Document [original]

International Journal of

Molecular Sciences

Article

Engineering Pyrrolysyl-tRNA Synthetase for the Incorporation

of Non-Canonical Amino Acids with Smaller Side Chains

Nikolaj G. Koch 1,2 , Peter Goettig 3, Juri Rappsilber 2,4 and Nediljko Budisa 1,5,*





Citation: Koch, N.G.; Goettig, P.;

Rappsilber, J.; Budisa, N. Engineering

Pyrrolysyl-tRNA Synthetase for the

Incorporation of Non-Canonical

Amino Acids with Smaller Side

Chains. Int. J. Mol. Sci. 2021,22,

11194. https://doi.org/10.3390/

ijms222011194

Academic Editor: Kensaku Sakamoto

Received: 19 September 2021

Accepted: 14 October 2021

Published: 17 October 2021

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4.0/).

1Institut für Chemie, Technische Universität Berlin, 10623 Berlin, Germany; [email protected]

2Institut für Biotechnologie-Bioanalytik, Technische Universität Berlin, 10623 Berlin, Germany;

[email protected]

3Structural Biology Group, Department of Biosciences, University of Salzburg, 5020 Salzburg, Austria;

peter[email protected]

4Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, UK

5Department of Chemistry, University of Manitoba, Winnipeg, MB R3T 2N2, Canada

*Correspondence: [email protected]; Tel.: +49-30-314-28821

Abstract:

Site-specific incorporation of non-canonical amino acids (ncAAs) into proteins has emerged

as a universal tool for systems bioengineering at the interface of chemistry, biology, and technology.

The diversification of the repertoire of the genetic code has been achieved for amino acids with long

and/or bulky side chains equipped with various bioorthogonal tags and useful spectral probes.

Although ncAAs with relatively small side chains and similar properties are of great interest to

biophysics, cell biology, and biomaterial science, they can rarely be incorporated into proteins. To

address this gap, we report the engineering of PylRS variants capable of incorporating an entire

library of aliphatic “small-tag” ncAAs. In particular, we performed mutational studies of a specific

PylRS, designed to incorporate the shortest non-bulky ncAA (S-allyl-L-cysteine) possible to date

and based on this knowledge incorporated aliphatic ncAA derivatives. In this way, we have not

only increased the number of translationally active “small-tag” ncAAs, but also determined key

residues responsible for maintaining orthogonality, while engineering the PylRS for these interesting

substrates. Based on the known plasticity of PylRS toward different substrates, our approach further

expands the reassignment capacities of this enzyme toward aliphatic amino acids with smaller side

chains endowed with valuable functionalities.

Keywords:

genetic code expansion; pyrrolysyl-tRNA synthetases; non-canonical amino acids;

aliphatic amino acids; bioorthogonal reactive handles; azidohomoalanine; photo-methionine; protein

engineering; stop codon suppression; S-allyl-L-cysteine

1. Introduction

Research in the field of reprogrammed protein translation has now reached experi-

mental and intellectual maturity: More than 200 non-canonical amino acids (ncAAs, i.e.,

a diversity that is an order of magnitude higher than that of the canonical amino acid

repertoire) were introduced into proteins via various genetic code expansion routes: Se-

lective pressure incorporation, stop codon suppression (SCS), fragment condensation,

protein semisynthesis, and peptidomimetics [

]. It has been shown that AAs with non-

proteinogenic functional groups can be used to manipulate, design, and elucidate protein

structure, dynamics, function, allosterism, interactions, catalysis, folding, synthesis, traf-

ficking, degradation, and aggregation [2–8].

Engineering aminoacyl-tRNA synthetase (aaRS)/tRNA pairs capable of recogniz-

ing, activating, and loading ncAAs onto their cognate tRNAs is now a well-established

strategy. It enables the site-specific ribosomal incorporation of ncAAs in response to a

reprogrammed codon. The most commonly used approach for this purpose is in-frame

stop codon suppression, targeting the amber stop codon [

–

]. Hereby, the ncAA is

Int. J. Mol. Sci. 2021,22, 11194. https://doi.org/10.3390/ijms222011194 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021,22, 11194 2 of 17

incorporated in response to an in-frame stop codon placed at a predefined position in the

protein coding sequence, ribosomally expressed either

in vivo

in vitro

[

–

]. Most aaRS

variants used for SCS so far are derived from Methanosarcina mazei/barkeri pyrrolysyl-tRNA

synthetases (MmPylRS/MbPylRS) or Methanocaldococcus jannaschii tyrosyl-tRNA synthetase

(MjTyrRS) [

–

]. The archaeal origin and therefore distant phylogeny is responsible for

their orthogonality in bacterial and eukaryotic cells [12].

