Enzyme stabilization via computationally guided protein stapling

Article abstract of DOI:10.1073/pnas.1708907114

Thermostabilization represents a critical and often obligatory step toward enhancing the robustness of enzymes for organic synthesis and other applications. While directed evolution methods have provided valuable tools for this purpose, these protocols are laborious and time-consuming and typically require the accumulation of several mutations, potentially at the expense of catalytic function. Here, we report a minimally invasive strategy for enzyme stabilization that relies on the installation of genetically encoded, nonreducible covalent staples in a target protein scaffold using computational design. This methodology enables the rapid development of myoglobin-based cyclopropanation biocatalysts featuring dramatically enhanced thermostability (ΔT_m = +18.0°C and ΔT₅₀ = +16.0°C) as well as increased stability against chemical denaturation [ΔC_m (GndHCl) = 0.53 M], without altering their catalytic efficiency and stereoselectivity properties. In addition, the stabilized variants offer superior performance and selectivity compared with the parent enzyme in the presence of a high concentration of organic cosolvents, enabling the more efficient cyclopropanation of a water-insoluble substrate. This work introduces and validates an approach for protein stabilization which should be applicable to a variety of other proteins and enzymes.

Full text of DOI:10.1073/pnas.1708907114

Enzyme stabilization via computationally guided
protein stapling
Eric J. Moorea, Dmitri Zorineb,c, William A. Hansenb,c,d, Sagar D. Khareb,c,d,1, and Rudi Fasana,1
aDepartment of Chemistry, University of Rochester, Rochester, NY 14627; bDepartment of Chemistry and Chemical Biology, Rutgers University, Piscataway,
NJ 08854; cCenter for Integrative Proteomics Research, Rutgers University, Piscataway, NJ 08854; and dQuantitative Biomedicine Graduate Program, Rutgers
University, Piscataway, NJ 08854
Edited by David A. Baker, University of Washington, Seattle, WA, and approved October 6, 2017 (received for review May 29, 2017)
stabilization (23). Unfortunately, the redox instability of disulfide
bridges make this approach unsuitable for many proteins and en-
zymes that are meant to function in reducing environments, such as
the intracellular milieu and/or in the presence of reductants (24–26).
Moreover, the reversibility and homotypic nature of disulfide bridges
can complicate folding of variants with multiple cross-links due to
disulfide scrambling (27). Thus, the development of computational
strategies for protein stabilization by means of chemically stable
covalent bonds remains an unmet challenge.
Here, we report the development of Rosetta-guided protein sta-
pling (R-GPS), a method for enzyme/protein stabilization based on
the computational design of genetically encodable, covalent “staples”
in a protein of interest. This method utilizes the Rosetta enzyme
design framework to identify optimal sites for intramolecular cross-
linking of the protein scaffold via redox-stable and irreversible thio-
ether bonds generated upon the reaction between a cysteine and a
genetically encoded noncanonical amino acid containing a cysteine-
reactive side-chain group (O-2-bromoethyl tyrosine, or O2beY) (28).
The R-GPS approach was implemented and validated in the context
of a myoglobin (Mb)-based cyclopropanation biocatalyst, resulting in
the development of “cyclopropanases” with significantly increased
stability against thermal and chemical denaturation as well as im-
proved catalytic performance under harsh reaction conditions.
Thermostabilization represents a critical and often obligatory step
toward enhancing the robustness of enzymes for organic synthesis
and other applications. While directed evolution methods have
provided valuable tools for this purpose, these protocols are laborious
and time-consuming and typically require the accumulation of several
mutations, potentially at the expense of catalytic function. Here, we
report a minimally invasive strategy for enzyme stabilization that
relies on the installation of genetically encoded, nonreducible covalent
staples in a target protein scaffold using computational design. This
methodology enables the rapid development of myoglobin-based
cyclopropanation biocatalysts featuring dramatically enhanced ther-
mostability (ΔTm = +18.0 °C and ΔT50 = +16.0 °C) as well as increased
stability against chemical denaturation [ΔCm (GndHCl) = 0.53 M], with-
out altering their catalytic efficiency and stereoselectivity properties. In
addition, the stabilized variants offer superior performance and selec-
tivity compared with the parent enzyme in the presence of a high
concentration of organic cosolvents, enabling the more efficient cyclo-
propanation of a water-insoluble substrate. This work introduces and
validates an approach for protein stabilization which should be appli-
cable to a variety of other proteins and enzymes.
