Reversible protein affinity-labelling using bromomaleimide-based reagents

Article abstract of DOI:10.1039/c3ob40239h

Reversible protein biotinylation is readily affected via conjugation with a bromomaleimide-based reagent followed by reductive cleavage. The intermediate biotinylated protein constructs are stable at physiological temperature and pH 8.0. Quantitative reve

Full text of DOI:10.1039/c3ob40239h

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Biomolecular Chemistry
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Reversible protein affinity-labelling using
bromomaleimide-based reagents†
Cite this: Org. Biomol. Chem., 2013, 11,
2408
Ramiz I. Nathani, Vijay Chudasama, Chris P. Ryan, Paul R. Moody, Rachel E. Morgan,
Richard J. Fitzmaurice, Mark E. B. Smith, James R. Baker and Stephen Caddick*
Received 2nd February 2013,
Accepted 26th February 2013
DOI: 10.1039/c3ob40239h
www.rsc.org/obc
Reversible protein biotinylation is readily affected via conjugation new bromomaleimide-based affinity-tagging reagent which
with a bromomaleimide-based reagent followed by reductive allows for the facile and reversible biotinylation of proteins.5c
cleavage. The intermediate biotinylated protein constructs are A buffered solution of a single point mutant (L111C) of the
stable at physiological temperature and pH 8.0. Quantitative SH2 domain of the Grb2 adapter protein was affinity-tagged,
reversibility is elegantly delivered under mild conditions of using and incubated, prior to cleavage in a large excess of beta-
a stoichiometric amount of a bis-thiol, thus providing an approach mercaptoethanol (BME, 100 equivalents, 25 mM). Although we
that will be of general interest in chemical biology and have found this protocol to be useful, we discovered that pro-
proteomics.
longed incubation of the affinity-tagged protein lead to some
unwanted maleimide hydrolysis (ca. 40% hydrolysis at 37 °C
after 4 h at pH 8.0) which rendered the labeling, at least par-
tially, irreversible, resulting in a sub-optimal yield of recovered
protein. Furthermore, the relatively harsh cleavage conditions
are unlikely to be compatible with proteins that contain sen-
sitive disulfide bonds, potentially resulting in unfavourable
protein unfolding, aggregation or disulfide scrambling. This
limitation is particularly important when one considers that
disulfide bonds are often very important for the structural
stability of proteins.6
To facilitate the study of a plethora of complex biological pro-
cesses, robust methods for the selective enrichment of tagged
proteins from complex biological mixtures is of primary
importance. Use of the biotin–avidin interaction, to facilitate
biotin-labeled protein enrichment or immobilisation, is a well-
established technique.1–3 However, classical approaches based
on this method are limited in that in order to ultimately
retrieve the protein of interest the very strong biotin–avidin
interaction (Ka = 1.7 × 1015
M
−1) must be disrupted. This is
often achieved under harsh, denaturing conditions that are
not experimentally useful.1 Strategies have been developed to
try and address this problem. The first approach requires the
design of analogues of biotin which bind with lower affinity to
avidin.4 Although useful, this approach is inherently hampered
as increased ease of elution comes at the price of weaker
initial binding. A second approach is to design affinity probes
which maintain the native biotin–avidin interaction but which
incorporate a cleavable linker.1,2
We have recently reported a new approach to cysteine bio-
conjugation through the use of bromomaleimides and bromo-
pyridazinediones.5 To date, our approach has provided access
to complex bioconjugates in high yields, without prior acti-
vation of reagents. As part of this programme, we reported a
We report herein a novel affinity tag reagent, which yields
bioconjugates that are stable to maleimide hydrolysis over
extended periods of time at physiological temperature and pH
8.0. Furthermore, we describe the subsequent liberation of
native protein from the bioconjugate under mild conditions, a
single equivalent of a dithiol, in both a model system and in
proof of concept biotin–streptavidin pull-down experiments.
The nature of the N-substituent of a maleimide is known to
effect the rate of hydrolysis to the corresponding maleamic
acid.7 Indeed, we have recently described the exploitation of
this effect for the irreversible labelling of proteins with hydro-
lytically unstable maleimides bearing electron withdrawing
groups on nitrogen, such as N-phenylmaleimides.5c Given that
electron withdrawing substituents on the maleimide nitrogen
promote hydrolysis,5c,7 we envisaged that incorporation of an
electron donating substituent on the maleimide nitrogen
would suppress such an effect.