The native substrate of the PylRS is the rare proteinogenic amino acid pyrrolysine (Pyl,

1a), a lysine analog with a 4-ethyl-pyrroline-5-carboxylate ring attached to the terminal

amino function of the side chain (Figure 1). The wild type enzyme can activate several

Pyl variants resembling ncAAs [

]. Moreover, catalytic promiscuity is widely exploited

in both native and genetically engineered classes of PylRS enzymes to enable recognition,

activation, and tRNA loading of the majority of all translationally active ncAAs. [

–

]. It

should be noted, however, that the majority of incorporable ncAAs with the PylRS system

are characterized with flexible, long-chained, and bulky pyrrolysine analogs [

] or

shorter but still bulky aromatic substrates, especially phenylalanine [

], tryptophan [

and histidine [

] analogs. Therefore, a new class of PylRS enzymes capable of recognizing,

activating, and tRNA loading with shorter chain ncAAs endowed with useful functional

groups is of great interest. Small ncAAs with shorter side chains containing azido, thioene,

fluoro, cyano, and nitroso groups can be particularly useful, e.g., for FTIR, NMR, crosslink-

ing, and spin labeling, because longer side chains are too flexible which usually results

in a loss of spectral information or the necessary proximity for specific bioorthogonal

reactions [

]. Moreover, still no efficient non-canonical counterparts are available for Glu

and Asp, which often form structurally important salt bridges or hydrogen-bond networks.

It would be useful to modify these acidic residues, e.g., by removing their hydrogen bond

donors or acceptors. Mimicking post-translational modifications of canonical amino acids

(cAAs) with their genetically encoded ncAA counterparts is also an attractive application

to elucidate their functions.

The main reason for the substrate promiscuity of PylRS is most likely the unique

substrate binding mode with relatively nonspecific hydrophobic interactions in the large

binding pocket of this enzyme. Therefore, it is not surprising that a multitude of ncAAs

can be recognized and activated with very few mutations (the majority has just 2–4) mainly

in the binding pocket [

]. For this reason, the PylRS is predestined for the implemen-

tation of new functions [

]. In general, an enzyme should possess two key features to

ensure successful recognition of new substrates. First, the target enzyme should have

low levels of the desired new activity, which in case of PylRS means enzymatic activ-

ity toward ncAAs that are highly divergent from the native substrate [

]. Second,

sufficient stability is required to buffer destabilizing mutations necessary for active site

remodeling

[23–25]

. Unfortunately, PylRS is marginally stable under standard cultivation

conditions in

Escherichia coli (E. coli)

[

], which is also reflected by the low

in vitro

solubil-

ity of the enzyme [

]. We demonstrated that this drawback can be partly remedied with

a solubility tag fused to the N-terminus of MbPylRS (manuscript in preparation) which

made this our enzyme of choice.

In our study, we performed mutational analyses to elucidate the structure activity

relationship of a PylRS designed to incorporate S-allyl-L-cysteine (Sac, 1). Based on this

knowledge, we engineered several new MbPylRS variants on a rational and semi-rational

basis, in order to incorporate a variety of small side chain ncAAs. We used an entire

library of small side-chain-containing ncAAs that can be structurally and functionally

categorized into five classes (Figure 1, detailed discussion in Appendix A). Briefly, they

include (i) aliphatic analogs of Sac (1); (ii) bio-orthogonal tags; (iii) small ncAAs with useful

spectroscopic probes; (iv) methionine analogs; and (v) substrates with a terminal alkene as

site-specific chemical cleavage site (being also bio-orthogonal tags).

Int. J. Mol. Sci. 2021,22, 11194 3 of 17

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 17

Figure 1. Survey of amino acids used in this study. Chemical structure of pyrrolysine (1a), S-allyl-L-cysteine (1), (S)-2-

aminoheptanoic acid (2), (S)-2-aminooctanoic acid (3), (S)-2-aminohept-6-enoic acid (4), (S)-2-aminohexanoic acid (5), (S)-

2-aminohex-5-enoic acid (6), (S)-2-aminopentanoic acid (7), (S)-2-aminopent-4-enoic acid (8), (S)-2-amino-3-cyclo-

propylpropanoic acid (9), (S)-2-aminobutyric acid (10), (S)-2-aminohept-6-ynoic acid (11), (S)-2-aminohex-5-ynoic

acid (12), (S)-2-aminopent-4-ynoic acid (13), (S)-2-amino-3-azidopropanoic acid (14), (S)-2-amino-4-azidobutanoic acid

(15), (S)-2-amino-5-azidopentanoic acid (16), (S)-2-amino-6-azidohexanoic acid (17), (S)-2-amino-3-cyanopropanoic acid

(18), (S)-2-amino-4-cyanobutanoic acid (19), (S)-2-amino-5,5’-azi-hexanoic acid (20), (S)-2-amino-4-methylpent-4-enoic

acid (21), L-methionine (22), L-methionine sulfoxide (23), L-methionine sulfone (24), L-ethionine (25), S-tert-butyl-L-cysteine

(26), S-propargyl-L-cysteine (27), S-benzyl-L-cysteine (28).

2. Results and Discussion

2.1. General MbPylRS and ncAA Incorporation Readout Setup

All PylRS variants used in this study are equipped with an N-terminal SmbP solubil-

ity tag [28]. As mentioned earlier, the tag restores activity by dramatically increasing the

abundance of soluble and active enzyme compared to the untagged aaRS. Most likely, this

phenomenon is due to an increase in kinetic stability and builds an improved and solid

starting point for our enzyme engineering efforts. This fusion enzyme is used throughout

this work (for sequence information see supplement part 2.4). The Y349F mutation was

included by default, as it is known to generally enhance aminoacylation and orthogonal

translation systems (OTS) efficiency [13,29].