protein thermostabilization computational protein design myoglobin
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noncanonical amino acids Rosetta macromolecular modeling
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Results
Choice of Target Enzyme and Protein Stapling Chemistry. A recently
reported Mb-derived cyclopropanation biocatalyst, Mb(H64V,
V68A) (29), was selected as the test bed for the development of
R-GPS. Compared with wild-type Mb, Mb(H64V,V68A) bears
nzymes play a major role in biotechnology and constitute at-
tractive catalysts for the implementation of efficient, selective,
E
and sustainable processes for the production of pharmaceuticals, fine
chemicals, and biofuels (1, 2). The marginal stability of proteins,
however, poses a fundamental challenge for the exploitation of en-
zymes for practical-scale syntheses and chemical manufacturing,
which often require harsh reaction conditions such as elevated
temperature and exposure to organic solvents (1). Due to these
limitations, protein stabilization against thermal and chemical de-
naturation has represented a long-standing goal in enzyme design
and engineering. In addition to higher robustness to operational
conditions, increasing the thermostability of a protein can enhance
its evolvability (i.e., its tolerance to mutagenesis for the acquisition
of new or improved functions) (3).
Well-known experimental strategies for enzyme stabilization in-
clude directed evolution and consensus mutagenesis (4–6). While
effective in many instances, these procedures remain laborious and
time-consuming, typically requiring several rounds of mutagenesis
and screening to obtain variants with significantly increased stability
(i.e., ΔTm > 5–10 °C) (7). Notable examples of enzyme stabilization
have been reported using computational design (8). In this area,
methods have focused on optimizing native state interactions, for
example through improving core packing (9–11), fragment contacts
(12), combined structure- and phylogeny-guided energy optimization
(13, 14), surface charge optimization (15, 16), and rigidification (17,
18). A complementary but comparatively less explored approach has
focused on decreasing the configurational entropy of the unfolded
state ensemble (19), primarily via the installation of nonnative
disulfide linkages (20). Indeed, the introduction of nonnative disul-
fide bridges (20) has proven useful for increasing the thermostability
of enzymes, although stability–activity trade-offs were also observed
(21, 22). Disulfide cross-linking can additionally provide kinetic
Significance
The marginal stability of most natural proteins presents a chal-
lenge for the exploitation of natural and engineered enzymes in
biotechnology and industrial biocatalysis. Protein stabilization
can be time- and labor-intensive and often involves extensive
amino acid substitutions, which may impair the activity and/or
selectivity of the enzyme. Here, we describe a computational
design method for enzyme stabilization that uses structure-based
modeling to introduce covalent ‟staples” in a protein scaffold via
a genetically encodable noncanonical amino acid. This method
was applied to obtain stapled variants of a stereoselective
cyclopropanation biocatalyst featuring greatly increased ther-
mostability and robustness to high concentrations of organic
cosolvents. This minimally invasive strategy for protein stabili-
zation should be applicable to a variety of enzymes and proteins.
Author contributions: E.J.M., S.D.K., and R.F. designed research; E.J.M., D.Z., and W.A.H.
performed research; E.J.M., D.Z., W.A.H., S.D.K., and R.F. analyzed data; and S.D.K. and
R.F. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Published under the PNAS license.
1To whom correspondence may be addressed. Email: sagar.khare@rutgers.edu or rfasan@
ur.rochester.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1708907114/-/DCSupplemental.
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two active site mutations that confer enhanced catalytic activity as positions of the cross-linking residues were obtained (Fig. 1B),
well as high diastereo- and enantioselectivity (>90–99% de and ee)
in the cyclopropanation of styrenes and vinylarenes with ethyl
α-diazoacetate as the carbene donor (29). On one hand, stabilization
of Mb(H64V,V68A) was desirable for enhancing its robustness for
synthetic applications, which include the synthesis of cyclopropane-
containing drugs and other carbene transfer reactions (30, 31). On
the other hand, the high stereoselectivity of Mb(H64V,V68A) was
envisioned to provide a sensitive probe for evaluating the impact of
the computationally designed covalent staples on subtle functional
properties such as stereoinduction. Indeed, previous studies indicated
that the stereoselectivity of Mb-based cyclopropanation catalysts is
highly sensitive to small structural variations in their active sites (30).
The thioether bond-forming reaction between cysteine and the
genetically encodable O-2-bromoethyl-tyrosine (O2beY) (28, 32) was
selected as the protein cross-linking strategy (Fig. 1A). Based on these
previous studies, the O2beY/Cys reaction was anticipated to offer
several promising features for the purpose of protein stapling, namely
(i) the ability to mediate the spontaneous formation of the thioether
cross-link at the posttranslational level, (ii) high chemoselectivity of
O2beY toward cysteine-mediated alkylation in the presence of other
nucleophilic residues (e.g., Lys and His), and (iii) “spatially con-
trolled” reactivity, whereby the O2beY and Cys undergo the nucle-
ophilic substitution reaction only when located in close proximity.
