Local Deuteration Enables NMR Observation of Methyl Groups in Proteins from Eukaryotic and Cell-Free Expression Systems

Article abstract of DOI:10.1002/anie.202016070

Therapeutically relevant proteins such as GPCRs, antibodies and kinases face clear limitations in NMR studies due to the challenges in site-specific isotope labeling and deuteration in eukaryotic expression systems. Here we describe an efficient and simpl

Full text of DOI:10.1002/anie.202016070

Angewandte
Communications
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How to cite:
International Edition: doi.org/10.1002/anie.202016070
German Edition: doi.org/10.1002/ange.202016070
Protein Isotope Labelling
Hot Paper
Local Deuteration Enables NMR Observation of Methyl Groups in
Proteins from Eukaryotic and Cell-Free Expression Systems
Abhinav Dubey+, Nikolay Stoyanov+, Thibault Viennet+, Sandeep Chhabra, Shantha Elter,
Jan Borggrꢀfe, Aldino Viegas, Radosław P. Nowak, Nikola Burdzhiev, Ognyan Petrov,
Eric S. Fischer, Manuel Etzkorn,* Vladimir Gelev,* and Haribabu Arthanari*
Abstract: Therapeutically relevant proteins such as GPCRs,
antibodies and kinases face clear limitations in NMR studies
due to the challenges in site-specific isotope labeling and
deuteration in eukaryotic expression systems. Here we describe
an efficient and simple method to observe the methyl groups of
leucine residues in proteins expressed in bacterial, eukaryotic
or cell-free expression systems without modification of the
expression protocol. The method relies on simple stereo-
selective 13C-labeling and deuteration of leucine that alleviates
the need for additional deuteration of the protein. The
spectroscopic benefits of “local” deuteration are examined in
detail through Forbidden Coherence Transfer (FCT) experi-
ments and simulations. The utility of this labeling method is
demonstrated in the cell-free synthesis of bacteriorhodopsin
and in the insect-cell expression of the RRM2 domain of
human RBM39.
characterize their interactions and dynamics.[3] However, for
such large protein complexes, observation of the methyl signal
requires replacement of surrounding protons with deuterons
to reduce signal loss due to dipole-dipole relaxation.[4] The
most common method for achieving specific labelling at
methyl positions is to grow bacteria in perdeuterated medium
supplemented with the appropriate 13C1H3-methyl-labeled
biosynthetic precursors of the amino acids targeted for
observation.[5] However, using this method, significant
dipole-induced relaxation can also arise from intra-residue
proton-proton interactions, for example, between the geminal
methyl groups in Leu or Val.[4,6] The introduction of pro-chiral
amino acid precursors[1b] eliminated intra-residue dipole-
dipole relaxation in bacterially expressed proteins.
Yet, biologically relevant targets, including most protein
kinases, integral membrane proteins (GPCRs, ABC trans-
porters and TCRs), and therapeutic antibodies often cannot
be functionally expressed in bacteria. In vitro protein
expression with ribosomal extracts represents an increasingly
viable alternative for obtaining functional proteins; however
the [13C,1H,2H]-labeled amino acids required for production
of methyl-labeled proteins by this method remain expensive
(especially for the Leu precursor)[7] and/or require elaborate
experimental protocols[7b,8] to achieve methyl labeling. Fur-
thermore, expressing functional G-protein coupled receptors
(GPCRs) in vitro remains a challenge.[9] Recently, yeast
strains able to produce functional GPCRs, survive deutera-
tion, and process the metabolic precursor from Ile have been
developed.[10] Met,[11] Ala[12] and methylated lysines[13] and
cysteines[14] have been used to probe GPCR dynamics, and the
N
MR spectroscopy provides a rich source of information
about the dynamics and interactions of proteins that are
essential for function. The side-chain methyl groups of Ile,
Leu and Val form critical hydrophobic contacts and stabilize
the structural cores and interaction grooves of proteins,
making them powerful probes in therapeutically relevant
systems.[1] The symmetry of the methyl group ensures that all
three hydrogens contribute to a single intense signal, further
enhanced by the favourable relaxation properties due to rapid