Figure 1.

Survey of amino acids used in this study. Chemical structure of pyrrolysine (1a), S-allyl-L-cysteine (1), (S)-

2-aminoheptanoic acid (2), (S)-2-aminooctanoic acid (3), (S)-2-aminohept-6-enoic acid (4), (S)-2-aminohexanoic acid

(5), (S)-2-aminohex-5-enoic acid (6), (S)-2-aminopentanoic acid (7), (S)-2-aminopent-4-enoic acid (8), (S)-2-amino-3-

cyclopropylpropanoic acid (9), (S)-2-aminobutyric acid (10), (S)-2-aminohept-6-ynoic acid (11), (S)-2-aminohex-5-ynoic

acid (12), (S)-2-aminopent-4-ynoic acid (13), (S)-2-amino-3-azidopropanoic acid (14), (S)-2-amino-4-azidobutanoic acid (15),

(S)-2-amino-5-azidopentanoic acid (16), (S)-2-amino-6-azidohexanoic acid (17), (S)-2-amino-3-cyanopropanoic acid (18),

(S)-2-amino-4-cyanobutanoic acid (19), (S)-2-amino-5,5

-azi-hexanoic acid (20), (S)-2-amino-4-methylpent-4-enoic acid (21),

L-methionine (22), L-methionine sulfoxide (23), L-methionine sulfone (24), L-ethionine (25), S-tert-butyl-L-cysteine (26),

S-propargyl-L-cysteine (27), S-benzyl-L-cysteine (28).

2. Results and Discussion

2.1. General MbPylRS and ncAA Incorporation Readout Setup

All PylRS variants used in this study are equipped with an N-terminal SmbP solubility

tag [

]. As mentioned earlier, the tag restores activity by dramatically increasing the

abundance of soluble and active enzyme compared to the untagged aaRS. Most likely, this

phenomenon is due to an increase in kinetic stability and builds an improved and solid

starting point for our enzyme engineering efforts. This fusion enzyme is used throughout

this work (for sequence information see supplement part 2.4). The Y349F mutation was

included by default, as it is known to generally enhance aminoacylation and orthogonal

translation systems (OTS) efficiency [13,29].

Int. J. Mol. Sci. 2021,22, 11194 4 of 17

To test the efficacy of ncAAs incorporation, we used superfolder-GFP (sfGFP) as a

model protein. The sfGFP-based fluorescence readout is the simplest approach for this

purpose as the fluorescence intensity of intact cells is directly correlated to the amount

of protein produced. The sfGFP reporter construct comprises an in-frame stop codon

at position 2 (instead of an arginine triplet; sfGFP(R2 amber)). This construct has been

routinely used as a readout vehicle for the

in vivo

suppression efficiency of the in-frame

amber stop codon [

]. This reporter construct is an integral part of the OTS established

in E. coli BL21(DE3) expression host strain. To avoid deficiency of ncAA in the cell and to

detect even very low incorporation activity, we used high concentrations of ncAAs (10 mM,

unless otherwise stated) in this study.

2.2. Testing MbSacRS for Aliphatic Substrates

We hypothesized that the previously reported SacRS could accommodate close struc-

tural aliphatic analogs of Sac (1). Therefore, this variant would be a good starting point for

the evolution of SacRS toward similar small-tag substrates. To scrutinize this hypothesis, a

SacRS variant (hereafter referred to as MbSacRS) was created based on a codon optimized

MbPylRS sequence by introducing the two crucial active site mutations C313W:W382S. In

addition, two previously identified advantageous N-terminal mutations T13I:I36V ([

cf. Figure S1) were introduced as well and maintained them for all other constructs. Sur-

prisingly, we found very low incorporation of the aliphatic substrates for the MbSacRS

(

Figure S2

). Since this specificity among very close structural analogs is relatively uncom-

mon for both native and mutant PylRS enzymes, we set out to investigate the structure–

activity relationships of the MbSacRS.

2.3. Elucidating the Structure–Activity Relationships of MbSacRS via Rational Mutation Studies

To date, no high-resolution crystal structure of SacRS is available. Since there are

only two PylRS mutations responsible for altering the substrate specificity to Sac (1), we

decided to perform rational mutation studies to elucidate the role of each residue in Sac-

incorporation activity. An overview of all relevant residues, whose mutations were guided

by crystal structures, can be found in Figure 2. Figure 2B was especially helpful because it

contains the same C313W mutation as the MbSacRS.

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 17

To test the efficacy of ncAAs incorporation, we used superfolder-GFP (sfGFP) as a

model protein. The sfGFP-based fluorescence readout is the simplest approach for this

purpose as the fluorescence intensity of intact cells is directly correlated to the amount of

protein produced. The sfGFP reporter construct comprises an in-frame stop codon at po-

sition 2 (instead of an arginine triplet; sfGFP(R2 amber)). This construct has been routinely

used as a readout vehicle for the in vivo suppression efficiency of the in-frame amber stop

codon [30]. This reporter construct is an integral part of the OTS established in E. coli

BL21(DE3) expression host strain. To avoid deficiency of ncAA in the cell and to detect

even very low incorporation activity, we used high concentrations of ncAAs (10 mM, un-

less otherwise stated) in this study.