RosettaDesign (38) was performed to identify additional sequence
changes to minimize steric clashes with the modeled staple. To assess
the method, we selected for experimental characterization a diverse
set of nine designs based on (i) the solvent accessibility of the staple
in the protein structure, (ii) total number of amino acid substitutions,
and (iii) Rosetta energies (Table 1). These designs were termed
sMb1 through sMb9 and had differences in scores relative to the
parent protein Mb(H64V,V68A) ranging from −6.7 (sMb1) to +16.6
(sMb9) Rosetta energy units (Reu), and intervening segment lengths
ranging from 73 (sMb2) to 121 (sMb5) residues. Seven of designed
variants feature a surface exposed thioether linkage, whereas for the
remaining two (sMb6 and sMb8) the cross-link is buried (Fig. 1C).
The number of designed amino acid substitutions, excluding the Cys
and O2beY residues, ranged from zero (sMb2 and sMb5) to two
(sMb3, sMb6, sMb7, sMb8, and sMb9).
Expression and Characterization of the sMb Designs. All of the designed
Mb(H64V,V68A)-derived variants could be expressed in soluble
form from Escherichia coli cells containing an orthogonal aminoacyl-
tRNA synthetase/tRNACUA pair for O2beY incorporation (28) via
amber stop codon suppression (39). In addition, the constructs were
all able to bind heme and fold properly, as evinced from the char-
acteristic Soret band (∼410 nm) in the corresponding UV-visible
spectra (SI Appendix, Fig. S4).
Computational Design of Stapled Mb(H64V,V68A) Variants Using
R-GPS. As a first design principle we reasoned that cross-linking res-
idues that are distal in the primary sequence but proximal in space in
the folded state would maximize thermostabilization by decreasing
the chain entropic cost of folding (33–36). As a second criterion we
envisioned the need to identify compatible backbone positions for
placing the haloalkane (O2beY) and thiol (Cys) side-chain groups so
that the cross-link is accommodated in a strain-free configuration
while making energetically favorable interactions with its surround-
ing residues (33). Following these guiding principles, the N-terminal
helices A and B and the C-terminal helices G and H of the target
enzyme, Mb(H64V,V68A), were chosen as the target regions for the
installation of the thioether bridges (Fig. 1B), as cross-links between
these structural elements would bear the highest contact order. The
RosettaMatch algorithm (37) was then applied to identify positions
where the O2beY and Cys residues can be accommodated for for-
mation of the thioether staple. Once geometrically feasible backbone
SDS/PAGE analysis revealed an increase in electrophoretic mo-
bility for five out of the nine protein constructs, namely sMb2, sMb3,
sMb4, sMb5, and sMb7, compared with Mb(H64V,V68A) (Fig. 2A
and SI Appendix, Fig. S5). This behavior is indicative of a more
compact structure of the protein under denaturing and reducing
conditions (SDS + DTT), which is consistent with the presence of the
nonreducible thioether (O2beY/Cys) cross-link. These conclusions
were corroborated by MALDI-TOF MS (Fig. 2B and SI Appendix,
Fig. S6), which showed a single signal corresponding to expected
mass of these proteins minus 82 Da, deriving from the loss of HBr as
a result of the O2beY/Cys cross-linking reaction. For sMb4, two m/z
signals consistent with the stapled and unstapled form of the protein
indicated the O2beY/cysteine cross-linking reaction has occurred
only partially. For the remaining designs, the only observable species
in the MS spectra corresponded to the O2beY-containing protein
lacking the thioether cross-link. Notably, the integrity of the O2beY
residue in these unstapled constructs demonstrates the lack of un-
desirable reactivity toward hydrolysis or abundant thiol-containing
intracellular metabolites such as glutathione. These results also
show how placing the O2beY/Cys pair in close spatial proximity in
the context of a folded protein is a necessary but not sufficient
condition for productive stapling. The positional dependence of
the stapling reaction could be rationalized based on analysis of the
rotamers of Cys and O2beY residues (Fig. 1B) in models of the
sMb constructs in their unstapled forms. In particular, a good
agreement was found between the experimental results and the
accessibility of near-attack conformations (NACs) compatible with
a bimolecular nucleophilic substitution reaction between the thiol
group of the cysteine and the alkyl bromide group in O2beY (see
SI Appendix for further discussion).