À
rotation around the C C bond. In combination with the
methyl-TROSY experiment,[2] which selects 13C-1H3 coher-
ences that relax much slower, methyl resonances can be
observed even in megadalton-size proteins and used to
[*] Dr. A. Dubey,[+] Dr. S. Chhabra, Dr. R. P. Nowak, Dr. E. S. Fischer,
Dr. H. Arthanari
zentrum Jꢁlich GmbH
52425 Jꢁlich (Germany),
Cancer Biology, Dana-Farber Cancer Institute
450 Brookline Avenue LC-3311, Boston MA, 02215 (USA)
and
and
JuStruct: Jꢁlich Center for Structural Biology, Forschungszentrum
Jꢁlich GmbH
Department of Biological Chemistry and Molecular Pharmacology,
Harvard Medical School
240 Longwood Avenue, Boston MA, 02215 (USA)
E-mail: hari@dfci.harvard.edu
N. Stoyanov,[+] N. Burdzhiev, Dr. O. Petrov, Dr. V. Gelev
Faculty of Chemistry and Pharmacy, Sofia University
1 James Bourchier Blvd., 1164 Sofia (Bulgaria)
E-mail: doktor.gelev@gmail.com
Dr. T. Viennet,[+] S. Elter, J. Borggrꢀfe, Dr. A. Viegas, Dr. M. Etzkorn
Institute of Physical Biology, Heinrich-Heine-University
Universitꢀtsstr. 1, 40225 Dꢁsseldorf (Germany)
and
52425 Jꢁlich (Germany)
E-mail: manuel.etzkorn@hhu.de
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202016070.
ꢂ 2021 The Authors. Angewandte Chemie International Edition
published by Wiley-VCH GmbH. This is an open access article under
the terms of the Creative Commons Attribution Non-Commercial
License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited and is not used
for commercial purposes.
Institute of Biological Information Processing (IBI-7), Forschungs-
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NMR signal was boosted by judicious “local inter-residue
deuteration”, that is, addition of deuterated amino acids of
the types that occur in the spatial vicinity of the methyl group
of interest. Alternatively, insect cell culture is frequently the
method of choice for the production of proteins that have
stringent requirements for proper folding and post-transla-
tional modifications. However, higher eukaryotic cells are not
2
viable in H2O concentrations higher than 30%. Still up to
75% protein deuteration has been achieved by the addition of
deuterated amino acids to the growth medium.[11c,15] Local
deuteration surrounding the desired [13C,1H]-methyl signals
was found to improve spectral quality significantly.[11c,15a]
Despite progress, there remains an unmet need for simple
and broadly applicable labeling of protein methyl groups, for
example, through the use of affordable stereo-specifically
labeled amino acids.[11a,12]
The stereo-selective isotope enrichment of branched
amino acids is amply documented in the chemistry litera-
ture.[16] An impressive set of [13C,15N,2H]-labeled amino acids
optimally designed for automated NMR structure determi-
nation was developed[7a] and applied to obtain stereo-specific
Leu labeled 150 kDa IgG2b glycoprotein produced in mouse
hybridoma cells,[1d] which produced better spectra than its
protonated counterpart in an otherwise protonated back-
ground. However, due to the complex chemistry and expen-
sive starting materials, these amino acids remain prohibitively
expensive. In light of this, we set out to produce a more
affordable version of Leu containing the essential features
needed for observing methyl resonances in large proteins.
Here we report a straightforward synthesis of Leu in
which the desired methyl group is [13C,1H]-labeled while some
of the proximal hydrogens are deuterium-enriched (Figure 1).
The “locally” deuterated, methyl 13C-labeled leucine (5,
Figure 1) is hereafter referenced as Leu-methLD. We present
an analysis of the benefits of this labeling configuration and
report significantly increased spectral resolution and sensi-
tivity even in an otherwise protonated background. We
incorporate Leu-methLD into the 42-kDa maltose binding
protein, the 12-kDa human RBM39245–332 encapsulating
RRM2 domain, and the 25-kDa membrane protein bacterio-
rhodopsin using E. coli, insect cell and cell-free protein
expression, respectively, without modification of the original
expression protocols. This method of labeling is flexible,
simple and promises to considerably expand the range of
isotopically labeled proteins available to NMR-based struc-
tural studies.