2.2. Testing MbSacRS for Aliphatic Substrates

We hypothesized that the previously reported SacRS could accommodate close struc-

tural aliphatic analogs of Sac (1). Therefore, this variant would be a good starting point

for the evolution of SacRS toward similar small-tag substrates. To scrutinize this hypoth-

esis, a SacRS variant (hereafter referred to as MbSacRS) was created based on a codon

optimized MbPylRS sequence by introducing the two crucial active site mutations

C313W:W382S. In addition, two previously identified advantageous N-terminal muta-

tions T13I:I36V ([31], cf. Figure S1) were introduced as well and maintained them for all

other constructs. Surprisingly, we found very low incorporation of the aliphatic substrates

for the MbSacRS (Figure S2). Since this specificity among very close structural analogs is

relatively uncommon for both native and mutant PylRS enzymes, we set out to investigate

the structure–activity relationships of the MbSacRS.

2.3. Elucidating the Structure–Activity Relationships of MbSacRS via Rational Mutation

Studies

To date, no high-resolution crystal structure of SacRS is available. Since there are only

two PylRS mutations responsible for altering the substrate specificity to Sac (1), we de-

cided to perform rational mutation studies to elucidate the role of each residue in Sac-

incorporation activity. An overview of all relevant residues, whose mutations were

guided by crystal structures, can be found in Figure 2. Figure 2B was especially helpful

because it contains the same C313W mutation as the MbSacRS.

Figure 2. Microenvironment of the active sites derived from the crystal structures of MmPylRS and the MmOmeRS mutant.

These structures guided the rational mutation approach. Shown are critical residues forming the active site. Since only

structures of M. mazei are available, these were used in a homology model for M. barkeri. Residue numbers in brackets

reflect the numbering of M. barkeri, while numbers not in brackets refer to M. mazei. (A) Wild-type MmPylRS (PDB ID:

2Q7H)[20] with bound Pyl-AMP. (B) Mutant MmOmeRS (PDB ID: 3QTC)[32] with bound O-Methyl-tyrosine-AMP-PNP

(O-Met-tyr-AMP-PNP).

Figure 2.

Microenvironment of the active sites derived from the crystal structures of MmPylRS and the MmOmeRS mutant.

These structures guided the rational mutation approach. Shown are critical residues forming the active site. Since only

structures of M. mazei are available, these were used in a homology model for M. barkeri. Residue numbers in brackets

reflect the numbering of M. barkeri, while numbers not in brackets refer to M. mazei. (

) Wild-type MmPylRS (PDB ID:

2Q7H) [

] with bound Pyl-AMP. (

) Mutant MmOmeRS (PDB ID: 3QTC) [

] with bound O-Methyl-tyrosine-AMP-PNP

(O-Met-tyr-AMP-PNP).

Int. J. Mol. Sci. 2021,22, 11194 5 of 17

Starting with residue S382, we reverted this position to wild-type Trp and tested

less bulky amino acids. As serine was located at position 382 in the original SacRS, we

investigated whether polar functional groups are necessary for Sac (1) incorporation. As

shown in Figure 3A, all constructed mutants resulted in comparable Sac incorporation, with

the exception of the C313W:W382F and C313W constructs. This was quite surprising since

the authors in the original SacRS report found only three variants for Sac (1) incorporation

and just one variant possessed the C313W mutation [

]. This finding highlights the

enormous importance of quality control when constructing libraries and the need for

sufficient analytics when analyzing newly found variants after screening. Figure 3A does

not provide a clear picture of whether a hydroxyl group at position 382 is an advantage,

since the C313W:W382A variant performs at comparable levels. In contrast, small size

clearly plays an important role, with the Phe and Trp mutations being the two most

inefficient variants in the group so far. Interestingly, the Trp mutant is found to have a

strong cAA background incorporation. This phenomenon is perfectly in line with the

literature reports. It is known that PylRS enzymes with C313W mutations and a small

residue at position N311 incorporate Phe [

]. The original finding of SacRS was based on

positive and negative selection rounds. The inclusion of the negative step in the selection

against variants with high background incorporations clearly shows why the variant W382

was overlooked. Considering the gathered data, the W382 mutation, to smaller residues, is

most likely important for restoring orthogonality of MbSacRS.

We tested the four best variants as depicted in Figure 3A in a concentration-dependent

manner to gain detailed information about the OTS performance

in vivo

. The best con-

structs were (C313W:W382T/Y) and showing similar activity, but twice the OTS efficiency

(at 0.6 mM Sac (1)) compared to the original SacRS (Figure S3). To determine whether this

was a specific property of the MbPylRS, we transferred the mutations to the MmPylRS,

the variant resulting in the SacRS. For this variant, the equivalent of the (C313W:W382T)

mutations was also observed as the best outcome, indicating a robust result (Figure S4).