A
B
Thermostability of the sMb Designs. The thermostability of the Mb
variants was examined by measuring their melting temperatures
(Tm) using CD (SI Appendix, Fig. S7). Mb(H64,V68A) was found to
exhibit an apparent Tm of 66.0 °C (Fig. 2C), which is about 14 °C
lower than that of wild-type Mb. Notably, all of the sMb constructs
containing the thioether staple showed an increase in thermosta-
bility compared with the parent protein, with ΔTm values ranging
from 3.9 °C to 10 °C (Table 1). The largest thermostabilization
effect was observed for sMb5 (ΔTm: +10.0 °C), followed by sMb2
(ΔTm: +7.2 °C). In contrast, the sMb variants lacking the cross-link
show comparable (sMb1 and sMb9) or lower Tm values (sMb4,
sMb6, and sMb8) compared with Mb(H64V,V68A) (ΔTm: +1.5 to
–16.8 °C; Table 1). To further examine the impact of the thioether
staple, analogs of the two most stable sMb variants, sMb2 and sMb5,
were prepared by substituting O2beY for O-propargyl-tyrosine
C
Fig. 1. Computational design approach. (A) Stapling reaction between the
noncanonical amino acid O-2-bromoethyl tyrosine and cysteine resulting in a
chemically stable thioether bond. (B) Structure of Mb (Protein Data Bank ID
code 1JP9) highlighting helices A–H (Left). The active-site heme group and
metal-coordinating histidine residue are shown as sticks. Conformational
ensemble of the modeled thioether linkage used to find compatible loca-
tions for stapling (Right). (C) Models showing locations of covalent staples
between A–H and B–G helices in designs sMb1 through sMb9.
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Moore et al.

Table 1. Computational and experimental values for Mb(H64V,V64A) and its variants
ΔGcomp
Reu
,
Location
staple*
Attack
Staple
(MS)
Tm °C
T50, °C
Cm (M)
(ΔCm)
‡
§
{
Variant
Mutations
angle, deg†
(ΔTm
)
(ΔT50
)
Mb(H64V,V68A)
—
0
None
B–G
n.a.
102
142
126
98
n.a.
No
66.0 1.0 62.3 0.6 1.55 0.02
(0.0)
67.5 1.3 59.1 0.6
(+1.5) (−3.2)
73.2 0.2 63.8 0.3
(0.0)
(0.0)
n.d.
sMb1
sMb2
sMb3
sMb4
sMb5
sMb6
sMb7
sMb8
sMb9
sMb10
sMb11
sMb12
sMb13
R31(O2beY), S35K, E109C
−6.73
−5.05
−2.66
−0.76
−0.68
+1.70
+2.34
+3.05
+16.6
n.a.
H36(O2beY), E109C
B/C–G
Yes
Yes
n.d.
n.d.
n.d.
(+7.2)
72.2 1.4
(+6.2)
(+1.5)
L9R, H12C, D122T, A127(O2beY)
D27(O2beY), H113C, V114G
G5(O2beY), D126C
A–H
60
3
(−2.3)
B–G
Yes
(partial)
Yes
56.1 1.1
50
2
(−9.9)
(−12.3)
A–H
169
132
156
108
112
n.a.
n.a.
n.a.
n.a.
76.0 2.0 71.5 0.9 1.79 0.02
(+10.0)
(+9.2)
(+0.24)
G25C, I28A, L69A, I111(O2beY)
D20S, G23C, D27A, R118(O2beY)
H24(O2beY), I28S, L69S, I111C
K16C, H119A, G121(O2beY), D122S
G5(O2beY), H36(O2beY), E109C, D126C
B–G (core)
B-G
No
Yes
No
No
53.0 1.0 51.2 0.3
n.d.
(−13.0)
69.9 1.4
(+3.9)
(−11.1)
54
(−8.3)
1
n.d.
n.d.
n.d.
B–G (core)
A-G/H
49.2 1.0 43.3 0.6
(−16.8)
64.3 1.3
(−1.7)
82.8 0.8
(+16.8)
n.d.
(−19.0)
52
(−10.3)
73
(+10.7)
61
2
B/C-G + A–H
B/C–G + A–H
B/C–G + A–H
B/C–G + A–H
Yes
(double)
Yes
(double)
n.d.
2
2.01 0.05
(+0.46)
n.d.
G5(O2beY), H36(O2beY), F106A,
E109C, D126C
G5(O2beY), H36(O2beY), E109C,
D126C, G129E
G5(O2beY), H36(O2beY), E109C,
H113E, D126C
n.a.
1
(−1.3)
66.6 0.8
(+4.3)
n.a.
n.d.
n.d.
n.a.
Yes
(double)
84.0 0.2 78.3 0.8 2.08 0.03
(+18.0) (+16.0) (+0.53)
deg, degrees; n.a., not applicable; n.d., not determined.
