Synthesis of Multi-Protein Complexes through Charge-Directed Sequential Activation of Tyrosine Residues

Article abstract of DOI:10.1021/jacs.1c03079

Site-selective protein-protein coupling has long been a goal of chemical biology research. In recent years, that goal has been realized to varying degrees through a number of techniques, including the use of tyrosinase-based coupling strategies. Early publications utilizing tyrosinase from Agaricus bisporus(abTYR) showed the potential to convert tyrosine residues into ortho-quinone functional groups, but this enzyme is challenging to produce recombinantly and suffers from some limitations in substrate scope. Initial screens of several tyrosinase candidates revealed that the tyrosinase from Bacillus megaterium (megaTYR) is an enzyme that possesses a broad substrate tolerance. We use the expanded substrate preference as a starting point for protein design experiments and show that single point mutants of megaTYR are capable of activating tyrosine residues in various sequence contexts. We leverage this new tool to enable the construction of protein trimers via a charge-directed sequential activation of tyrosine residues (CDSAT).

Full text of DOI:10.1021/jacs.1c03079

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Article
Synthesis of Multi-Protein Complexes through Charge-Directed
Sequential Activation of Tyrosine Residues
Casey S. Mogilevsky,$ Marco J. Lobba,$ Daniel D. Brauer, Alan M. Marmelstein, Johnathan C. Maza,
Jamie M. Gleason, Jennifer A. Doudna, and Matthew B. Francis*
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ABSTRACT: Site-selective protein−protein coupling has long been
a goal of chemical biology research. In recent years, that goal has
been realized to varying degrees through a number of techniques,
including the use of tyrosinase-based coupling strategies. Early
publications utilizing tyrosinase from Agaricus bisporus(abTYR)
showed the potential to convert tyrosine residues into ortho-quinone
functional groups, but this enzyme is challenging to produce
recombinantly and suffers from some limitations in substrate
scope. Initial screens of several tyrosinase candidates revealed that
the tyrosinase from Bacillus megaterium (megaTYR) is an enzyme
that possesses a broad substrate tolerance. We use the expanded
substrate preference as a starting point for protein design experiments and show that single point mutants of megaTYR are capable
of activating tyrosine residues in various sequence contexts. We leverage this new tool to enable the construction of protein trimers
via a charge-directed sequential activation of tyrosine residues (CDSAT).
challenging to install on the surface of full proteins.9 For
many enzymatic coupling techniques, such as sortase, butelase,
INTRODUCTION
■
Site-selective post-translational modification of proteins has
and farnesyltransferase, coupling can only occur at the N- and
long been an object of study for both chemists and biologists.
C-termini.6,11−14 More recent work has demonstrated that a
The augmentation of the native properties of these
combination of sortase and butelase can be used to
biomolecules to contain fluorophores,1,2 drug molecules,3
sequentially conjugate multiple coupling partners onto a single
and other signaling moieties has been of tremendous value
protein.15 A similar method for synthesizing multiprotein
to both our understanding of biology and the creation of new
conjugates was also developed that uses an asparaginyl
therapeutics.3−6 While much work has been done in the
endopeptidase instead of the butelase and sortase combina-
attachment of small molecule cargoes, the facile attachment of
tion.16 Yet another approach to terminal protein ligation is the
additional whole proteins has proven more difficult. The
use of intein-based splicing which enables the facile
primary challenge of protein−protein coupling lies in the steric
combination of peptides and proteins through thioester
inhibition that must be overcome in the joining of large
intermediates.10,17 While powerful, these approaches are still
molecules as well as the difficulty in the generation of suitable
limited to conjugation via N or C-termini, which limits the
selective reaction handles due to the frequent occurrence of
relative orientation of the conjugate. Most other coupling
most native amino acids. Current methods for the site-selective
approaches such as SpyCatcher/SpyTag require the insertion
synthesis of protein conjugates involve the introduction of