In our hands the most economical route to methyl-labeled
leucine was a simplified version of a synthetic route by
Siebum et al.[17] (Figure 1a). The isopropyl oxazolidinone (1)
was acylated with deuterated propionyl chloride, and the
product was stereo-selectively methylated with small excess
of 13CH3-methyl iodide, with an enantiomeric excess of 94%.
Reductive cleavage with LiAlH4 (LiAlD4 can be used for
deuteration of the b positions), followed by treatment with
PPh3/Br2 yielded the stereoselective methyl-labeled isobutyl
bromide (4). Chiral alkylation of activated glycine (6) yielded
L-Leu, but the reaction proved sluggish unless stoichiometric
quantities of expensive Maruoka catalyst[18] were used. To
save cost we opted for the racemic DL-Leu which retained the
Figure 1. Simple and inexpensive synthesis of “locally” deuterated
13CH3-methyl leucine. A) Synthesis route of Leu-methLD, (5); b) The
pro-R-13CH3 or pro-S-13CH3 methyl configuration of 5 is obtained with
94% stereoselectivity starting from the corresponding Evans’ chiral
auxiliary 1.
stereochemistry of the labeled methyl groups (Figure 1a).
The final Leu-methLD (5-13C-5,5,5,4-d4-DL-Leu, 5) contained
94% of the desired 13C1H3-methyl DL-leucine and 6% of the
methyl-inverted configuration (Figure 1b). We synthesized
and tested the pro-R isotopomer (R-methLD) in overall yield
of 24% from d5-propionic acid and 18% from 13CH3-methyl
iodide. The cost of reagents per milligram of R-methLD was
less than 1 E. By comparison, a labeled ketoacid used to
generate an analogous Leu labeling pattern in cell-free
expression was reportedly obtained for 20 EmgÀ1.[7a]
A
summary of the cost of Leu-methLD (5) used to label the
proteins in this study is given in the supplementary informa-
tion.
Using theoretical calculations, we compared the contri-
1
bution of intra- and inter-residue H to dipole-dipole relax-
ation of Leu-methyl protons in the 42-kDa protein MBP. We
used the atom coordinates from a previously deposited NMR
structure (PDB ID: 1EZO) and calculated all inter-proton
distances between leucine methyl protons and the rest of the
protons in MBP (averaged over all the conformations). The
details of the computation are described in the supplementary
information. Deuterating four of the closest intra-residue
protons, namely Hd21, Hd22, Hd23 and Hg, translates to
a 37% reduction in dipole-dipole interactions (Figure S1)
leading to narrower linewidths. This is comparable to the
dipole-dipole relaxation caused by all the remaining inter-
residue protons ( ꢀ 38%). This suggests that local deutera-
tion, including stereo-specific methyl deuteration could
sufficiently reduce the rate of transverse relaxation and
2
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improve resolution and sensitivity in the absence of complete
for more details). bR was refolded and purified in n-dodecyl-
protein deuteration.
b-D-maltoside (DDM) micelles, leading to a final protein-
detergent complex of approximately 100 kDa.[21] Heteronu-
clear multiple quantum correlation (HMQC) spectra were
acquired on both samples (see Figure 3a black vs. red).
Similarly, for RBM39245–332, medium prepared simply by
replacing uniformly labeled Leu with 75 mgLÀ1 of R-methLD
was used and heteronuclear single quantum correlation
(HSQC) spectra (Figure 3 f) were measured. Notably, the
quality of the RBM39245–332 spectrum was sufficient to also
detect the much weaker signals arising from the residual 6%
pro-S (d2) 13C1H3-methyl groups. In both cases (bR and
To experimentally validate these findings and quantify the
effect of local deuteration, we used forbidden coherence
transfer (FCT) experiments.[19] FCT experiments translate
into a build-up curve where the slope reports on local
dynamics (order parameter S2) and the plateau on the local 1H
density that contributes to relaxation. Fits show that deviation
from the control is higher for 1H density than for S2
parameters, as expected (Figure S4, supplementary informa-
tion). As examples, we highlight Leu7 and Leu121 in MBP
that was (i) d1,2-[13C,1H]-methyl-labeled and fully protonat-
ed, and (ii) d1-[13C,1H]-methyl, d2-[12C,2H]-methyl, g-[2H]-
labeled in an otherwise fully protonated background. The
samples were produced in E. coli with the addition of (i) 13C-
a-ketoisovalerate[20] or (ii) R-methLD (Figure 1b) to the aque-
ous M9 minimal medium 1 h before induction (Figure S4).