Department of Chemistry, University College London, 20 Gordon Street, London,
WC1H OAJ, UK. E-mail: VPEnterprise@ucl.ac.uk; Fax: +44 (0)20 7679 7463;
Thus, n-butyl and methoxy substituents (inductively and
mesomerically electron donating groups, respectively), were
incorporated into bromomaleimides to form compounds 1
and 2 for model hydrolysis studies. We also synthesised
Tel: +44(0)20 3108 5071
†Electronic supplementary information (ESI) available: 1H and 13C spectra for all
new and known compounds, and ES-MS spectra for all reactions with proteins
described herein. See DOI: 10.1039/c3ob40239h
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ethylglycol methyl ether bromomaleimide 3 to investigate diamine, rather than butyl-1,4-diamine or 4-(aminomethyl)-
whether the hydrolysis of our previously reported PEG-biotin cyclohexanamine, was chosen as the spacer to minimise any
linker was intimately linked to the ethyl glycol ether function- interference between the protein and the biotin–streptavidin
ality. Cyclohexylmethyl bromomaleimide 4 was also syn- interaction. Bromomaleimide 6 was synthesized by coupling
thesised to assess whether a N-cyclohexylmethyl group, as in mono Boc protected decane-1,10-diamine with activated biotin,
the case reported by Hermanson,8 would reduce hydrolysis in followed by TFA deprotection of the Boc group and reaction
this system. Bromomaleimides 1–4 were readily synthesised by with bromomaleic anhydride in acetic acid (see ESI† for details).
refluxing bromomaleic anhydride with the corresponding
The reactivity of biotinylated bromomaleimide 6 with Grb2
amine in acetic acid for 3 h. The crude reaction was purified SH2 (L111C) 5 was tested and, as anticipated, reaction at 0 °C
by column chromatography to yield pure bromomaleimides for 1 h resulted in the formation of bioconjugate 8 in quanti-
1–4 with yields ranging from 54% to 72% (see ESI† for details). tative conversion (Scheme 1). In order to evaluate whether the
Single point mutant (L111C) of the SH2 domain of Grb2 efficiency of bioconjugation with bromomaleimide 6 was
adaptor protein, 5, which otherwise does not contain cysteine specific to Grb2 SH2 (L111C) 5, reaction was also carried out
residues, was chosen as a model protein. This protein was with GFP (S147C) 7.5b Use of a fluorescent protein would also
treated with one equivalent of bromomaleimide reagent for provide a parameter by which to quantify the release of protein
1 h at 0 °C (these bioconjugation reactions may also be per- in our reversible biotinylation methodology (vide infra). Gratify-
formed at room temperature or 37 °C). Analysis of the crude ingly, bioconjugation with GFP (S147C) 7 also proceeded
reaction mixtures by LCMS indicated that, despite varying the efficiently, forming bioconjugate 9 in quantitative conversion
N-substituent, reactions with bromomaleimides 1–4 proceeded (Scheme 1). Conjugates 8 and 9 were then tested for stability
with quantitative conversion. The hydrolytic stability of the by heating them at 37 °C, pH 8.0 for 4 h. Most pleasingly, as
conjugates was then assessed by incubation at 37 °C for 4 h at observed with the n-butyl and cyclohexylmethyl model
pH 8.0 (Table 1). Although the conjugates of bromomaleimides systems, no hydrolysis was observed.‡
2 and 3 showed significant hydrolysis, the conjugates of 1 and
Having established that adducts 8 and 9 are stable to
4 were completely stable. On this basis, it was concluded that a hydrolysis at physiological temperature at pH 8.0 over a pro-
non-functionalised alkyl chain (i.e. an inductively electron longed period of time, and in view of our goal of providing a
donating group) on the maleimide nitrogen should be general protocol for the reversible biotinylation of proteins, we
sufficient to minimise hydrolysis.
In light of the results with bromomaleimides 1 and 4, a bio-
tinylated-bromomaleimide derivative with a non-functionalised
alkyl chain, dodecane-biotin bromomaleimide 6, was syn-
thesized and its hydrolytic stability evaluated. Decane-1,10-
Table 1 Reaction of Grb2 SH2 (L111C) 5 with bromomaleimides 1–4 and sta-
bility of the formed conjugates at 37 °C for 4 h at pH 8.0.a
Hydrolysis of
Conversion of Grb2 conjugate at 37 °C
Maleimide R group
SH2 (L111C) 5
for 4 h
1
2
>99%
>99%
<1%
40%
3
4
>99%
>99%
45%
<1%
Scheme 1 Reaction of dodecane-biotin bromomaleimide 6 with Grb2 SH2
(L111C) 5 and GFP (S147C) 7, and release of the free proteins using BME (100×)
or EDT (1×).
a Detailed experimental details are given in the ESI.
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turned our attention to developing a mild set of cleavage con- dithiane 11 and bicyclic bisthioether 12 in a 65 : 35 ratio after
ditions for the liberation of a protein of interest from the affinity 90 min at 37 °C (Scheme 2(c)). However, after prolonged incu-
label. In our previous report, we described that Grb2 SH2 bation at 37 °C, bicyclic bisthioether 12 was completely con-
(L111C) maleimide conjugates react with a single equivalent of verted to the thermodynamic product, dithiane adduct 11.
