Genetic Incorporation of Olefin Cross-Metathesis Reaction Tags for Protein Modification

Article abstract of DOI:10.1021/jacs.8b09433

Olefin cross-metathesis (CM) is a viable reaction for the modification of alkene-containing proteins. Although allyl sulfide or selenide side-chain motifs in proteins can critically enhance the rate of CM reactions, no efficient method for their site-selective genetic incorporation into proteins has been reported to date. Here, through the systematic evaluation of olefin-bearing unnatural amino acids for their metabolic incorporation, we have discovered S-allylhomocysteine (Ahc) as a genetically encodable Met analogue that is not only processed by translational cellular machinery but also a privileged CM substrate residue in proteins. In this way, Ahc was used for efficient Met codon reassignment in a Met-auxotrophic strain of E. coli (B834 (DE3)) as well as metabolic labeling of protein in human cells and was reactive toward CM in several representative proteins. This expands the use of CM in the toolkit for "tag-and-modify" functionalization of proteins.

Full text of DOI:10.1021/jacs.8b09433

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Communication
Genetic Incorporation of Olefin Cross-
Metathesis Reaction Tags for Protein Modification
Bhaskar Bhushan, Yuya A. Lin, Martin Bak, Anuchit Phanumartwiwath, Nan Yang, Matthew Bilyard, Tomonari
Tanaka, Kieran Hudson, Lukas Lercher, Monika Stegmann, Shabaz Mohammed, and Benjamin G. Davis
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 16 Oct 2018
Downloaded from http://pubs.acs.org on October 16, 2018
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Genetic Incorporation of Olefin Cross-Metathesis Reaction Tags for
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Bhaskar Bhushan,1 Yuya A. Lin,1† Martin Bak,1 Anuchit Phanumartwiwath,1 Nan Yang,1 Matthew
K. Bilyard,1 Tomonari Tanaka,1 Kieran L. Hudson,1 Lukas Lercher,1 Monika Stegmann,2 Shabaz
Mohammed1,2 and Benjamin G. Davis1,*
1Department of Chemistry, University of Oxford, Chemistry Res. Laboratory, Mansfield Rd, Oxford OX1 3TA UK
2Department of Biochemistry, University of Oxford, OX1 3QU Oxford, UK
Supporting Information Placeholder
reassignment of the Met codon exploits the flexibility of
native methionyl-tRNA synthetase (MetRS) in accepting
these analogs as substrates for tRNA loading.14 Here, by
probing the flexibility of MetRS, we show metabolic labelling
of proteins with an unnatural allyl chalcogen-containing
amino acid (Met analogue S-allylhomocysteine (Ahc)) that is
also metathesis-reactive.
ABSTRACT: Olefin cross-metathesis (CM) is
a viable
reaction for the modification of alkene-containing proteins.
Although allyl sulfide or selenide side-chain motifs in
proteins can critically enhance the rate of CM reactions, no
efficient method for their site-selective genetic incorporation
into proteins has yet been reported. Here, through the
systematic evaluation of olefin-bearing unnatural amino
acids for their metabolic incorporation, we have discovered
S-allylhomocysteine (Ahc) as a genetically encode-able Met
analogue that is both processed by translational cellular
machinery and is also a privileged CM substrate residue in
proteins. In this way, Ahc was used for efficient Met-codon
reassignment in a Met-auxotrophic strain of E. coli (B834
(DE3)), as well as metabolic labeling of protein in human
cells and was reactive towards CM in several representative
proteins. This expands the use of CM in the tool kit for ‘tag-
and-modify’ functionalization of proteins.
The incorporation efficiency of amino acids and their analogs
in vivo is controlled, to a significant extent, by their
activation by the corresponding aminoacyl-tRNA synthetases
(aaRS).4,15,16 Fersht & Dingwall first demonstrated that a Met
analogue, L-ethionine, may be mischarged by the MetRS
without being metabolically edited.17 By contrast, L-
homocysteine, a natural competitor of Met, is transformed to
the corresponding thiolactone to avoid misreading in
translation.18 Side-chain length and heteroatom position can
therefore be critical determinants in ‘metabolic recognition’
of such analogs. Therefore, in our design (Fig. 1) of putative
analogs we explored these aspects: tolerance of MetRS
towards positioning of the side-chain heteroatom required
for CM plus concomitant variation of side-chain length.