Interestingly the second-best variant of Mb and MmSacRS were different. This result

highlights the fact that the best mutation found in one species is not necessary the best

in another, although mutations are transferable between the different PylRS systems of

different species. The C313W:W382T variant was also reported to incorporate S-propargyl-

L-cysteine (27) [

]. Therefore, we also tested the top four Sac (1) incorporating constructs

also with substrate 27 and found that the (C313W:W382T/Y) constructs gave comparable

results (Figure S5). All gathered data for the W382 mutations suggest that this residue

only needs to be smaller than Trp to restore orthogonality. However, the efficiency of Sac

incorporation differs for different residues at this position. In particular, variants with a

polar OH- or SH-group at W382 position perform best. Examining the effect of the C313W

mutation in our system, the data displayed in Figure 3B clearly indicates that the size of

the residue at this position is the most prominent factor, albeit not the only one. No variant

with a small amino acid at this position was able to incorporate Sac. By contrast, the bulkier

Phe allows Sac incorporation, though at a lower level compared with all C313W mutants.

Even with Met at this position, very low incorporation is detectable. The inactive C313H

variant, comparable in size to C313F, suggests that a non-polar residue is necessary to be

present in this microenvironment. Overall, the data collected suggest that a reduced size of

the binding pocket containing the C313W mutation is essential for Sac incorporation.

Int. J. Mol. Sci. 2021,22, 11194 6 of 17

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 17

Starting with residue S382, we reverted this position to wild-type Trp and tested less

bulky amino acids. As serine was located at position 382 in the original SacRS, we inves-

tigated whether polar functional groups are necessary for Sac (1) incorporation. As shown

in Figure 3A, all constructed mutants resulted in comparable Sac incorporation, with the

exception of the C313W:W382F and C313W constructs. This was quite surprising since the

authors in the original SacRS report found only three variants for Sac (1) incorporation

and just one variant possessed the C313W mutation [33]. This finding highlights the enor-

mous importance of quality control when constructing libraries and the need for sufficient

analytics when analyzing newly found variants after screening. Figure 3A does not pro-

vide a clear picture of whether a hydroxyl group at position 382 is an advantage, since the

C313W:W382A variant performs at comparable levels. In contrast, small size clearly plays

an important role, with the Phe and Trp mutations being the two most inefficient variants

in the group so far. Interestingly, the Trp mutant is found to have a strong cAA back-

ground incorporation. This phenomenon is perfectly in line with the literature reports. It

is known that PylRS enzymes with C313W mutations and a small residue at position N311

incorporate Phe [34]. The original finding of SacRS was based on positive and negative

selection rounds. The inclusion of the negative step in the selection against variants with

high background incorporations clearly shows why the variant W382 was overlooked.

Considering the gathered data, the W382 mutation, to smaller residues, is most likely im-

portant for restoring orthogonality of MbSacRS.

Figure 3. Comparison of Sac incorporation efficiency for MbPylRS constructs (A) MbPylRS(C313W)

and variants mutated at position W382 and (B) MbPylRS(W382S) with variants mutated at position

C313. The fluorescence was measured for intact E. coli BL21(DE3) cells expressing the SUMO-

sfGFP(R2amber) reporter protein. The data (incl. standard deviation) represent the mean of three

biological replicates.

Figure 3.

Comparison of Sac incorporation efficiency for MbPylRS constructs (

)MbPylRS(C313W)

and variants mutated at position W382 and (B)MbPylRS(W382S) with variants mutated at position

C313. The fluorescence was measured for intact E. coli BL21(DE3) cells expressing the SUMO-

sfGFP(R2amber) reporter protein. The data (incl. standard deviation) represent the mean of three

biological replicates.

2.4. Rationalizing Sac Incorporation Data and Creating Aliphatic Substrate Activating

MbPylRS Variants

It was previously proposed that the C313W mutation is critical for activation of smaller

substrates [

]. This hypothesis was also fully recapitulated in this work. Having thus

established that the C313W mutation is critical for the incorporation of Sac (1) and probably

also smaller aliphatic Sac (1) analogs, all these mutants were tested for incorporation of

substrates 2, 3, 5, 7, and 10 (Figure 4). These fluorescence data showed that all variants,

except the C313W mutant, did not incorporate the tested substrates.

Unfortunately, the construct that incorporated the aliphatic ncAAs also exhibited a

considerable level of cAA background incorporation. As mentioned previously, bulky C313

mutations of MbPylRS lead to background Phe incorporation [

]. We hypothesized

that the key to restoring the lost orthogonality associated with the C313W mutation would

lie in the mutation of MbPylRS at position N311, which is also known to be one of the

two gatekeeper residues for Pyl activation. Our working hypothesis was to restore the

orthogonality by increasing the size of the amino acid at this position to interfere with the

Phe substrate accommodation while creating a catalytic pocket suitable for the short-chain

aliphatic ncAAs. As shown in Figure 5A, the orthogonality also increases by increasing the

AA size at the N311 position, until it is fully restored for the N311L:C313W variant.