of bulky recognition domains and pose additional risks of
uniquely reactive unnatural amino acids (UAAs) into the
immunological rejection.18
backbone sequence,7,8 maleimide-cysteine linkages,9 intein-
Tyrosinases, a broadly distributed class of enzymes involved
based strategies,10 and enzymatic coupling reactions.6,11−14
in the melanin synthesis pathway in every kingdom of life,
Although these methods have addressed many important
provide promising new alternatives. Tyrosinase enzymes
challenges in chemical biology, limitations still exist. The site
selective incorporation of unnatural amino acids into proteins
Received: March 23, 2021
relies on stop codon repurposing, which requires special
growth media and often lowers protein yield.7,8 Thiol-reactive
cross-linkers, such as maleimides, are specific to cysteine
residues on the protein surface that are rare enough to afford
selectivity and can be readily inserted via single base
mutagenesis. However, the maleimide reactive handle is
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Figure 1. Structure and activity of tyrosinase enzymes from eukaryotic, prokaryotic, and archaeal sources. (A) Protein structure for abTYR from
Agaricius bisporus with acidic residues in red and basic residues in blue. PDB ID: 2Y9X. (B) Protein structure for megaTYR from Bacillus
megaterium with acidic residues in red and basic residues in blue. PDB ID: 6EI4. (C) Protein structure for cnkTYR from Candidatus Nitrosopumilus
koreensis with acidic residues in red and basic residues in blue. Structure was generated using the I-TASSER Web server. (D) Peptide reaction
screen showing percent conversion of Y200C sfGFP with the indicated peptide. All reactions carried out at 150 μM Y200C sfGFP, 250 μM peptide,
and 12 U/L equivalent of either abTyr (dark blue), megaTYR (blue), or cnkTyr (light blue) in 50 mM pH 6.5 phosphate buffer for 30 min before
quench with 4 mM tropolone and analysis via ESI-TOF-MS. Deconvoluted data were analyzed on www.chartograph.com with percent conversion
calculated as percentage of peak area for the modified vs unmodified samples. Asterisks indicate where negligible amounts of protein modification
were detected.
normally catalyze the oxidation of tyrosine to a dopaquinone
intermediate, which then cyclizes to form dopachrome.19−23
Recent studies have repurposed the commercially available
mushroom tyrosinase (abTYR) to catalyze site specific
oxidative coupling reactions at N- and C-terminal tyrosine
residues using different types of coupling partners. Strained
alkynes24 and nucleophilic coupling partners, such as
secondary amines,25 anilines,26 and surface exposed cysteine
residues,27 have expanded the possible sites of enzymatic
protein bioconjugation past the N- and C-termini. This
methodology is versatile and can take place at temperatures
ranging from 4 °C to room temperature and near-neutral pH
to preserve the function of sensitive proteins.27
protein L, regardless of linker length.26 Both abTYR and
protein L are over 100 kDa in mass and are negatively charged,
suggesting that electrostatic repulsion and steric hindrance may
be the factors preventing the activation of this particular
substrate by abTYR.28,29 A smaller, 35.5 kDa tyrosinase from
Bacillus megaterium (megaTYR, referred to as bmTYR in ref
26) was found to activate C-terminal tyrosines on protein L
regardless of linker length.21,26 In this study, we characterize
the substrate preferences of these tyrosinases and show that
abTYR is unable to oxidize tyrosines in negatively charged
substrates, whereas megaTYR and the recently reported
tyrosinase from the archaeon, Candidatus Nitrosopumilus
koreensis (cnkTYR),30 are much more promiscuous. In this
report, we generate new versions of megaTYR selective for
tyrosine residues in specific charge contexts. Moreover, we
leverage this new tool to enable the construction of protein
In spite of the recent success and convenience of abTYR, it
is not without limitations. In a recent investigation, abTYR was
unable to activate tyrosine residues at the C terminus of
B
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Figure 2. Evolutionary conservation of amino acids in megaTYR (bmTYR). (A) Multiple sequence alignment of megaTYR backbone to its six
nearest homologues. Residues are colored by conservation score calculated by using the evolutionary rate of 100 sequence homologues on the
ConSurf web server. Highly conserved copper-binding residues are marked with an asterisk. Engineering sites are indicated with orange triangles.