*Corresponding to alpha helices A through H as labeled in Fig. 1.
†Angle for nucleophilic attack of Cys thiol group to O2beY side-chain alkyl bromide group (θattack) as described in SI Appendix, Fig. S1A.
‡Apparent melting temperatures as determined via circular dichroism (SI Appendix, Fig. S7).
§Half-maximal denaturation temperature of holoprotein (Soret band) after 10-min incubation.
{Midpoint guanidinium chloride concentration from chemical denaturation curves measured by CD.
(OpgY) (40). OpgY is an isostere of O2beY but is unable to react
the covalent staples from the two most promising variants, namely
with cysteine to form the thioether cross-link, as confirmed by SDS/ sMb2 and sMb5, resulting in the sMb10 design. Upon production
PAGE (Fig. 2A) and MALDI-TOF MS analyses (SI Appendix, Fig. in E. coli, MS analysis confirmed the successful formation of both
S6). The resulting OpgY-containing variants, sMb2(OpgY) and
sMb5(OpgY), displayed significantly lower Tm values than their
stapled counterparts (ΔTm: –12.2 °C and –11.9 °C, respectively),
confirming the stabilizing effect of the thioether cross-link, as
encoded in computational design.
cross-links within this protein (Fig. 2B). Further characterization of
sMb10 showed a nearly additive effect of the two thioether staples
toward increasing thermostability of the hemoprotein in terms of
both Tm and T50 (ΔTm: +16.8 °C; ΔT50: +10.7 °C; Table 1).
Comparison of the CD spectra corresponding to sMb5, sMb10, and
Mb(H64V,V68A) revealed no major changes in the protein sec-
ondary structure as a result of the presence of a single (sMb5) and
double staple (sMb10) (SI Appendix, Fig. S8).
Evaluation and further stabilization of sMb10 was then pur-
sued via structure-guided point mutagenesis. Investigation of the
F106A substitution in sMb11 confirmed the energetic impor-
tance of the computed π–π stacking interaction between the
O2beY residue in position 36 and the phenyl ring of Phe106 (see
SI Appendix for further discussion). Next, we noticed that in-
stallation of the 36–109 cross-link (from sMb2) and 5–126 cross-
link (from sMb5) replaces in each case a solvent-exposed
negatively charged residue (i.e., Glu109 and Asp126, respectively)
with the less-polar O2beY/Cys staple. Accordingly, two addi-
tional constructs (sMb12 and sMb13) were designed to in-
corporate “compensating” neutral-to-negatively charged amino
acid substitutions (i.e., G129E and H113E, respectively) at a
compatible position in proximity of each staple. Whereas sMb12
showed reduced thermostability (T50) compared with sMb10,
sMb13 was found to exhibit higher Tm (ΔTm: +1.2 °C) as well as
significantly increased stability against temperature-induced
heme loss (ΔT50: +5.3 °C) compared with sMb10. The presence
of the two O2beY/Cys cross-links in sMb13 was confirmed by MS
As a second measure of thermostability, half-maximal de-
naturation temperatures (T50) were determined by monitoring
heme loss (λmax: 408 nm) upon incubation of the hemoproteins
(10 min) at variable temperatures (Fig. 2D). This is a more
stringent assay of thermal stability since it monitors the ability of
the sMb variants to remain associated with the heme cofactor,
which is essential for their activity as cyclopropanation bio-
catalysts. As thermal denaturation of holomyoglobin is irrevers-
ible, an increase in T50 is also a measure of kinetic stabilization
upon cross-linking. In this assay, Mb(H64V,V68A) showed a T50
value of 62.3 °C (Table 1). Whereas sMb3 and sMb7 showed
reduced T50 values compared with Mb(H64V,V64A) (Table 1),
both sMb5 and sMb2 exhibited improved thermal stability
compared with the parent protein (ΔT50: +9.2 °C and +1.5 °C,
respectively; Fig. 2C). These results demonstrate that the ther-
mostabilization effect induced by the O2beY/Cys cross-links in
both sMb2 and sMb5 is not restricted to the protein secondary
structure, as determined by the CD melting curves, but extends
to the heme-bound (“holo”) forms of these proteins.
Design and Characterization of Doubly Stapled Mb Variants. Further
stabilization of Mb(H64V,V68A) was then pursued by combining
Moore et al.