thiol to form bisthioethers.5a,c In an environment of an excess of
The EDT cleavage strategy was evaluated by incubation of
thiol, however, these bioconjugate thioethers are unstable, indi- biotin–bromomaleimide conjugates 8 and 9 with a single
cating that the process is under equilibrium (Scheme 2(a)). To equivalent of EDT for 4 h at 37 °C, pH 8.0. Analysis of the
date, this reversibility has been exploited by adding a large crude reaction mixtures by LCMS revealed complete regene-
excess of thiol (i.e. 100× of BME or Glutathione) to facilitate the ration of free unmodified proteins with no evidence for the sig-
release of free protein. However, a major disadvantage of this nificant formation of side-products (Scheme 1).‡ As a control,
technique is that it requires a large excess of thiol, which, as dis- our previously reported cleavage conditions were also applied
cussed above, could limit the applicability of this approach.
to biotinylated conjugates 8 and 9.5c Although reaction of 9
To improve upon our capture and release methodology, it with excess BME (100 equivalents) for 4 h at 37 °C, pH 8.0
was essential to facilitate the release of free protein without yielded GFP (S147C) 7 in quantitative conversion, sub-optimal
using an excess of thiol (Scheme 2(a)). We rationalised that yield (ca. 90%) was observed for release of Grb2 SH2 (L111C) 5
dithiosuccinimides are unlikely to be stable because of the low from adduct 8 as a result of the persistence of succinimide
pKa of the succinimide proton – and that this is key for the formed from mono-addition of BME to 8; thus highlighting a
reversibility of this system. We envisaged that a tether-type further advantage of our novel dithiol cleavage strategy.
approach using bidentate thiol nucleophiles which could form
Having identified an affinity tag which is stable to hydro-
bi- or spirocyclic adducts by trapping the reactive dithio- lysis at 37 °C for several hours and a reagent which enables
succinimides into a stable, unreactive adduct, would liberate clean release of protein with only a single equivalent of redu-
free protein without the need for an excess of thiol. Thus, we cing thiol (EDT), we sought to evaluate these optimised proto-
embarked upon a model study using thiomaleimide 10. Pleas- cols in proof of concept pull down experiments (Scheme 3).
ingly, reaction of 10 with a single equivalent of 1,2-ethane-
This also provided an opportunity to compare our pre-
dithiol (EDT) afforded spirocyclic dithiane 11 in 95% yield viously reported PEG maleimide reagent5c with our new
after 24 h (Scheme 2(b)). Further insight into the mechanism decane bromomaleimide reagent 6. Thus, GFP (S147C) 7 was
of cleavage of thiomaleimide with ethanedithiol was provided modified with PEG and decane maleimide reagents at 0 °C for
by treatment of N-methylbromomaleimide with EDT, which 1 h, forming bioconjugates 13 and 9 in quantitative conver-
revealed the formation of two major products, spirocyclic sion. Bioconjugates 13 and 9 were then incubated directly with
streptavidin beads at 37 °C for 4 h (Scheme 3(a)). In each case,
after the incubation period, unbound GFP-bioconjugate
was removed by washing the beads with PBS buffer solution
(3 × 10-fold dilution). Incubation of the washed GFP (S147C)
loaded streptavidin beads (14 and 15) with EDT at 37 °C for
4 h resulted in release of GFP (S147C) 7 (Scheme 3(a)); this was
determined by fluorescence analysis (vide supra) of the super-
natant (Scheme 3(b)). Gratifyingly, a significantly increased
level of release from the decane conjugate 9 was observed,
likely due to superior hydrolytic stability. This clearly demon-
strates a substantial advantage of 6 over our previously
reported PEG linked affinity tag reagent and the applicability
of our mild cleavage conditions to reversible protein affinity
labelling using streptavidin beads; thus highlighting the
potential of our optimised cleavable tag/EDT strategy.
Conclusions
The continuing study of the interaction of proteins with other
biomolecules is vital in furthering our understanding of
human physiology. The development of proteomic techno-
logies will facilitate the discovery of new diagnostics and drugs
that will underpin 21st century medicine.9,10 Key to this is the
development of novel conjugation technologies that allow for
Scheme 2 (a) Reaction of Grb2 SH2 (L111C) maleimide bioconjugate with
the enrichment, manipulation and analysis of proteins using
an excess of thiol; (b) Reaction of cysteine–maleimide conjugate with EDT;
(c) Reaction of N-methylbromomaleimide with EDT, and rearrangement of 12 to 11. mild conditions that are compatible with maintaining native
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Scheme 3 (a) Binding of 13 and 9 to Streptavidin beads, and release of GFP (S147C) 7 with EDT (1×); (b) Fluorescence spectra of GFP (S147C) 7 released from
bead washes after incubation of 14 and 15 with EDT (1×) at 37 °C for 4 h.
protein structure and function.11 We have described an
elegant approach that enables the reversible affinity-labelling
of proteins. We envisage that this technology will have appli-
cations in a variety of affinity applications but particularly in
proteomic applications where the capture, release and analysis
of native proteins and their complexes is highly desirable.
The authors are grateful to Dr D. Papaioannou and
Prof. G. Waksman for donation of the Grb2 SH2 plasmid. We
also gratefully acknowledge the EPSRC, BBSRC, Wellcome
Trust, UCL, UCLB and GSK for support of our programme.
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Notes and references
‡Analogous results were observed for 4 h at 37 °C, pH 7.4.
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