Int. J. Mol. Sci. 2021,22, 11194 7 of 17

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 17

We tested the four best variants as depicted in Figure 3A in a concentration-depend-

ent manner to gain detailed information about the OTS performance in vivo. The best

constructs were (C313W:W382T/Y) and showing similar activity, but twice the OTS effi-

ciency (at 0.6 mM Sac (1)) compared to the original SacRS (Figure S3). To determine

whether this was a specific property of the MbPylRS, we transferred the mutations to the

MmPylRS, the variant resulting in the SacRS. For this variant, the equivalent of the

(C313W:W382T) mutations was also observed as the best outcome, indicating a robust

result (Figure S4). Interestingly the second-best variant of Mb and MmSacRS were differ-

ent. This result highlights the fact that the best mutation found in one species is not nec-

essary the best in another, although mutations are transferable between the different

PylRS systems of different species. The C313W:W382T variant was also reported to incor-

porate S-propargyl-L-cysteine (27) [35]. Therefore, we also tested the top four Sac (1) in-

corporating constructs also with substrate 27 and found that the (C313W:W382T/Y) con-

structs gave comparable results (Figure S5). All gathered data for the W382 mutations

suggest that this residue only needs to be smaller than Trp to restore orthogonality. How-

ever, the efficiency of Sac incorporation differs for different residues at this position. In

particular, variants with a polar OH- or SH-group at W382 position perform best. Exam-

ining the effect of the C313W mutation in our system, the data displayed in Figure 3B

clearly indicates that the size of the residue at this position is the most prominent factor,

albeit not the only one. No variant with a small amino acid at this position was able to

incorporate Sac. By contrast, the bulkier Phe allows Sac incorporation, though at a lower

level compared with all C313W mutants. Even with Met at this position, very low incor-

poration is detectable. The inactive C313H variant, comparable in size to C313F, suggests

that a non-polar residue is necessary to be present in this microenvironment. Overall, the

data collected suggest that a reduced size of the binding pocket containing the C313W

mutation is essential for Sac incorporation.

2.4. Rationalizing Sac Incorporation Data and Creating Aliphatic Substrate Activating

MbPylRS Variants

It was previously proposed that the C313W mutation is critical for activation of

smaller substrates [32,33]. This hypothesis was also fully recapitulated in this work. Hav-

ing thus established that the C313W mutation is critical for the incorporation of Sac (1)

and probably also smaller aliphatic Sac (1) analogs, all these mutants were tested for in-

corporation of substrates 2, 3, 5, 7, and 10 (Figure 4). These fluorescence data showed that

all variants, except the C313W mutant, did not incorporate the tested substrates.

Figure 4. Comparison of the efficiency of aliphatic ncAA incorporation for MbPylRS(C313W) constructs mutated at posi-

tion W382. The fluorescence was measured for intact E. coli BL21(DE3) cells producing the SUMO-sfGFP(R2amber) re-

porter protein. The data (incl. standard deviation) represent the mean of three biological replicates.

Figure 4.

Comparison of the efficiency of aliphatic ncAA incorporation for MbPylRS(C313W) constructs mutated at position

W382. The fluorescence was measured for intact E. coli BL21(DE3) cells producing the SUMO-sfGFP(R2amber) reporter

protein. The data (incl. standard deviation) represent the mean of three biological replicates.

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 17

Unfortunately, the construct that incorporated the aliphatic ncAAs also exhibited a

considerable level of cAA background incorporation. As mentioned previously, bulky

C313 mutations of MbPylRS lead to background Phe incorporation [20,34]. We hypothe-

sized that the key to restoring the lost orthogonality associated with the C313W mutation

would lie in the mutation of MbPylRS at position N311, which is also known to be one of

the two gatekeeper residues for Pyl activation. Our working hypothesis was to restore the

orthogonality by increasing the size of the amino acid at this position to interfere with the

Phe substrate accommodation while creating a catalytic pocket suitable for the short-chain

aliphatic ncAAs. As shown in Figure 5A, the orthogonality also increases by increasing

the AA size at the N311 position, until it is fully restored for the N311L:C313W variant.

An incorporation pattern of the tested aliphatic substrates was noticeable, most likely

corresponding to the size of the newly created catalytic pocket. The signal intensity for

the N311L:C313W variant suggests that substrate 2 is the optimal size for this pocket. This

substrate is the aliphatic equivalent to Sac (1). The data supported the hypothesis that it

was possible to restore orthogonality by replacing the residue at position N311 to bulkier

AAs. This finding encouraged us to test all amino acids larger than valine at this position.

The additional functional variants found are shown in Figure 5B. Figure 5 shows all the

obtained active variants. Two of them (N311M/Q:C313W) can incorporate some of the

substrates even better than the N311L:C313W construct. Interestingly, these two addi-

tional variants exhibit a different ncAA incorporation profile. The N311M construct favors

substrates with carbon chain length C6, while the N311Q favors C7. The incorporation

profile of the N311Q variant is similar to that of the N311L.

Figure 5. Comparison of aliphatic ncAA incorporation efficiency for MbPylRS(C313W) constructs mutated at position

N311. A) Construct N311A with increased binding pocket size and additional mutations that stepwise decrease the size

of the binding pocket . B) Four additional active variants, found after screening of all 19 (all cAAs besides glycine) possible

constructs. The fluorescence was measured for intact E. coli BL21(DE3) cells producing the SUMO-sfGFP(R2amber) re-

porter protein. The data (incl. standard deviation) represent the mean of three biological replicates.