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Fig. 2. Characterization of stapled Mb(H64V,V68A) variants (sMb variants). (A and B) SDS/PAGE gel (A) and MALDI-TOF MS spectrum (B) of Mb(H64V,V68A)
and representative sMb variants. c, cross-linked; c(2×), doubly cross-linked; nc, not cross-linked. Calculated masses: Mb(H64V,V68A): 18,474 Da; sMb2 (c):
18,500 Da; sMb4 (c): 18,472 Da; sMb5 (c): 18,594 Da; sMb10 [c(2×)]: 18,621 Da; sMb13 [c(2×)]: 18,612 Da. (C) Thermal denaturation curves for Mb(H64V,V68A)
and selected stapled variants as measured via CD at 220 nm (Tm determination). (D) Heat-induced inactivation curves (heme loss) for the same proteins as
determined by decrease of Soret band signal (408 nm) after incubation (10 min) at variable temperatures (T50 determination). See SI Appendix, Figs. S5–S7 for
additional data. (E and F) Catalytic activity (E) and enantioselectivity (F) of Mb(H64V,V68A) and stapled variants in styrene cyclopropanation reactions with
EDA in buffer only and in the presence of 30% vol/vol ethanol. Relative activities refer to normalized catalytic turnovers (TON) relative to TON measured with
Mb(H64V,V68A) in buffer only reactions. See SI Appendix, Figs. S10–S12 for related data with other organic cosolvents. Rel Int, relative intensity.
Increased Stability Against Chemical Denaturation. Enhanced ro-
bustness to chemical denaturation and organic solvents is a
highly desirable trait of enzymes to be employed for synthetic
applications (44, 45). To examine this aspect, the most promising
sMb constructs (sMb5, sMb10, and sMb13) were subjected to
denaturation experiments in the presence of guanidinium chlo-
ride (Gnd·HCl) (SI Appendix, Fig. S9). Reflecting the trend
emerging from the thermostability assays, a progressive stability
increase in the presence of the chaotropic agent was observed
going from the singly stapled sMb5 (Cm: 1.79 M) to the doubly
stapled sMb10 and sMb13 (Cm: 2.01 M and 2.08 M, respectively),
compared with the parent enzyme (Cm: 1.55 M).
Next, we investigated the effect of the staples toward improving
the performance of the sMb variants in organic solvents. In the
presence of 30% (vol/vol) ethanol, Mb(H64V,V68A) catalyzes the
cyclopropanation of styrene with reduced activity (48% yield; 2,420
TON; 51% relative activity) and lower enantioselectivity (92% ee)
compared to the same reaction in buffer (Fig. 2 E and F; entry 6 vs.
1, Table 2). Compared with Mb(H64V,V68A), both sMb10 and
sMb13 better tolerate the presence of the organic cosolvent,
affording the cyclopropanation product 3a in higher yields (78–82%;
3,900–4,100 TON; entry 7, Table 2) and with higher diastereo-
(1:400 vs. 1:130 diastereomeric ratio for trans:cis) and enantiose-
lectivity (97% vs. 92% ee) (entry 7 vs. 6, Fig. 2F and Table 2). As
stereoselectivity is highly sensitive to structural perturbation within
the active site of these biocatalysts (30), these results suggest that
stabilization of the Mb scaffold by covalent stapling minimizes the
disruptive effects induced by the organic solvent. A similar trend was
observed upon comparing the activity and selectivity of sMb5,
sMb10, and sMb13 with those of Mb(H64V,V68A) in cyclo-
propanation reactions in the presence of high concentrations
(30% vol/vol) of other organic solvents such as methanol, dime-
thylformamide (DMF), and DMSO (SI Appendix, Figs. S10–S12). In
comparison, tetrahydrofuran (THF) was tolerated equally well by
both the parent protein and the stapled variants (entry 9, Table 2). In
analysis, which also revealed the presence of a small fraction of
singly stapled protein (Fig. 2B). The CD spectra of sMb13 and
sMb10 were found to be superimposable, indicating that the
“charge-compensating” mutation H113E had minimal impact on
the protein structure (SI Appendix, Fig. S8). Thus, as a result of
merely five solvent-exposed mutations in sMb13, the thermosta-
bility (Tm) of the original Mb(H64V,V64A) biocatalyst could be
augmented by +18 °C. These modifications also translated into a
significantly increased thermal stability of the heme-bound form of
the protein (ΔT50: +16 °C).