Alkenes can be installed into proteins by incorporation of
some unnatural amino acids.1-4 However, although aliphatic
alkene-containing amino acids such as homoallylglycine
(Hag)4 are reactive in both self-metathesis and cross-
metathesis reactions as monomeric, protected amino acids in
organic solvents5 and can be metabolically incorporated into
proteins, they are unreactive in CM reactions in aqueous
media.6 On the other hand, chemically-installed7 unnatural
amino acids such as S-allylcysteine (Sac)6 and Se-
allylselenocysteine (Seac)8 have been demonstrated to be
CM-reactive under aqueous conditions, with the allylic
chalcogen heteroatom S (and even more so Se), providing
crucial coordination to the metal catalyst centre.6,8,9 Schultz
and co-workers have reported the incorporation of O-crotyl
serine in yeast via amber stop-codon suppression, and have
demonstrated its reactivity in on-protein intramolecular ring
closing metathesis.10 However CM with genetically-
incorporated residues has yet to be demonstrated.
Figure 1. Design of -heteroatom Met analogs S-
allylcysteine (Sac, 1), Se-allylselenocysteine (Seac, 2), and
δ-heteroatom analogs S-allylhomocysteine (Ahc, 3) & Se-
allyl homoselenocysteine (Ahs, 4).
An alternative approach to ‘non-sense’ codon reassignment
involves the direct commandeering of ‘sense’ codons for
amino acids such as methionine (Met) and subsequent
reassignment to incorporate unnatural amino acids (e.g. Hag,
norleucine, trifluoromethionine, homopropargylglycine &
azidohomoalanine) as Met analogs.4,11-13 This effective
Two types of substrates were designed & synthesized; those
with a γ-heteroatom (1, 2) and those with a δ-heteroatom (3,
4). Previous work in our group has demonstrated that the
chemical installation of Seac in proteins enhances the rate of
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on-protein CM and allows for an increased breadth of
ligands and an AMBER-derived forcefield (Figure 2 and SI).20
Pleasingly, in their minimized poses all UAA analogs were
found to occupy the Met-binding site of MetRS (Figure 2C).
Consistent with previous studies,16 these revealed similar
major contributors for binding of native Met (e.g.
electrostatic interaction of Met-Nα with the sidechain of
Asp52) to those known. Notably, in all cases (Met and uAAs
1-4) the heteroatom (S / Se) of the sidechain was held by
hydrogen bonding to the backbone amide NH of Leu13 at the
heart of the Met-binding site. However, flexibility in the
hydrophobic ‘end-wall’ of the site (determined by Tyr260),
led to accommodation of a range of sidechain termini (Me or
allyl, Fig. 2): heteroatom-to-Tyr260-O distances were
displace by up to ∼0.5 Å. Moreover, primary binding contact
with Asp52 was lost or distorted. Notably, for Ahc (3)
compensatory interactions were predicted between Ahc-
C(=O)O and both of the backbone amide NHs of Tyr15 &
Pro14 (Fig. 2B). Together these data highlighted both the
critical role of sidechain heteroatom positioning and
encouragingly suggested sufficient flexibility of MetRS (in
accommodating extended termini Me / allyl) as well as
compensatory binding modes in certain cases (i.e. Ahc).
metathesis partners compared to Sac.8,9 Thus, the metabolic
incorporation efficiencies of selenium analogues of 1 & 3 (2 &
4, respectively) were also considered. We reasoned too that
since none of these uAAs possess a free side-chain SH / SeH
(unlike e.g. L-homocysteine), they would not be ‘edited’
through conversion to corresponding chalcogen lactones. 1,2
were prepared according to our prior methods.19 3 & 4 were
synthesized in homochiral form (L-3, L-4) from L-Met and L-
SeMet respectively using a demethylative allylation strategy
that proved direct and efficient (Scheme 1). 3 could also be
readily synthesized from the commercially available DL-
homocysteine thiolactone as a racemate (DL-3, see SI).
