Assessing multiple conformations of lanthanide binding tags for proteins using a sensitive19F-reporter

Article abstract of DOI:10.1039/d1cc00791b

Quantifying the isomeric species of metal complexes in solution is difficult.¹⁹F NMR herein was used to determine the abundance of isomeric species and dynamic properties of lanthanide binding tags. The results suggest that¹⁹F is an efficient reporter in assessing and screening paramagnetic tags suitable for protein NMR analysis.

Full text of DOI:10.1039/d1cc00791b

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Assessing multiple conformations of lanthanide
binding tags for proteins using a sensitive
19F-reporter†
Cite this: Chem. Commun., 2021,
57, 4291
Received 10th February 2021,
Accepted 24th March 2021
Jia-Liang Chen, Ben-Guang Chen, Bin Li, Feng Yang and Xun-Cheng Su
*
DOI: 10.1039/d1cc00791b
rsc.li/chemcomm
Quantifying the isomeric species of metal complexes in solution is chemical exchange broadening and the large paramagnetic
difficult. 19F NMR herein was used to determine the abundance of shift and paramagnetic relaxation enhancement nearby the
isomeric species and dynamic properties of lanthanide binding tags. paramagnetic center.6 Here, we used 19F-NMR to discriminate
The results suggest that 19F is an efficient reporter in assessing and the different conformations of paramagnetic species.
screening paramagnetic tags suitable for protein NMR analysis.
The lanthanide (Ln) complexes of 1,4,7,10-tetraazacyclodode-
cane-N,N0,N00,N00 0-tetraacetic acid (DOTA) and its derivatives are
Relying on the dipolar interactions between the unpaired kinetically inert but show a diverse range in the magnetic anisotropy,
electron and nuclei spins in biomolecules or ligands, paramag- and these complexes are widely used in paramagnetic NMR of
netic effects manifested in NMR spectroscopy have been widely proteins.3 DOTA–Ln complexes generally present two main isomeric
used in characterizing the structures and dynamics of proteins states (four enantiomeric pairs), the square antiprism (SAP) and
and protein complexes, nucleotides, and oligosaccharides.1,2 twisted square antiprism (TSAP) conformations, in solution due to
To achieve these rich sources of structural restraints, site- the ring inversion and arm rotation.7 The abundance of the two
specific labeling of proteins with a paramagnetic tag is gener- isomers highly depends on the ionic radius of Ln3+.7 These two
ally required. Lanthanide ions (Ln3+) are preferred in the isomers differ greatly in water exchange rates and magnetic
generation of long-range distance and angular restraints of anisotropy.8 The difficulty in using DOTA–Ln complexes as para-
biomolecules in structural biology,1 and chemically synthesized magnetic tags in structural biology is the ease of forming multiple
lanthanide binding tags represent a mainstream in site-specific paramagnetic species in solution.3,5b–e To achieve a guideline in
labeling of proteins with a paramagnetic tag.3
designing suitable DOTA–Ln like tags for applications in biological
Because of the magnetic anisotropy and high coordinating systems, we used 19F NMR to quantify the isomer populations in the
numbers of lanthanide ions, multiple paramagnetic species are free DOTA–Ln like tag and testify this concept in the protein–tag
generally present in the protein–tag conjugates as evidenced in conjugates.
the high resolution NMR spectra. It is indeed in the following
We synthesized two DOTA–Ln like tags, 4PS-5F-Py-DO3MA(S)-
cases, if the tag generates a new chiral center once attached to a Ln (T1-Ln) and 4PS-5F-Py-DO3A-Ln (T2-Ln) (Ln = Lu, Yb, Tm, Tb,
protein4a or the coordination core of metal complexes contains Dy) following the previous protocol with some modifications
a chiral center4b–d or multiple conformation states in slow (Fig. 1) (detail in the ESI†).9 Each tag contains a thiol reactive
exchange in the NMR timescale.5 In contrast, only one diamag- phenylsulfone group (PS) at the fourth position in pyridine, which
netic species is generally observed in the protein–tag conju- can be conjugated to a solvent cysteine in a protein.10 To reduce
gates. Therefore, the chirality has to be carefully considered in
the design of suitable paramagnetic tags for applications in
biological systems. However, elucidation of multiple paramag-
netic species in the paramagnetic tags is difficult by proton
based NMR spectra, because the chemical shift assignments of
the individual protons are complex and difficult due to the
Fig. 1 Chemical structures of DOTA derived paramagnetic tags, 4PS-5F-
Py-DO3MA(S)-Ln (A), and 4PS-5F-Py-DO3A-Ln (B). Each tag contains a
thiol-specific reaction moiety, phenylsulfonated pyridine (ref. 9 and 10),
and a 19F NMR reporter.