Catalytic Activity and Selectivity of Stapled Mb(H64V,V68A) Variants
in Cyclopropanation Reactions. In the absence of selective pressure
to maintain function, thermostabilization via protein engineering
is often accompanied by a reduction in catalytic efficiency and/or
selectivity of the target enzyme (41–43). To assess the impact of
the thioether staples on the catalytic properties and stereo-
selectivity of the thermostabilized sMb variants these biocatalysts
were tested in a model cyclopropanation reaction with styrene
(1) and α-diazoacetate (EDA, 2) (Table 2). Under the applied
conditions [10 mM styrene, 20 mM EDA, 2 μM Mb variant
(0.02 mol%)] Mb(H64V,V68A) was found to produce (1S,2S)-ethyl
2-phenylcyclopropanecarboxylate (3a) in 94% yield (4,710 turnovers
or TON) and high diastereomeric (99.4% de) and enantiomeric
excess (99.1% ee) (entry 1, Table 2). Under identical conditions,
both sMb10 and sMb13 were able to catalyze the formation of 3a
with equally high catalytic activity (92–94% yields; 4,710–4,615
TON) and stereoselectivity (>99% de and ee; entries 4–5, Table 2).
These data clearly indicated that thermostabilization induced by the
stapling procedure had no negative impact on the carbene transfer
reactivity of these hemoproteins, nor did it perturb the asymmetric
environment provided by the distal heme pocket, which is
critical for inducing the (1S,2S)-enantioselectivity during the
cyclopropanation reaction (29).
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Table 2. Catalytic activity and selectivity of Mb(H64V,V68A) and its stapled variants for cyclopropanation of styrene
(1a) and pentafluorostyrene (1b) with ethyl 2-diazo-acetate (2) in the absence and in the presence of organic cosolvents
Mb variant
R
R
O
0.02-0.2 mol%
R
R
R
R
O
+
S
S
O
KPi (pH 7.0)
+ solvent
R.T.
N2
O
R
R
R
R
1a (R = H)
1b
3a (R = H)
3b
2
(R = F)
(R = F)
Entry
Variant
Product
Solvent
Yield, %
TON
% de
99.4
99.3
99.1
99.4
99.2
98
99.3
99.8 (99)
97 (99)
99.8 (99.8)
99.9 (99.9)
99.8
% ee
99.1
99.2
99.1
99.3
99.2
92
97
99.6 (99)
95 (94)
99.6 (99.4)
98.5 (98.5)
99.9
1
2
3
4
5
6
7
8*
9*
10*
11*
12
13
14
Mb(H64V,V68A)
sMb2
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3b
3b
3b
—
94
94
94
94
92
4,710 180
4,740 100
4,690 25
4,710 250
4,610 140
2,420 280
4,120 270
4,220 140 (3,480)
4,360 90 (4,660)
3,220 280 (3,000)
1,990 190 (1,020)
—
sMb5
sMb10
sMb13
—
—
—
Mb(H64V,V68A)
sMb10
30% EtOH
30% EtOH
30% DMF
30% THF
30% DMSO
45% DMSO
45% DMSO
45% DMSO
45% DMSO
48
82
sMb10
sMb13
sMb10
sMb10
84 (64)
81 (92)
64 (60)
39 (20)
54
Mb(H64V,V68A)
sMb10
270
395
380
5
5
5
80
77
99.8
99.8
99.9
99.9
sMb13
Reaction conditions for 3a: 2 μM protein (0.02 mol%), 10 mM 1a, 20 mM EDA (2), 10 mM sodium dithionite in 50 mM potassium
phosphate (pH 7), room temperature. Reaction conditions for 3b: 10 μM protein (0.2 mol%), 5 mM 1b, 10 mM EDA (2), 10 mM sodium
dithionite in 50 mM potassium phosphate (pH 7), room temperature.
*Values in brackets correspond to reactions performed in the presence of Mb(H64V,V68A) as the catalyst.
the presence of these organic cosolvents, both sMb10 and sMb13 are
able to produce 3a in high yields (64–88%; 3,200–4,440 TON) and
high stereoselectivity (99.5–99.8% de; 99.2–99.6% ee) (entries 8–10,
Table 2). Furthermore, sMb10 and sMb13 maintain good catalytic
activity (1,080–1,990 TON) as well as excellent stereoselectivity
(>99% de; 97–98.5% ee) even in the presence of a nearly 1:1 mixture
(45% vol/vol) of buffer with DMF or DMSO (entry 11, Table 2
and SI Appendix, Figs. S11 and S12). These results are re-
markable considering that most heme-dependent enzymes are
readily inactivated by low concentrations (>5–10% vol/vol) of
these organic solvents (46, 47). Given their enhanced performance
at high DMSO concentrations, sMb10 and sMb13 could be applied
to afford the cyclopropanation of a water-insoluble substrate, that
is, pentafluorostyrene (1b), with higher efficiency than possible
using the parent Mb variant (Table 2, entries 13 and 14 vs. 12).