(B) Conservation coloring mapped to the megaTYR structure. Bound substrate is colored in green (PDB ID: 4P6T).44 (C) Active site of megaTYR
with conservation coloring, copper ions shown in gold. Variable residues R209 and D55 surrounding the highly conserved active site were selected
as engineering targets to alter substrate scope.
trimers via charge-directed sequential activation of tyrosine
residues (CDSAT).
abTYR was incapable of activating phenols attached to a single
stranded DNA (ssDNA) substrate. In contrast, our first
experiment with megaTYR showed significant levels of
coupling between GFP and the phenol-DNA (Figure S1).
This reaction occurs as two distinct steps: the initial activation
of the phenol to the ortho-quinone followed by addition of the
nucleophile. These results suggested that some aspect of the
DNA substrate was preventing activation by abTYR but was
tolerated by megaTYR (Figure S1). We reasoned that this was
likely the high degree of negative charge on the nucleotide
substrate.
We next examined the substrate preference of the new
tyrosinases, specifically with respect to their charge and steric
tolerance. A peptide library with tyrosine residues at their C-
termini was obtained, shown in Supporting Information Table
1, which contained a variety of charged and neutral sequence
elements. C-terminal tyrosine peptides were chosen as they
most closely resemble the protein constructs previously shown
to be effective substrates for the tyrosinase oxidation. Future
studies will be needed to examine the substrate activation on
internal tyrosine residues as previous studies have shown
tyrosinase activity in unstructured peptide substrates.33,34
However, our previous work has shown that even highly
exposed tyrosine residues in folded proteins are not readily
targeted unless they are adjacent to the N- or C-terminal
RESULTS AND DISCUSSION
■
In our search for more sequence permissive enzymes than
abTYR (Figure 1A) we identified tyrosinases from Bacillus
megaterium (megaTYR) and Candidatus Nitrosopumilus kore-
ensis (cnkTYR) as optimal candidates for recombinant
expression. These particular tyrosinases were chosen as they
are both monomeric tyrosinases that have been shown to be
easily expressible in E. coli.30,31 Moreover, megaTYR was
selected because several previous studies showed that the
enzyme tolerated point mutations while retaining high activity
to free tyrosine.21,31,32 The published crystal structures for
megaTYR were beneficial for protein engineering, as they
allowed rational design of point mutants and facilitated
computational analysis of the protein (Figure 1B).31 In
contrast, cnkTYR was recently shown to have excellent
monophenol monooxygenase activity and to tolerate low
temperatures without significant loss in catalytic efficiency
which posed an attractive candidate despite the lack of
published crystal structure (homology model presented in
Figure 1C).23,30
In addition to the previously published work on protein L,26
the motivation for these explorations was the realization that
C
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Figure 3. Electrostatic calculations on tyrosinase mutants. (A) Surface potential maps of abTYR and several megaTYR mutants with negative
charge in red and positive charge in blue. (B) Table showing results of charge potential calculations for the various tyrosinase mutants. For each
tyrosinase a point charge was placed 14 Å from the copper atoms within the active site and potential charge and pI were calculated using the Epik
̈
and Macromodel modules of Maestro developed by Schrodinger. Δ potential is calculated as the change in potential between the wild type and the
listed mutant.
position.26,27 The peptides from the C-terminal library were
coupled to Y200C GFP in 30 min reactions and analyzed via
ESI-TOF MS to determine conversion to the modified
peptide−protein conjugate. To ensure that peptide coupling
was not biased by electrostatic interactions between GFP (pI =
6.2) and the peptides, control reactions were run on a p-
aminophenylalanine modified MS2 capsid, which has a pI of
8.0. These reactions reached similar levels of modification (SI
Figure S2). In a comparison of abTYR to the non-eukaryotic
tyrosinases, it is readily apparent that abTYR is significantly
more sensitive to negative charge (Figure 1D). In fact, abTYR
shows no activity toward any peptide with 3 or more acidic
residues adjacent to the terminal tyrosine. Also of note is the
fact that megaTYR shows high activity toward all peptide
substrates, with a drop in activity only in the case of the
RRRRY peptide. These results supported the earlier challenge
activating ssDNA substrates using abTYR and suggest that
megaTYR may serve as a highly permissive tyrosine activator.