State Key Laboratory of Elemento-organic Chemistry, College of Chemistry,
Nankai University, Tianjin 300071, China. E-mail: xunchengsu@nankai.edu.cn
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1cc00791b
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Table 1 19F chemical shifts and populations (P) of the two isomers in T1-
Ln and T2-Ln complexes determined by 1D 19F NMR spectra, and DdAB is
the absolute value of the chemical shift differences between the two
isomeric species
the paramagnetic relaxation enhancement effect, one fluorine was
anchored at the 5-position of the pyridine ring, which is distant
from the paramagnetic center (Fig. 1). The two tags were assessed
by 1D 19F NMR, and the isomeric exchange rates were extracted by
2D 19F-EXSY spectra.
Isomer A (SAP)
Isomer B (TSAP)
In the 1D 19F NMR spectra, one single 19F signal was
observed for the T1-Lu and T1-Yb complexes (Fig. 2A). In
contrast, two sets of 19F signals in varied populations were
determined for the T1 complexes with Tm3+, Tb3+ and Dy3+. The
abundance of minor species was about 10%, 18%, and 15% for
Tm3+, Dy3+, and Tb3+ complexes, respectively (Table 1). Differ-
ent from T1-Ln, both diamagnetic and paramagnetic complexes
of T2-Ln presented two species in solution (Fig. 2B). The minor
species in T2-Ln complexes was about 25%, 22%, 15%, 10%,
and 18% for Lu3+, Yb3+, Tm3+, Dy3+, and Tb3+, respectively
(Table 1). To characterize the dynamic exchange properties
between the two isomeric species, 2D 19F-EXSY experiments
were performed (Fig. S2 and S3, ESI†). Notably, the chemical
shift difference between two isomeric species increased from
about 2 ppm to tens of ppm (Table 1). The reaction product of
L-cysteine with tag–Ln complexes (Ln = Lu, Yb, Tm) preserves
similar populations for the two isomers in solution (Fig. S4 and
Table S1, ESI†), indicating that the phenylsulfone group in the
pyridine has no significant effect on the isomeric diversity.
These data suggest that the 1D and 2D 19F-NMR spectra are
efficient to discriminate and quantify the isomeric species of
DOTA–Ln like complexes in solution. The EXSY data showed a
minor species with a population of about 5% and 4% for T1-Yb
and T1-Lu complexes that was not visible in the 1D NMR
spectrum of the 1.0 mM complex (Fig. 2 and Fig. S5, ESI†).
Ln
d (ppm)
P (%)
d (ppm)
P (%)
DdAB
T1-Ln
T2-Ln
Lu
Yb
Tm
Dy
Tb
Lu
Yb
Tm
Dy
Tb
ꢀ121.38
ꢀ92.99
96
95
90
82
85
25
22
15
10
18
ꢀ123.60
ꢀ108.70
ꢀ98.65
4a
2.22
15.71
64.05
25.41
20.51
1.75
13.02
49.88
21.55
16.23
5a
ꢀ34.60
10
18
15
75
78
85
90
82
ꢀ246.06
ꢀ206.56
ꢀ121.41
ꢀ96.76
ꢀ220.65
ꢀ186.05
ꢀ123.16
ꢀ109.78
ꢀ101.56
ꢀ187.00
ꢀ172.00
ꢀ51.68
ꢀ208.55
ꢀ188.23
a
The minor species in T1-Lu and T1-Yb was determined by an EXSY
experiment.
In the T1-Ln complexes, the major species has larger lantha-
nide induced shifts (LIS) than the minor one, whereas T2-Ln
complexes present an opposite trend (Fig. 2 and Fig. S6, ESI†).