The key residues found here, which are important for maintaining the orthogonality

of the PylRS system will facilitate future enzyme engineering efforts for the incorporation

of structural analogs. The information will enable the creation of smarter PylRS-libraries;

in particular, now a reduction in library size is feasible and will increase the likelihood of

finding desired enzymes. This will facilitate the establishment of ready-made enzyme

Figure 5.

Comparison of aliphatic ncAA incorporation efficiency for MbPylRS(C313W) constructs mutated at position

N311. (

) Construct N311A with increased binding pocket size and additional mutations that stepwise decrease the size of

the binding pocket. (

) Four additional active variants, found after screening of all 19 (all cAAs besides glycine) possible

constructs. The fluorescence was measured for intact E. coli BL21(DE3) cells producing the SUMO-sfGFP(R2amber) reporter

protein. The data (incl. standard deviation) represent the mean of three biological replicates.

An incorporation pattern of the tested aliphatic substrates was noticeable, most likely

corresponding to the size of the newly created catalytic pocket. The signal intensity for the

N311L:C313W variant suggests that substrate 2 is the optimal size for this pocket. This

substrate is the aliphatic equivalent to Sac (1). The data supported the hypothesis that it was

possible to restore orthogonality by replacing the residue at position N311 to bulkier AAs.

This finding encouraged us to test all amino acids larger than valine at this position. The

Int. J. Mol. Sci. 2021,22, 11194 8 of 17

additional functional variants found are shown in Figure 5B. Figure 5shows all the obtained

active variants. Two of them (N311M/Q:C313W) can incorporate some of the substrates

even better than the N311L:C313W construct. Interestingly, these two additional variants

exhibit a different ncAA incorporation profile. The N311M construct favors substrates

with carbon chain length C6, while the N311Q favors C7. The incorporation profile of the

N311Q variant is similar to that of the N311L.

The key residues found here, which are important for maintaining the orthogonality

of the PylRS system will facilitate future enzyme engineering efforts for the incorporation

of structural analogs. The information will enable the creation of smarter PylRS-libraries;

in particular, now a reduction in library size is feasible and will increase the likelihood

of finding desired enzymes. This will facilitate the establishment of ready-made enzyme

arsenals to test a wide variety of natural and synthetic ncAAs for translational activity.

In addition, for close structural analogues, the number and type of selection cycles (e.g.,

negative selection) can be reduced, making the overall selection process simpler and

more feasible.

2.5. Semi-Rational Engineering of PylRS Constructs for Small Aliphatic Substrate Incorporation

The previously described effort led to five constructs with efficient incorporation of

aliphatic ncAAs. We selected two of these constructs (N311M:C313W and N311Q:C313W)

for further engineering. The goal was to improve the incorporation efficiency and/or

specificity of the aliphatic substrates. For this reason, we targeted residues potentially in

close proximity to them. After inspecting the crystal structure (Figure 2), we planned to

randomize residues A267, V366, Y349, and W382 in the active site by site-saturation muta-

genesis (SSM) with NNK primers (N = A/T/C/G; K = G/T). We started with position V366

because it is located opposite to the N311 residue. Thereby we hypothesized that altering

V366 might have the strongest tuning effect with respect to aliphatic ncAA recognition.

The randomization of this position resulted in two new variants for each parent construct,

with mutations V366A/K (Figure 6).

Figure 6A shows the OTS performance of these constructs compared to the starting

N311M:C313W construct. The V366K variant did not show a strong difference in the

incorporation profile compared to the starting enzyme. Interestingly, the V366A mutation

resulted in a specificity shift toward long-chain aliphatic ncAAs in the incorporation profile.

This phenomenon seems plausible based on the MmPylRS crystal structures (Figure 2), since

this mutation potentially increases the space of the binding pocket, which should facilitate

the incorporation of longer substrates. Figure 6B shows that the two variants found, based

on the N311Q:C313W construct, performed comparably to the parent enzyme. Similar

to the N311M:C313W:V366A construct, there is also a slight shift in the incorporation

profile toward longer ncAAs observable for the V366A mutant. Since this variant already

favors ncAAs with C7 chain length over C6, the shift is smaller than for the N311M:C313W

construct. Unfortunately, screening of randomizations A267, Y349, and W382 did not yield

better performing variants. However, a variant with a markedly different incorporation

profile was found (N311M:C313W:W382H). This mutant preferably incorporated the longer

chained substrate 3 (Figure S6).

2.6. Evaluating the Incorporation of Biochemically Useful Aliphatic ncAA Analogs

We screened all generated MbPylRS constructs for incorporation of the aliphatic

ncAA/AA analogs listed in Figure 1(besides 2, 3, 5, 7 and 10). Substrates 1–9, 20–26, and 28

were highly incorporated (Figure 5, Figures S7–S9) and could be expressed in a standard E.

coli BL21(DE) protein production strain to higher levels (up to 21 mg/L protein per culture,

Table 1). Interestingly, several constructs also incorporated Sac (1) and substrate 27, with

the best Sac (1) incorporating construct being N311M:C313W:V366A (Figures S10–S12).

This result clearly illustrates that multiple substrate-recognizing enzyme topologies can

be based on the same scaffold. Since this variant was also able to incorporate 26, we were

encouraged to perform another round of randomization to find out if its performance is

Int. J. Mol. Sci. 2021,22, 11194 9 of 17

enhanced or if other interesting Cys-based amino acid derivatives were incorporated. This

approach led to the identification of N311M:C313W:V366A:W382N/T/Y mutants that were

able to successfully incorporate substrate 28.