One additional observation of this study was that cnkTYR
samples showed no discernible pattern of substrate specificity.
The structures of the three tyrosinases were compared to
determine the origin of their selectivities, with cnkTyr
represented by a homology model created using the I-TASSER
server.29,30,35−38 abTYR is found as a protein complex and
shows significant steric occlusion of the active sites (Figure
1A),29 while megaTYR and cnkTYR are monomeric and show
a much more open active site (Figure 1B,C).30,35 Due to their
more predictable behaviors and established crystal structures,
we focused our work on megaTYR and abTYR in subsequent
experiments. However, further exploration or engineering of
cnkTyr could yield a more specific phenol monooxygenase
capable of acting with higher precision.
In the interest of improving megaTYR for even broader
activity, we performed an evolutionary conservation analysis of
the megaTYR backbone in order to determine target positions
for engineering. Using the ConSurf web server, the 100 closest
nonredundant homologues of megaTYR were clustered and a
multiple sequence alignment was prepared (Figure 2A).39−42
A
normalized conservation score was calculated for each position
using an empirical Bayesian methodology, which accounts for
phylogenetic relationships between homologues in its con-
struction of an evolutionary model.43 The evolutionary
conservation was mapped onto the megaTYR structure and
used to identify mutable anionic residues with low
conservation scores (Figure 2B, Table S2). Position R209
was also chosen as a position of interest, despite its mild
conservation, due to its established role in potentiating
diphenolase activity and its proximity to the active site (Figure
2C).31 We also chose to study R280 as a more distal mutable
cationic residue with a low conservation score (Table S2).
Previously studied mutants at positions V218 and F197 were
not further investigated, as their impact on megaTYR activity
was not beneficial to our purpose of producing a more
electrostatically targeted tyrosinase.21,32
The utility of modulating tyrosinase charge was explored in
abTYR and megaTYR. Mutants of megaTYR identified from
literature and the conservation screen were modeled in
Maestro using the charge mapping tool from the Epik and
D
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Figure 4. Point mutants of megaTYR and their impact on substrate preference. (A) Structural comparison of abTYR vs megaTYR with locations of
point mutations highlighted. R209 is shown in yellow, D55 in blue. Structures show acidic (Asp and Glu) residues in red and basic (Lys and Arg)
residues in blue. (B) Comparison of reactivity of abTYR vs single mutants of megaTYR on peptides EEEEY and RRRRY. Reactions were run for 30
min at RT in 50 mM pH 6.5 phosphate buffer, 5 U/mL tyrosinase, 150 μM Y200C sfGFP and 250 μM peptide. Reactions were quenched with 4
mM tropolone. (C) Competition reactions between RRRRY and EEEEY peptides to assess substrate preference of tyrosinase mutants. Reactions
were run for 30 min at RT in 50 mM pH 6.5 phosphate buffer, 5 U/mL tyrosinase, 150 μM Y200C sfGFP and 250 μM of each peptide (500 μM
total peptide concentration) . Reactions were quenched with 4 mM tropolone. (D) Peptide reaction screen showing percent conversion of Y200C
sfGFP with the indicated peptide. All reactions were run at 10 μM Y200C sfGFP, 250 μM peptide, and 12 U/mL equivalent of either wt megaTYR
(blue), R209H megaTYR (dark blue), or D55R megaTYR (light blue) in 50 mM pH 6.5 phosphate buffer for 30 min before quenching with 4 mM
tropolone and analysis via ESI-TOF-MS. Deconvoluted data were analyzed on www.chartograph.com with percent conversions calculated as
percentage of peak area percentages for the modified vs unmodified species.