The different isomers for the major species between the T1-Ln
and T2-Ln complexes is due to the effect of the additional
methyl group in the arm. In the DOTA–Ln complexes, the SAP
isomer adopts a large torsion angle between the N4 and O4
planes (B391) and a more compact coordinating cage than the
TSAP isomer.7 The SAP isomer presents a stronger ligand field
than the TSAP isomer.8c,11 The ligand field parameters can be
determined by Reilley analysis of LIS,12 and the contact (dC) and
pseudocontact (dPCS) terms under the axial symmetry assump-
tion can be written as
ꢀ
ꢁ
ꢀ
ꢁ
3 cos2 y ꢀ 1
12pr3
m0mB2CJB20 3 cos2 y ꢀ 1
dPCS ¼ Dwax
¼
(1)
2
10ðkTÞ
12pr3
LIS = dpara ꢀ ddia = dC + dPCS = hSZiF + CJG
(2)
(3)
LIS
CJ
¼ F þ
G
hSZi
hSZi
where Dwax is the axial component of the magnetic suscepti-
bility tensor, r and y are polar coordinates of the nucleus
relative to the anisotropic Dw-tensors, m0 is the permeability
of vacuum, mB is the Bohr magneton, B02 is the second rank
ligand field parameter. hSZi and CJ are lanthanide-dependent
constants, F is the observed nucleus dependent value, and G is
the value dependent on the ligand field parameters and the
geometric location of the observed nucleus.12 As shown in
eqn (3), the plot of LIS/hSZi with respect to CJ/hSZi generates a
linear relationship and the slope represents the strength of the
ligand field.
The major species of T1-Ln complexes presents a larger
slope than the minor species from the Reilley plot according
to eqn (3) (1.33 for major species and 0.67 for minor species)
(Fig. 2C), suggesting that the major species adopts the SAP state
and the minor species is in the TSAP state. This result is
consistent with the previous observations in LnDOTA,13a
Fig. 2 1D 19F NMR spectra of the T1-Ln (A) and T2-Ln (B) (Ln = Lu, Yb, Tm,
Dy, Tb) complexes. The arrow denotes the minor species. The Reilley plot
of Dd-19F/hSZi vs. CJ/hSZi was performed for the major (black) and minor
(red) species in T1-Ln (C) and T2-Ln complexes (D). Dd-19F is the chemical
shift difference between the paramagnetic species and diamagnetic spe-
cies. Experimental details are in the ESI† and the values of CJ and hSZi for
Ln3+ are from ref. 12.
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LnDO3A-SA,13b and LnNB-DOTA.13c In contrast, the major higher conformational stability than T2-Ln complexes due to
species of T2-Ln complexes is in the TSAP state, since its slope the introduction of a methyl group at the coordination arms.14c
(0.56) is smaller than that of the minor species (1.08) from the
To evaluate whether the isomeric species in the free tag is
Reilley plot (Fig. 2D). The population of isomeric species in held true in its protein conjugates, we used human ubiquitin
T2-Ln complexes is similar to those in the LnHPDO3A11d and G47C as a target protein to assess these two tags. In the G47C-
LnDOTMA(R).13d We note that T2-Ln (Ln = Tb, Dy, Tm, Yb, and T1-Ln (Ln = Tm, Yb) conjugates, one major paramagnetic
Lu) complexes differ from the PyDO3A-Eu, which has 66% SAP species with large PCSs was observed in the 2D 15N-HSQC
state.13e This is probably due to the variations in the ion spectra (Fig. 4 and Fig. S7, ESI†). A minor paramagnetic
radius.5e,7,11d To summarize, the LIS plot of the 19F nucleus is species with population of 12% ꢁ 3% for G47C-T1-Tm and
an efficient reporter to determine the individual isomers of 5.4% ꢁ 1.5% for the G47C-T1-Yb conjugate were also observed,
DOTA–Ln like complexes in solution.