Altogether, these results demonstrate the value of R-GPS toward
enhancing the robustness and performance of a biocatalyst in the
presence of chemical denaturants and organic cosolvents.
Two computationally designed staples could be effectively com-
bined in the final designs (sMb10 and sMb13) to achieve additive
effects for stabilization against thermal and chemical denaturation.
While the introduction of computationally designed nonnative
disulfide bridges may result in misfolded variants due to disulfide
scrambling (27), no evidence of incorrect cross-linking was noted for
the sMb variants containing the two thioether staples. We attribute
this result to the heterotypic nature of the O2beY/Cys thioether
linkage, which reduces the number of wrong cross-links that can be
formed compared with the homotypic disulfide bridge. In addition,
the presence of specific geometric requirements for successful for-
mation of the O2beY/Cys cross-link, as revealed by the present
studies (SI Appendix, Fig. S1), likely contributes to further disfavor the
formation of incorrect linkages. At the same time, a noticeable re-
duction in the protein yields was observed upon incorporation of
multiple copies of O2beY via amber stop codon suppression [2 mg/L
culture for sMb10 compared with 10–15 mg/L for sMb2/sMb5
and 30 mg/L for Mb(H64V,V68A)]. While this result is not
surprising given the less-efficient incorporation of noncanonical
amino acids (ncAAs) compared with natural amino acids, various
strategies have recently enabled the expression of proteins con-
taining multiple ncAAs (53–55) and these protocols should increase
the accessibility of proteins containing multiple staples designed
with the present methodology.
R-GPS was able to identify a small set of structural modifications
(i.e., four and five mutations for sMb10 and sMb13, respectively)
capable of conferring these carbene transferase enzymes signifi-
cantly increased stability against thermal and chemical denaturation
without impairing their catalytic activity and subtle functional
properties such as stereoselectivity. The improved stability proper-
ties of these biocatalysts translated into improved performance and
stereoselectivity in reactions conducted in high concentrations of
organic solvents, also enabling the more efficient transformation of
a water-insoluble olefin substrate. Since R-GPS involves a small set
of amino acid substitutions, most of the protein residues remain
available for stabilization based on optimization of interactions
in the native state. Thus, it should be possible to combine R-GPS
with other computational design (13, 14, 17) and/or experimental
Discussion
As demonstrated through the design and characterization of highly
stabilized variants of an Mb-based cyclopropanase, R-GPS enables
the rapid identification of optimal sites within a target protein
scaffold for structural stabilization via “stapling” through nonnative
covalent bridges. In its current implementation, R-GPS utilizes a
bioorthogonal reaction between cysteine and the genetically
encodable noncanonical amino acid O2beY to produce chemically
stable and nonreducible thioether cross-links. Key advantages of
this chemistry include its chemoselectivity, spatially controlled re-
activity, and efficient and spontaneous formation of the cross-link
at the posttranslational level and in the intracellular space. The
latter feature allows for the production of the stapled proteins
inside cells and eliminates the need for protein manipulation
during or after purification (48). Whereas R-GPS has been vali-
dated using the O2beY/Cys cross-linking method, we expect this
approach can be extended to other noncanonical amino acids
and/or other chemistries useful for protein cross-linking (49–52).
Moore et al.
PNAS Early Edition
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Fig. S6), thermal and chemical denaturation curves (SI Appendix, Figs. S7 and
S9), and reaction characterization data (SI Appendix, Figs. S11–S13).
methods (4–6) to achieve additive or synergistic effects in pro-
tein/enzyme stabilization. The minimally invasive approach to
protein stabilization described here is expected to provide a
valuable strategy for greatly enhancing the stability of enzymes
and proteins without impacting their functional properties.
ACKNOWLEDGMENTS. We thank David Vargas for help with the character-
ization of 3b, Daniel Greenfield for technical assistance, and Dr. Jermaine
Jenkins (Structure Biology & Biophysics Facility, University of Rochester) for
assistance with the CD instrumentation. This work was supported by Na-
tional Institute of Health Grant GM098628 (to R.F.) and National Science
Foundation Grants MCB1716623 and MCB1330760 (to S.D.K.). MS instrumen-
tation at the University of Rochester was supported by National Science
Foundation Grants CHE-0840410 and CHE-0946653. E.J.M. acknowledges
support from NIH Graduate Training Grant T32GM118283.
Materials and Methods
See SI Appendix for detailed descriptions of computational methods and
experimental procedures and for additional data: NAC analyses (SI Appen-
dix, Figs. S1 and S2 and Table S1); UV-visible and CD spectra (SI Appendix,
Figs. S4 and S8), protein gels (SI Appendix, Fig. S5), MS spectra (SI Appendix,
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