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(i) AcCl, MeOH
(ii) Boc2O, NEt3, CH2Cl2
X
X
BocHN
X = S: 94 %
X = Se: 84 %
CO2Me
H2N
X = S, L-Met
X = Se, L-SeMet
CO2H
I
TBAI, K2CO3
(i) TFA, CH2Cl2
(ii) 5M LiOH, THF
(iii) DOWEX(H+)
counts min-1
X
Demethylative
Allylation
X
H2N
CO2H
X = S, Ahc (3): 74%
X = Se, Ahs (4): 58%
BocHN
CO2Me
X = S: 55 %
X = Se: 69 %
Met Sac Seac Ahc Ahs H2O
Scheme 1. Synthesis of S-allylhomocysteine (Ahc, 3) and
Se-allylhomoselenocysteine (Ahs, 4) & relative processing
of Met and analogues 1-4 by MetRS determined through
ATP-PPi exchange; endpoint 20 min (see SI).
First, to evaluate chalcogen-assisted CM reactivity of these
motifs, protected forms of amino acids 1-4 were tested as
small-molecule models (Scheme 2), as previously.6,8
Pleasingly, all proved CM-reactive towards allyl alcohol,
under aq. conditions typical6-9 of prior successful protein CM
reactions in reasonably short reaction times; little or no
homodimerization was observed. Encouraged by these
results, we next explored incorporation of 1-4 into proteins.
Figure 2. Interactions of docked amino acids with MetRS
for A) Met, B) Ahc (see Fig S1 for further poses of other
analogs 1-4). C) Docked Met (blue backbone) adopts a
similar pose in the same Met pocket of MetRS identified
by crystallography (pdb 1PG0, white backbone).
To experimentally test these predictions, E. coli MetRS was
expressed19 and the relative activities for loading of Met, and
analogs 1-4 onto tRNA determined using ATP-PPi exchange
(see Scheme 1 and SI).21 Met, as natural substrate, proved
most effective, as expected. However, from the panel of
olefinic uAAs 1-4, only the S-allyl uAAs 1 & 3 showed
significant activity above background; Ahc 3 showed the
greatest activity (∼6-fold > 1, Scheme 1). Determination and
comparison of the Michaelis-Menten parameters for Met & 3
(Table 1, SI and Fig S2) suggests that both display similar
binding to MetRS (as judged by KM) although 3 has a lower
kcat. Thus, despite the clear differences in structure, the
turnover (as judged by kcat/KM) of Ahc (3) by MetRS was less
than an order-of-magnitude lower than for native substrate
Met (Table 1). Following these in silico & in vitro predictions
Scheme 2. Cross-metathesis on model amino acids Sac 1,
Seac 2, Ahc 3 & Ahs 4.
Molecular mechanics analysis was used to explore structural
constraints in interactions with and processing by MetRS.
Docking (see SI) of 1-4, and their corresponding adenylates,
into the active site of E. coli MetRS (derived from Met-bound
MetRS structure pdb 1PG0) used gradient-descent
minimization of the energy of conformationally randomized
&
validations of putative uAA processing, in vivo
incorporation of each Met analog was investigated. The
nucleosome constituent protein histone H322,23 was tested as
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the first target model protein using the Met-auxotrophic
conversions ranging from 55% (for bulkier 6) to >95% for
allyl alcohol, including for the intact Qβ virus-like particle
bearing 180 reaction sites (Scheme 3), and for SarZ, which
has two reaction sites. SsβG was found to retain its functional
activity following CM reactions (see Table S4).
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2
3
4
5
6
7
8
B834(DE3) E. coli strain. Efficient incorporation (> 95%) was
only observed for Ahc 3, by both LCMS and tryptic digest-
MSMS (Fig. 3 and SI).
