13C-HSQC and 13C-HMBC spectra recorded for the
reaction mixture and starting materials, we confirmed that a
thiol ether bond was formed between L1 and L-cysteine, and
phenylsulfinate was produced during the reaction (Fig. S1–S5,
ESI†). Notably, the nucleophilic attack to the phenyl ring with
release of 4-sulfinatopyridine-2,6-dicarboxylate was not observed.
Incubation of the 4-(phenylsulfonyl) pyridine moiety with other
amino acids containing potential nucleophiles including hydroxyl
and amine groups (up to 48 hours) produced no chemical shift
perturbations, suggesting that the phenylsulfonated pyridine is
highly thiol-specific (Fig. S6–S12, ESI†).
The high reactivity and chemoselectivity of the 4-phenylsulfonyl
pyridine moiety to free thiols as demonstrated above suggests that
the 4-phenylsulfonate pyridine can be a versatile building block in
protein modifications. As the chemical environment for individual
residues in structured biomolecules may affect the chemical
reactivity and selectivity in protein bioconjugations, we thus under-
took the selectivity assay of microenvironment effects on the
reactions of phenylsulfonated pyridine derivatives towards protein
thiols. The chemical reaction process was monitored by 15N-HSQC
spectra for a mixture of 1.0 mM 15N-labelled G47C mutant of
human ubiquitin and 5.0 mM L1 in 20 mM Tris buffer at pH 7.6
and 298 K. The reaction was completed within 10 hours. The
chemical shift perturbations showed that the residues with large
chemical shift changes were those vicinal to the ligation site of
G47C, and no chemical modifications of Met 1, K6, K11, K27, K29,
K33, K48 and K63 residing in different structural environments
were determined in the 15N-HSQC spectrum (Fig. 2). This was
further demonstrated by addition of Gd3+ ion into the solution of
the G47C–L1 conjugate, which was purified after a small desalting
PD10 column. Gd3+ is the strongest paramagnetic metal ion, and it
generates very strong paramagnetic effects (PREs) on the vicinal
nuclear spins close to its binding area (Fig. S13, ESI†).1a,c These
results clearly show that the phenylsulfonated pyridine moiety can
be used as a thiol-specific reaction reagent in site-specific labelling
of proteins.
Fig. 2 (A) Superimposition of 15N-HSQC spectra of a 0.1 mM solution of
uniformly 15N-labeled G47C ubiquitin (black) and G47C–L1 conjugate
(red). (B) Chemical-shift differences between G47C and G47C–L1 con-
jugate, calculated as d = ((DdH)2 + (DdN/10)2)1/2. (C) Ribbon drawing of
human ubiquitin (PDB code: 1UBI).11 The mutation site of G47 was high-
lighted in red and side-chains of lysine residues in blue. The spectra were
recorded at 298 K in 20 mM MES buffer, pH 6.4.
Fig. 3 (A) Superimposition of 15N-HSQC spectra of a 0.1 mM solution
of uniformly 15N-labeled G47C–R72A–R74A ubiquitin (black) and G47C–
R72A–R74A–L2 conjugate (red). The NMR spectra were recorded under the
same conditions as in Fig. 2C. (B) Chemical-shift differences between G47C–
R72A–R74A and its L2 conjugate (black sphere), and protein–L2 and its Y3+
Since L1 has only three coordinating atoms for the lanthanide
ion, its protein conjugate requires the coordination of protein to
immobilize the lanthanide ion. L2, a ligand with seven coordi-
nating sites and very high-binding-affinity for lanthanide ions,
was explored in the subsequent study. Quantitative ligation of the
G47C–R72A–R74A triple mutant of human ubiquitin with L2 was
complex (red triangle), respectively, calculated as d = ((DdH)2 + (DdN/10)2)1/2
.
achieved by incubating the protein with a five-fold excess of L2 at larger chemical shift changes in the 15N-HSQC spectra (Fig. 4 and
room temperature and pH 7.6 for 16 hours. Low-molecular Fig. S15, ESI†) in comparison with those obtained with G47C-
weight compounds were subsequently separated from the ligated 4vinylPyMTA.5b Despite the very short thiolether tether in the
product by ion-exchange chromatography (yield 4 85%). Small protein construct, only a single paramagnetic species was observed
chemical shift perturbations close to G47C were observed after as the molar ratio of lanthanide to protein was increased from
ligation with L2 (Fig. 3). The chemical shift mapping indicates 0.2 to 1.2. Large PCSs of up to ꢀ0.6 ppm were observed even in the
that only G47C was modified in the protein–L2 conjugate (also in presence of the weakly paramagnetic lanthanide Yb3+. Four sets of
Fig. S14). The resulting ligation product formally resembles the PCSs were measured for Tb3+, Dy3+, Tm3+, and Yb3+, and the
previously described ubiquitin G47C–4vinylPyMTA complex,5b respective Dw-tensor parameters were determined using the pro-
except that two methylene groups are absent. Titration of G47C– gram Numbat12 to fit the crystal structure of ubiquitin11 (Table 1).
R72A–R74A–L2 with paramagnetic lanthanide ions produced very Good correlations between the experimental and backcalculated
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Fig. 5 Ribbon drawing of ubiquitin showing the metal position (magenta
sphere) determined by fitting the PCSs of the ubiquitin G47C–R72A–
R74A–L2–lanthanide complexes to the ubiquitin structure (PDB ID: 1UBI),11
and the distance to the Ca atom (blue sphere) of G47.
and stable thiolether tether between the protein and the func-
tional tag, which is resistant to the reducing reagents, is simple;
and it does not need optimization of the ligation site in protein.