The ratio of values to which the two FCT curves in Figure 2b
plateaus gives us an estimate of the linewidth reduction
( ꢀ 50%) due to the reduced dipole-dipole interaction from
the geminal methyl protons. We present additional FCT
curves for other Leu residues in MBP in supplementary
Figures S2,S3.
These results show that “local deuteration” of Leu will
reduce transverse relaxation sufficiently to produce usable
NMR spectra from large, otherwise non-deuterated protein
complexes. It represents a new avenue for proteins that
cannot faithfully be expressed in bacteria. To validate this
hypothesis, we expressed the seven-transmembrane-helix
protein bacteriorhodopsin (bR) in an E.-coli-based cell-free
system and the 12-kDa human RBM39245–332 encapsulating
RRM2 domain in Sf-9 cells. For bR, uniformly 13C-labeled
non-deuterated L-Leu (8.5 mg) or R-methLD (Figure 1b)
(17 mg) was used, and no difference in expression yield was
observed between the samples (see supplementary methods
1
RBM39), the JCC couplings in the [U-13C,1H]-Leu sample
were absent in the Leu-methLD sample, obviating the 13C
constant-time evolution component of the pulse sequence[22]
and enabling applications of non-uniform sampling. A
projection of the 2D spectra (along 1H dimensions, Fig-
ure 3c,e,h,j) showed a 30%–50% increase in resolution and
on average an approximately 6-fold increase in intensity.
While an intensity gain of 2-fold is expected due to the
absence of 13C-13C 1J-couplings (Figure 3d,i), the reduced 1H-
1H dipolar couplings in the Leu-methLD sample contribute to
the further enhanced spectral features. Notably, for bR in
DDM micelles the residue-specific intensity gains can be well
above 10-fold and depend on dynamic range and local
protonation levels of the system favouring peaks that also
require signal enhancements the most (see Figure S10 for
more details). Overall, use of Leu-methLD in the otherwise
natural abundance expression system improved the quality of
methyl spectra sufficiently to tip the scales in favour of NMR
studies of binding interactions, dynamic and allosteric mech-
anisms in numerous challenging systems.[3a,13a]
The method reported here provides a relatively inexpen-
sive route to produce stereospecific methyl labeling of Leu
residues, which is directly applicable for most commonly used
protein production systems, including insect cells and in vitro
expression without a need for modification of the existing
expression protocols. Our simulations and experiments fur-
ther clarify the effects of local deuteration on transverse
relaxation rates. While the frequency of Leu in the eukaryotic
proteome is around 9%, making it one of the most abundant
amino acids, analogous site-selective [13C,1H/2H] enrichment
of Val and Ile should produce similar gains in the quality of
protein NMR spectra. Further improvements in the protein
spectrum are expected if surrounding residues and/or envi-
ronment would be additionally deuterated or if Leu-methLD is
additionally deuterated at the beta protons, for example, by
using LiAlD4 (an additional 20–40% increase in the cost of
reagents) in step 3 (Figure 1). However, the significant
improvement in the absence of further deuteration makes
this approach particularly appealing for the wide range of
systems that so far eluted detection due to stringent require-
ments on the expression system. We therefore expect this
methodology to enable further characterizations of challeng-
ing protein targets, including also the use of solid-state
NMR.[23] The simplified protein spectra, the enhanced S/N,
and the flattened dynamic range favor the use of non-uniform
data sampling,[24] which will lead to further gains in sensitivity
for protein complexes up to the megadalton regime.