Table 1. Michaelis-Menten Parameters for MetRS
S
S
R
catalyst 5
Met
Ahc (3)
5 M GdmCl, 50 mM NaPi
MgCl2, 30% t-BuOH
or PBS (+/- DMSO)
kcat (s-1)
KM (M)
kcat/KM (s-1M-1)
23.2 ± 2.7
3.1 ± 2.7
R
+
R = OH or
6
870 ± 188
0.026 ± 0.006
1030 ± 235
RT/37 oC, pH 7.4-8.0, 3 h
9
0.0030 ± 0.0007
S
R
S
OH
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S
OH
a See also Figure 3 B,C and SI for further details.
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49
120
Se
X
n
Se
H3-Ahc120, >90%a
OH
SsG-Ahc49, 55%b
16
H2N
COOH
Ahs (4)
Np276-Ahc61, > 95%a
+H3N
CO2
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-
S
180
S
X
Feed,
Induce
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S
R
1
S
43
120
S
H2N
COOH
Ahc (3)
4
E. coli
OH
R
H2N
COOH
Sac (1)
Histone
H3-Ahc120
S
S
120
120
Ubq-Ahc1, 84%a
SarZ-Ahc4-Ahc43, 73%b
Q-Ahc16, 82%a
H3
Calc. = 15222
Obs. = 15223
X
Scheme 3. CM on Ahc-incorporated proteins: single-site
Np276, Histone H3, SsβG, Ubq and multi-site
incorporated SarZ and Qβ. Conversion based on aMS with
allyl alcohol, or bfluorescence assay (Figs S3-5) with 6.
Met
Se
Auxotroph
H2N
COOH
Se
Seac (2) X
Finally, direct Ahc incorporation in mammalian cells could
have useful application in the generation of probes in cellulo
and in chemical proteomic strategies (such as noncanonical
amino acid tagging28,29). We next tested and demonstrated
such incorporation using Ahc in human (HEK293T) cells
(Fig. 4 & SI). As a test protein from human cells, not only was
installation of Ahc into the Fc region of IgG determined by
both MS & MSMS (see SI) but the incorporated Ahc was also
found to be reactive, allowing direct CM-labeling of IgG-Fc
through reaction with olefinic-biotin 7.30 7 also proved
effective in reaction with other proteins (e.g. SarZ, SsβG, see
SI). Given the potential used of biotin tags in affinity
proteomic methods,28,29 this both highlights the capability of
Ahc to serve as a general Met analogue across different
translational systems / cell types and also suggests its utility
in the future interrogation of human proteomes.
Figure 3. Successful incorporation of Ahc, 3 into Histone
H3; Sac 1, Seac 2 and Ahs 4 were not incorporated (see SI).
These experiments highlighted 3 as the most suitable for
genetic incorporation. Proteins of varying structure and
function were chosen to test scope. Thus, in addition to H3,
single-site incorporations of Ahc were successfully carried
out in right-handed β–helix pentapeptide repeat protein
Np276,24 and in TIM-barrel β-glycosidase SsβG.25 Excellent
levels (>95%) of multi-site incorporation of Ahc were also
demonstrated: in monomeric α–helix bundle DNA-binding
protein SarZ,26 as well as multimeric bacteriophage coat
protein Qβ.27 For all proteins, site and level of incorporation
was confirmed by LCMS of intact protein and tryptic-MSMS
analysis. Ahc-incorporated proteins retained both secondary
& tertiary structure (as determined by CD, see SI) and
function (retention of glycosidase activity by SsβG, and DNA
binding by SarZ, see SI and Figs S6-8). Typical yields (mg/L)
for Ahc-incorporated proteins were: SarZ ~0.5 (in LBQB),
~2.9 (LB); SsG ~4.5 (LB); IgG-Fc ~1 (DMEM); H3 ~2.4 (LB);
Np276 ~1.1 (LB).