consistent with the results of free T1-Ln complexes (Fig. 4,
The inspiring results demonstrated by 19F-NMR encouraged Table 1 and Fig. S7, ESI†). The 1D 19F NMR of G47C-T1-Tm
us to determine the isomeric exchange rates in solution, which presented a similar result (Fig. 4). In addition, the G47C-T1-Tm
is highly important for site-specific tagging proteins.5f We conjugate showed strikingly different PCSs from the G47C-Py-
performed EXSY experiments for the T1-Yb and T2-Yb com- DO3MA(S)-Tm conjugate (Table S2 and Fig. S8, S9, ESI†),16
plexes with mixing time varying from 1 ms to 30 ms to extract indicating that the fluorine atom tunes the relative orientation
isomeric exchange parameters (details in the ESI†).7b,14 As of the tag–Ln complex to protein. In great contrast, two sets of
shown in Fig. 3, the fitted exchange rate between the two paramagnetic species with different PCSs were observed in the
isomers was 165 ꢁ 13 sꢀ1 for the T1-Yb complex. The abun- G47C-T2-Ln (Ln = Tm, Yb) conjugates (Fig. 4 and Fig. S7, ESI†).
dance of the TSAP and SAP states is 96% and 4%, respectively, The two paramagnetic species have similar populations of
which was not identified by 1D 19F NMR (Fig. 2A and Table 1). 48% ꢁ 2%, and 52% ꢁ 2% in the G47C-T2-Tm conjugate and
Similarly, the fitted exchange rate between the two isomers is 44% ꢁ 2.5%, and 56% ꢁ 2.5% in the G47C-T2-Yb conjugate,
276 ꢁ 16 sꢀ1 for the T2-Yb complex, which is faster than that of respectively, strikingly different from the free T2-Ln complexes
the T1-Yb complex (Fig. 3) because of the absence of the methyl (Table 1, Fig. 4 and Fig. S7, ESI†). The 1D 19F NMR of the
group in the arm. The relative population was 76% in the TSAP G47C-T2-Tm conjugate had a similar but slightly skewed result
state and 24% in the SAP state, consistent with the results of 1D due to the broad signals in the minor species (Fig. 4). The
19F NMR. Interestingly, a third conformation (about 1%) was inconsistency between the free T1-Ln and T2-Ln tags and the
also observed in the 2D 19F-EXSY spectra of the T2-Yb complex, protein conjugates suggests the impact of the local environ-
and this additional isomer was readily transformed to the ment surrounding the ligation site on the conformational
SAP rather than the TSAP isomer and the exchange rate was stability and isomeric interconversion of the tag–Ln complex,
131 ꢁ 1 sꢀ1 (Fig. 3). The value is similar to the arm-rotation rate especially for the dynamic-lability DO3A-Ln like tag (Fig. 4 and
but larger than the ring-inversion rates in the DOTA–Ln like Fig. S7, ESI†). The decreased isomeric exchange rate by the
complexes,13b,14 indicating that the third isomer in the T2-Yb
complex might stem from the 4PS-pyridine arm rotation
as found in LnDO3A-SA13b and some DO3A-Ln like
complexes.11d,15 To summarize, the T1-Ln complexes have
Fig. 4 Isomeric species of two DOTA–Ln like complexes evaluated in
ubiquitin G47C conjugates. 1D 19F NMR and 15N-HSQC spectra overlay of
0.2 mM Ub G47C-Tag–Tm conjugate (blue) and 0.2 mM Ub G47C (red)
Fig. 3 Exchange rates between different isomers determined by 2D
19F-EXSY spectra for the DOTA–Ln complexes. The 19F-EXSY spectra:
(A) T1-Yb, and (B) T2-Yb. The exchange rates between different isomers:
(C) T1-Yb, and (D) T2-Yb. The NMR spectra were recorded for the 20 mM
lanthanide complex with a mixing time of 20 ms.
recorded in 20 mM MES buffer at pH 6.4 and 298 K for (A) T1-Tm and
(C) T2-Tm, respectively. The arrow denotes the minor species. Compar-
ison of populations of two isomers in free tag–Tm (cyan) and Ub G47C-
Tag–Tm conjugate (15N-HSQC: orange, 19F NMR: gray) for (B) T1-Tm and
(D) T2-Tm, respectively.
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