Figure 2. Local deuteration reduces the transverse relaxation rate of
Leu methyl groups. (a) Enlarged view of the NMR structure of maltose
binding protein (MBP, PDB:1EZO) around Leu121. Leu121 is shown
as a ball and stick model with deuterated protons in black, Hb2 and
Hb3 in blue, and the methyl protons being studied in red. The orange
spheres are the inter-residue protons within 6 ꢃ of the Cd1 atom of
Leu121, and the grey spheres are the rest of the protons in MBP.
(b) Experimentally determined FCT curves of Leu7 d1[13CH3] and
Leu121 d1[13CH3] in natural hydrogen abundance MBP (green curve)
and MBP with d1[13CH3] that is “locally” d2 and g-deuterated with Leu-
methLD (blue curve).
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Figure 3. Stereo-specific CD3/13CH3-labeled Leu with Leu-methLD facilitates NMR characterization of proteins expressed in vitro and in insect cells.
(a) [13C,1H]-HMQC spectra of cell-free expressed bR in DDM micelles with [U-13C, 1H] Leu (black) or Leu-methLD (red). (b) Structure of
bacteriorhodopsin (bR, PDB ID:1R84) with [13C, 1H]-labeled Leu methyl groups highlighted as red spheres. One-dimensional slices at indicated
frequency in the 1H/13C dimension are shown in (c)–(e). (c) Signal intensity is increased by 5.5-fold. Comparison of normalized data shows that
a singlet is obtained instead of doublet facilitating high resolution acquisition in the 13C dimension (d) and that linewidth at half-height is reduced
by 29% in the 1H dimension (e). (f) [13C,1H]-HSQC spectra of the human RBM39245–332 encapsulating RRM2 domain expressed in insect cells with
[U-13C, 1H] Leu (black) or Leu-methLD (red). (g) Structure of the CAPER-RRM2 domain (PDB ID: 2JRS) with [13C,1H] labeled Leu methyl groups
highlighted as red spheres. Some of the residual 6% of inverted methyl labeled leucines are also observable (example marked by a blue arrow).
1
Analog as for bR (c–e), one-dimensional slices at indicated frequency in the H/13C dimension are shown in (h)–(j). (h) Signal intensity is
1
increased by 6-fold. (j) Linewidth at half height is reduced by 49% in the H dimension.
[3] a) J. L. Kitevski-LeBlanc, T. Yuwen, P. N. Dyer, J. Rudolph, K.
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Biomolecular NMR Center and funding through an Emmy
c) A. K. Schuetz, L. E. Kay, eLife 2016, 5, e20143.
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edges funding from the Claudia Adams Barr Program for
Innovative Cancer Research. E.S.F. acknowledges funding
from the Linde Family Foundation. E.S.F. is a Damon
Runyon-Rachleff Innovator supported in part by the
Damon Runyon Cancer Research Foundation (DR-50-18).
Open access funding enabled and organized by Projekt
DEAL.
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Vladimir Gelev is the founder or FB Reagents Ltd, a manu-
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Keywords: cell free protein expression ·
Forbidden Coherence Transfer · eucaryotic protein expression ·
methyl labeled leucine · methyl TROSY
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Manuscript received: December 3, 2020
Revised manuscript received: February 22, 2021
Accepted manuscript online: March 26, 2021
Version of record online: && &&, &&&&
Angew. Chem. Int. Ed. 2021, 60, 1 – 6
ꢀ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Protein Isotope Labelling
A. Dubey, N. Stoyanov, T. Viennet,
S. Chhabra, S. Elter, J. Borggrꢁfe,
A. Viegas, R. P. Nowak, N. Burdzhiev,
O. Petrov, E. S. Fischer, M. Etzkorn,*
V. Gelev,* H. Arthanari*
&&&& — &&&&
Methyl-based magnetization usage can
strongly benefit investigations of large
protein systems via NMR spectroscopy.
Here, a simple and cost-effective method
to obtain locally deuterated leucine suit-
able for methyl NMR and to quantify its
benefits is presented. The approach sub-
stantially expands the applicability of
methyl labeling in emerging expression
systems including cell-free and eucaryotic
systems.
Local Deuteration Enables NMR
Observation of Methyl Groups in Proteins
from Eukaryotic and Cell-Free Expression
Systems
6
www.angewandte.org
ꢀ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 1 – 6
These are not the final page numbers!