MacroModel modules to calculate the surface potential for
each of the proteins compared to a point charge placed 14 Å
from the active site copper atoms (Figure 3A, Table S2). We
reasoned that surface-exposed polar and ionic residues
proximal to the active site may be attractive targets, as they
would have larger effects on active site potential than more
distal residues, and modeled them in a similar manner (Table
S3). We found that abTYR monomers had a net potential of
−4.4 V due to high numbers of acidic residues on their
surfaces, corresponding to an isoelectric point (pI) of only
4.41. When the full active complex of abTYR is considered the
net potential drops to −15.7 V.29 In contrast, megaTYR was
shown to have a net potential of +4.4 V and a pI of 8.66,
correlating well with the observed charge preference.35
In accordance with our assumption that more proximal
residues would have a larger impact on active site potential, we
found that R209H had a potential of +3.0 V, a significant
reduction compared to the wild type, whereas R280H has a
much smaller impact on the electrostatics near the active site
(Figure 3B, Table S2). Similarly, D55K had the largest impact
on the active site potential among the D/E to K mutants,
corresponding to a potential of +7.33 V. Because of this, we
also chose to model D55R to compare the effects of mutating
anionic residues to lysine or arginine. While D55K had a
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Figure 5. Coupling of two proteins to a small molecule. (A) Scheme depicting the construction of a cysteine GFP (Y200C GFP), as well as a
negative tyrosine tagged cysteine GFP (GFP E4Y). (B,C) ESI-MS data for unmodified Y200C GFP and GFP E4Y respectively. (D) ESI-MS of
GFP E4Y modified with biotin maleimide. Biotin-maleimide coupled to GFP E4Y by combining 150 μM GFP E4Y with 500 μM biotin-maleimide
at pH 7.5 for 2 h. (E) GFP E4Y was coupled to biotin-phenol by combining 150 μM GFP E4Y with 200 μM biotin-phenol with 12 U/mL abTYR
in 50 mM phosphate pH 6.5 for 40 min at RT. Product mass expected = 29054 Da. (F,G) Reaction of 40 μM GFP-E4Y-biotin with 100 μM Y200C
GFP with 12 U/mL megaTYR for 45 min at RT. Expected mass for GFP + GFP + maleimide biotin = 56446 Da and for GFP+GFP phenol =
56618 Da.
potential of +7.33 V, D55R showed a slightly more positive net
potential of +7.5 V, due to the higher pKa of arginine (Figure
3B, Table S2).
was not capable of complete substrate differentiation, we chose
to focus on D55R as the higher performing mutant.
We then performed a full substrate screen on each megaTYR
variant (wild type, R209H, and D55R) at the same
concentration as the initial substrate screen (12 U/mL) and
found that these mutations only had a significant effect on the
enzymatic activity toward highly charged peptide substrates.
Interestingly, D55R megaTYR exhibited low conversion for the
RRRRY peptide but retained high levels of activity toward the
less highly charged cationic peptides (Figure 4D). Generation
of sequence-orthogonal tyrosinases could enable the facile
creation of complex multidomain proteins by coupling anionic
and cationic tagged subunits sequentially. This could be done
in a manner similar to peptide synthesis but using entire
proteins as building blocks. The controlled production of
multiprotein polymers would be an enabling technology for
protein based materials, biomedical technologies, and enzyme
engineering.
On the basis of data from the conservation screen and
potential modeling, we generated megaTYR with R209H,
D55K, and D55R mutations (structures with residues high-
lighted in Figure 4A). After initial screening at a tyrosinase
concentration of 5 U/mL, it was observed that R209H
decreased the charge preference of megaTYR, while D55K and
D55R were found to increase the preference for anionic
substrates as shown by the higher level of modification with the
EEEEY peptide (Figure 4B). To assess the degree of substrate
preference, we ran competition assays containing the EEEEY
and RRRRY peptides in equal concentrations. In these assays,
abTYR only showed activity toward RRRRY, while R209H
showed equal preference for the two (Figure 4C). Interest-
ingly, wild type megaTYR showed a 90% preference for
EEEEY, while the D55K mutant showed a 95% preference and
D55R a surprising >99% preference for the anionic peptide
(Figure 4C). As D55K experienced expression challenges and
Although the megaTYR mutants to date were not fully
prevented from acting on cationic substrates, the range in
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Figure 6. Use of tyrosinase charge selectivity for the synthesis of ternary protein complexes. (A) Reaction scheme showing the use of sequential
reactions with abTYR and megaTYR to couple 3 proteins together. (B) Protein sequence maps for S152C mCherry (mCherry), Y200C GFP with
EEEEY tag (GFP E4Y), and a MYGGS tagged nano Luciferase (nanoLuc). (C) Reaction of 120 μM GFP-E4Y with 200 μM nanoLuc and 20 U/
mL abTYR for 1 h. GFP + nanoLuc structure shown above chart. Expected mass = 48,728 D) GFP E4Y-nanoLuc (10 μM) purified by spin down
using a 50 kDa MWCO spin concentrator to a concentration of 50 μM and combined with 150 μM S152C mCherry and 20 U/mL D55R
megaTYR for 1 h at RT converted E4Y GFP-nanoLuc to the mCherry-GFP-nanoLuc trimer after purification using 50 kDa MWCO spin filter.