In conclusion, our results demonstrate that the previously
unexplored amino acid S-allyl homocysteine (Ahc) is an
effective Met surrogate that is incorporated into proteins not
only by the translational apparatus of Met-auxotrophic E. coli
(with good efficiency >95%) thereby allowing genetic control
of olefin cross metathesis in proteins using sense (Met)
codon reassignment but also even into human cells. Such
incorporation could be potentially be improved further by
use of MetRS variants.3 It should be noted that there remain
some limitations of the CM method; we observed here, for
example, that in one case the use of tBuOH for improving
the solubility of catalyst 5 led to the loss of activity of the
highly solvent-sensitive protein SarZ (see SI). In addition,
whilst reactions using 5 typically proceed with strong E-
selectivity,9 some E/Z-heterogeneity may also exist in
conjugates. Thus, with the continuing development of water-
soluble31,32 or Z-selective33 catalysts for cross-metathesis and
the genetically-controlled capabilities disclosed here, there is
now good motivation for the development of effective, in vivo
With Ahc-containing proteins in hand, their reactivity
towards CM was tested; allyl alcohol or fluorescein-olefin 6
were used as model metathesis partners. Evaluations of
reaction conditions and optimizations were conducted
through systematic variation of various parameters (SI Tables
S1 and S2) and highlighted: the utility of tBuOH or DMSO as
co-solvents (where needed) lacking alpha-protons; potential
utility of PEG-500 as co-solvent; strong benefit of MgCl2 as
an additive;6 phosphate-based buffers and mild denaturant
(e.g. through guanidinium) to increase solubility and
stability; and Ru scavenging during work-up for LCMS
monitoring. Following this process and, consistent with the
observed reactivity for Ahc in model amino acids, CM was
observed on all tested Ahc-containing proteins with
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Ahc-enabled CM in cellular systems, thereby enabling access
to a broad range of metal-mediated biological applications.
Florian Seebeck & Ritu Raj for discussions; and James
Wickens, Sonia de Munari for technical assistance.
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S
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32
H2N
COOH
Ahc (3)
IgG-Fc-Ahc32
human cells
O
O
NH
H
HEK293T@IgG-Fc
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catalyst 5
MgCl2, 30% t-BuOH
5 M GdmCl, 50 mM
NaPi pH 8.0,
HN
H
NH
S
7
RT, 3 h
S
NH
32
O
avidin
S
H
HN
pulldown
H
NH
digest
O
biotin-labeled
IgG-Fc
LC-MS/MS
Figure 4. Use of Ahc in human cells and CM-labelling of
Ahc-incorporated IgG-Fc with olefin-biotin 7. Insets show
MS/MS of isolated IgG-Fc29-36 peptide DTL-M*-IMSR
containing analog M*=3 and anti-biotin Western (non-
reducing, size markers, 20, 30, 40, 50, 60, 80, 110 kDa;
arrows show IgG-Fc monomer and dimer) demonstrating
incorporation and CM reaction with 7, respectively.
Dotted box shows potential affinity proteomic workflow.
ASSOCIATED CONTENT
Supporting Information
Full procedures and protein ESI-MS. Material is available free
of charge via the Internet at http://pubs.acs.org.
Corresponding Author
ben.davis@chem.ox.ac.uk
Present Addresses
†Department of Chemistry, National Sun Yat-sen University,
No.
70,
Lienhai
Rd,
Kaohsiung,
Taiwan
Department of Biobased Materials Science, Kyoto Institute
of Technology, Matsugasaki, Sakyo-ku, Kyoto, Japan.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We thank the Rhodes Trust (BB), EPA Cephalosporin
Scholarship (BB), EPSRC (YAL, LL) & CRUK (LL); Royal
Society Wolfson Research Merit Award (BGD) for funding;
(19) Shimizu, Y.; Inoue, A.; Tomari, Y.; Suzuki, T.; Yokogawa, T.;
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S-allylhomocysteine (Ahc)
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E. coli
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Hoveyda-Grubbs II
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HO2C
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Met
Auxotroph
Protein
Expression
Cross-Metathesis
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Ahc-incorporated
Protein
HEK
cell
Metathesis-Reactive
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