Moreover, 4PS-PyMTA is relatively small but has high binding
affinity for lanthanide ions. As PyMTA has higher affinity for
lanthanide ions than EDTA, the lanthanide complex of the
protein–L2 conjugate can be readily prepared by simply mixing
the modified protein and lanthanide ions. Excess of lanthanide
ions can be removed by addition of EDTA.
Fig. 4 Superimposition of 15N-HSQC spectra of uniformly 15N-labeled
G47C–R72A–R74A ubiquitin derivatized with L2 at G47C in the presence of
diamagnetic Y3+ (red) and paramagnetic Dy3+ or Tm3+ (black), respectively.
The molar ratio of lanthanide to protein was about 1.2 : 1. The spectra were
recorded at 298 K and pH 6.4 with a 1H NMR frequency of 600 MHz in
20 mM MES buffer.
Using the Module program,13 the alignment tensors were
determined by simulation the backbone amide RDCs of regularly
structured residues to the protein structure (PDB code: 1UBI).
Notably, the alignment tensors determined by RDCs are signifi-
cantly smaller than those back-calculated from PCSs based on
Dw-tensor using eqn (1),
PCSs were determined (Fig. S16, ESI†). The metal position was
found at a distance of 8.66 Å to carbon atom Ca of G47 (Fig. 5).
As show in Table 1, the Dw-tensors determined in G47C–
R72A–R74A–L2 are significantly larger than those obtained with
G47C–4vinylPyMTA.5b It indicates that the short thiolether
tether in the protein–L2 conjugate is sufficiently rigid to restrict
the paramagnetic tag. Both L2 and 4vinylPyMTA in reaction
with protein generate a highly stable thiolether tether, but L2,
4PS-PyMTA, is a more rigid paramagnetic tag. The magnetic
susceptibility anisotropies generated by the protein–L2 complex
are comparable with a number of rigid lanthanide binding tags
like 4MMDPA,7a TAHA,7e LBT7f–h,k and 4MTDA7i, but are generally
smaller than DOTA-tags5d,7b–d,j that are also varied to the ligation
site. The reaction of phenylsulfonated pyridine derivatives and
protein has several advantageous features: generation of a rigid
2
B0
Aax;rh
¼
Dwax;rh
(1)
15kTm0
where Aax,rh are the axial and rhombic components of the
alignment tensor, Dwax,rh the axial and rhombic components of
magnetic susceptibility anisotropy tensor, respectively (Table 2
and Fig. S17, ESI†). The different sensitivities of PCSs and RDCs
to protein local mobility, first reported in the K79A mutant of
cytochrome c under alkaline conditions,14 have been found in
many lanthanide loaded proteins.7 In the case of the ubiquitin
G47C–L2 conjugate, the much smaller RDCs are probably not
only caused by the local mobility of protein. The residual mobility
of the tag and non-specific transient contact of the tag with the
protein surface are also likely to average RDCs. It should be
pointed out that the hydrophobic patch formed by residues Leu8,
Ile44 and Val70 in ubiquitin dominates most interactions of
ubiquitin with its binding partners.15 Since G47C sits close to
the hydrophobic hot spot, any non-specific contact of L2 with
these hydrophobic residues will decrease the RDCs greatly. The
less sensitivity of PCSs to protein local mobility and probably also
residual fluctuations of tag than RDCs offers additional advan-
tages in structural biology by paramagnetic NMR spectroscopy.
The PCS defined rigidity of the protein–L2 conjugate provides the
possibility of high-resolution distance measurements by double
electron–electron resonance (DEER) and FRET methods, which
can be extended to high-molecule-weight systems.
Table 1 Dw-tensor parameters of ubiquitin G47C–R72A–R74A–L2 com-
plexed with Tb3+, Dy3+, Tm3+, and Yb3+, respectivelya
Tb3+
Dy3+
Tm3+
Yb3+
Dwax
Dwrh
a
ꢀ21.5 (3.4)
ꢀ5.1 (0.9)
91.4
ꢀ24.5 (4.3)
ꢀ7.1 (2.3)
91.2
16.6 (1.7)
3.9 (1.1)
91.4
6.7 (0.9)
1.4 (0.4)
91.6
b
122.2
121.7
123.2
121.2
g
113.9
109.9
116.8
112.8
a
The tensor parameters are in units of 10ꢀ32 m3. The tensor parameters
determined for the corresponding lanthanide complexes of ubiquitin
G47C–4vinylPyMTA5b are shown in brackets for comparison.
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Table 2 Comparison of alignment tensor and Dw-tensor parameters of
ubiquitin G47C–R72A–R74A–L2 complexed with Tb3+, Dy3+, Tm3+, and
Yb3+, respectivelya
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Aax (ꢁ104)b
1.4
1.6
Arh (ꢁ104)b
0.2
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Aax (ꢁ104)c
ꢀ5.6
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using eqn (1).
In conclusion, we report a new way of generating a stable,
short, and rigid thioether bridge between the target protein and
functional tag, which is also resistant to reducing reagents,
for paramagnetic NMR spectroscopy analysis. 4PS-PyMTA is a
small and high-affinity lanthanide binding tag, and the protein
ligation reaction is simple with high yield. As pyridine is capable
of binding lanthanide and transition metal ions, the 4-phenyl-
sulfonated pyridine derivative generates a shortest and stable
tether in the protein conjugates for paramagnetic NMR and also
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in preparation). With the high demand of simplicity and chemo-
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protein–tag conjugates for the study of DEER, FRET and para-
magnetic NMR, we anticipate that the phenylsulfonated pyridine
moiety will find broad applications in structural biology and
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