Trimer structure shown above spectrum. Expected mass = 76 153 Da.
selectivity between abTYR and D55R megaTYR proved a
tantalizing target. To test this, the cysteine-containing Y200C
sfGFP was modified with EEEEY and RRRRY C-terminal tags,
which should be unreactive with abTYR and D55R megaTYR,
respectively (Figures S3, S4). After purification, the Y200C
sfGFP-EEEEY (Y200C GFP-E4Y) was exposed to abTYR for
30 min in a variety of conditions. Notably, the mushroom
tyrosinase was able to append GGGGY and RRRRY peptides
to the Y200C GFP-E4Y without oxidizing the EEEEY tag
(Figures S3, S4). In addition, exposing the Y200C GFP-E4Y to
abTYR resulted in no discernible oxidation by ESI-MS. This
aligns with our previous peptide screens, and suggests that the
EEEEY tag is not succeptible to abTYR activation (Figure S3).
Similarly, Y200C sfGFP-RRRRY (Y200C GFP-R4Y) was
exposed to D55R megaTYR under similar conditions, with
the EEEEY peptide used in place of the RRRRY peptide.
Similarly to abTYR, D55R megaTYR was able to selectively
oxidize the added peptides without modifying the tyrosine tag
on the Y200C GFP-R4Y. Reaction selectivity was further
confirmed when exposure of Y200C GFP-R4Y to D55R
megaTYR alone did not result in any oxidation (Figure S4).
Together, these data suggest that abTYR and D55R megaTYR
can be used for CDSAT on proteins and peptides.
To validate reaction orthogonality further, we next
compared abTYR and maleimide mediated biotinylation of
Y200C GFP-E4Y (Figure 5A−C). As a positive control,
Y200C GFP-E4Y was selectively modified with maleimide
biotin to generate biotinylated Y200C GFP-E4Y (Figure 5D).
In parallel, we modified Y200C GFP-E4Y with a phenol-biotin
in the presence of abTYR and showed selective oxidation of
the small molecule to afford the Y200C GFP-E4Y-biotin
conjugate (Figure 5E). After purification using a 10 kDa
MWCO spin concentrator, both the maleimide and phenol
GFP-E4Y-biotin were combined with Y200C sfGFP and 12 U/
mL D55R megaTYR for 30 min and analyzed via ESI-TOF-
MS. We observed over 70% conversion to the trimer
composed of two GFP proteins and a biotin molecule, making
this one of the first techniques capable of creating 3-
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component fusions without the need for solid support
tethering or removable protective groups (Figure 5F,G). In
addition, some higher order modifications appeared, arising
from residual disulfide formation between unreacted thiols in
the first step. To combat the undesired formation of disulfide
bonds between thiols in the protein coupling partner, we
suggest pretreating these proteins with a 2x molar excess of
TCEP in pH 7.0 Tris-HCl buffer, followed by an exchange into
the reaction buffer via spin filtration. This pretreatment step
was used for all subsequent reactions.
AUTHOR INFORMATION
Corresponding Author
■
Matthew B. Francis − Department of Chemistry, University of
California, Berkeley, California 94720, United States;
Materials Sciences Division, Lawrence Berkeley National
Laboratories, Berkeley, California 94720, United States;
orcid.org/0000-0003-2837-2538; Email: mbfrancis@
berkeley.edu
Authors
To evaluate the potential of an iterative tyrosinase approach
for the creation of protein trimers, Y200C GFP-E4Y was
combined with a MYGGS-tagged nanoluciferase and 20 U/mL
abTYR to activate the neutral luciferase tag selectively (Figure
6A−C). Clean conversion to the Y200C GFP-E4Y-nano-
luciferase dimer was observed, with no excess oxidation. To
remove any unreacted starting material, the solution was
purified using a 50 kDa MWCO spin concentrator (Figure
6C). As a third component, the thiol-containing S152C-
mCherry was added to the dimer solution with 20 U/mL
D55R megaTYR to activate the EEEEY tag on the GFP. This
strategy led to smooth conversion to the mCherry-GFP-
luciferase trimer (Figure 6D). We saw 100% conversion from
the dimer to the final product, indicating that megaTYR is
capable of catalyzing tyrosinase oxidation on anionic tags even
in the presence of multiprotein complexes. The trifunctional
nature of the construct identity was further validated by
luminescence and fluorescence measurements. Upon introduc-
ing the nanoLuc substrate, FRET was observed between the
luciferase, GFP, and mCherry subunits. In addition, direct
excitation of the GFP fluorophore led to emission from the
mCherry segment via energy transfer (Figure S5). This new
capability to oxidize tyrosine residues in different charge
contexts iteratively highlights the potential of this system to
generate complex protein products for future applications.
Conclusion. Here, we show that recombinantly expressed
tyrosinases are successful activators of terminal tyrosine
residues on a variety of protein and peptide substrates. The
results from the megaTYR mutant screen show that bacterially
derived tyrosinases possess sufficient selectivity to mediate
protein−protein coupling reactions through tyrosine-cysteine
bond formation and can be engineered to alter their substrate
preferences. The success at modulating the charge preference
of this enzyme indicates that it is tolerant toward mutation and
can be engineered to achieve orthogonal reactivity preferences
to those of abTYR. The result of these efforts is the ability to
sequentially activate tyrosine residues based on their charge
context, or CDSAT. The protein trimers created using CDSAT
show that minimal engineering is required to produce a system
capable of creating higher-order protein constructs while
controlling their orientation via cyteine position. Such
multivalent conjugates have tremendous potential for materi-
als, therapeutic, and diagnostic applications.
Casey S. Mogilevsky − Department of Chemistry, University
of California, Berkeley, California 94720, United States;
orcid.org/0000-0002-0887-6862
Marco J. Lobba − Department of Chemistry, University of
California, Berkeley, California 94720, United States;
orcid.org/0000-0001-9473-7692
Daniel D. Brauer − Department of Chemistry, University of
California, Berkeley, California 94720, United States;
orcid.org/0000-0001-6974-3476
Alan M. Marmelstein − Department of Chemistry, University
of California, Berkeley, California 94720, United States;
orcid.org/0000-0003-2024-0602
Johnathan C. Maza − Department of Chemistry, University of
California, Berkeley, California 94720, United States;
orcid.org/0000-0003-2898-8770
Jamie M. Gleason − Department of Chemistry, University of
California, Berkeley, California 94720, United States
Jennifer A. Doudna − Department of Chemistry, Department
of Molecular and Cell Biology, and Howard Hughes Medical
Institute, University of California, Berkeley, California 94720,
United States; Gladstone Institutes, San Francisco, California
94158, United States; Innovative Genomics Institute,
University of California, Berkeley, California 94720, United
States
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c03079
Author Contributions
$These authors contributed equally
Notes
The authors declare the following competing financial
interest(s): M.B.F is the Chair of the UC Berkeley Department
of chemistry; a co-founder of Catena Biosciences; and a
scientific adviser to Catena Biosciences. M.J.L is a co-founder
of Catena Biosciences. The Regents of the University of
California have patents pending for the use of tyrosinase in the
manner described herein on which the authors are inventors.
ACKNOWLEDGMENTS
■
The authors thank the Zhang lab for making available the I-
TASSER server: https://zhanglab.ccmb.med.umich.edu/I-
TASSER/. The authors also thank Dr. Kathy Durkin and Dr.
Dave Small and the MGCF Facility for providing computa-
tional resources for the electrostatics calculations. Computa-
tional work was supported by NIH Grant: S10OD023532.
This work was supported by NIH Grant: R01 GM138693.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c03079.
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