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 17

arsenals to test a wide variety of natural and synthetic ncAAs for translational activity. In

addition, for close structural analogues, the number and type of selection cycles (e.g., neg-

ative selection) can be reduced, making the overall selection process simpler and more

feasible.

2.5. Semi-Rational Engineering of PylRS Constructs for Small Aliphatic Substrate Incorporation

The previously described effort led to five constructs with efficient incorporation of

aliphatic ncAAs. We selected two of these constructs (N311M:C313W and N311Q:C313W)

for further engineering. The goal was to improve the incorporation efficiency and/or spec-

ificity of the aliphatic substrates. For this reason, we targeted residues potentially in close

proximity to them. After inspecting the crystal structure (Figure 2), we planned to ran-

domize residues A267, V366, Y349, and W382 in the active site by site-saturation muta-

genesis (SSM) with NNK primers (N = A/T/C/G; K = G/T). We started with position V366

because it is located opposite to the N311 residue. Thereby we hypothesized that altering

V366 might have the strongest tuning effect with respect to aliphatic ncAA recognition.

The randomization of this position resulted in two new variants for each parent construct,

with mutations V366A/K (Figure 6).

Figure 6. Comparison of aliphatic ncAA incorporation efficiency for (A) MbPylRS(N311M:C313W)

and (B) MbPylRS(N311Q:C313W) constructs both mutated at position V366. The fluorescence was

measured for intact E. coli BL21(DE3) cells producing the SUMO-sfGFP(R2amber) reporter protein.

The data (incl. standard deviation) represent the mean of three biological replicates.

Figure 6.

Comparison of aliphatic ncAA incorporation efficiency for (

)MbPylRS(N311M:C313W) and

(

)MbPylRS(N311Q:C313W) constructs both mutated at position V366. The fluorescence was measured for intact E.

coli BL21(DE3) cells producing the SUMO-sfGFP(R2amber) reporter protein. The data (incl. standard deviation) represent

the mean of three biological replicates.

Substrates 11–19 with very low incorporation signals were additionally screened in release

factor 1 (RF1) knock-out strains JX33, B-95.

∆

A, and C321.

∆

A.exp (

Figures S13–S16

)

[36–38]

Usually, strains lacking RF1 produce higher amounts of full-length protein by amber

suppression. All of these RF1 knock-out strains were engineered in previous work of

our group to possess the lambda DE3 lysogen encoding the T7 polymerase compatible

with our reporter protein setup. Although background incorporation increased in all RF1

knockout strains (as previously observed see [

]), some setups resulted in an increased

ratio of ncAA/AA incorporation compared to background incorporation levels. The

best performing strain and MbPylRS combinations were selected for larger scale protein

production (Table 1).

Int. J. Mol. Sci. 2021,22, 11194 10 of 17

Table 1.

Optimal reporter protein production setup, calculated and observed molecular weights of the reporter proteins

His

-SUMO-sfGFP(R2AA)-strep (a) /SUMO-sfGFP(R2AA)-His

(b) and protein production yields per liter of culture. The

masses were determined by ESI-MS of intact proteins.

AA E. coli

Strains 1

MbPylRS

Construct

Reporter

Construct

Calculated

Mass [Da]

Observed

Mass [Da] ∆Mass [Da] Protein Yield

[mg·L−1]2

1BL21

N311M:C313W:V366A

a 40,194.9 40,196 1.1 10.8

2BL21 N311Q:C313W a 40,178.8 40,180 1.2 5.1

3BL21

N311M:C313W:V366A

a 40,192.9 40,194 1.1 1.6

4BL21

N311M:C313W:V366A

a 40,176.8 40,179 2.2 1.7

5BL21 N311M:C313W a 40,164.8 40,166 1.2 1.9

6BL21

N311M:C313W:V366K

a 40,162.8 40,164 1.2 1.4

7BL21 N311M:C313W a 40,150.8 40,153 2.2 1.2

8BL21 N311M:C313W a 40,148.8 40,150 1.2 0.7

9BL21 N311M:C313W a 40,162.8 40,164 1.2 1.4

10 BL21

N311M:C313W:V366A

a 40,136.8 40,195 58.2 0.8

11 C321.∆A.exp N311M:C313W b 38,990.9 38,992 1.1 5.1

12 C321.∆A.exp N311M:C313W b 38,976.9 38,979 2.1 4.9

13 C321.∆A.exp N311M:C313W b 38,962.8 38,965 2.2 11.3

14 JX33 N311M:C313W b 38,979.8 38,996 16.2 4.3

15 C321.∆A.exp N311M:C313W b 38,993.8 38,994 0.2 14.2

16 C321.∆A.exp N311M:C313W b439,007.9 39,007 0.9 5.3

17 C321.∆A.exp N311M:C313W b 39,021.9 38,997 24.9 6.4

18 C321.∆A.exp

N311Q:C313W:V366K

b 38,963.8 39,015 51.2 19

19 C321.∆A.exp

N311Q:C313W:V366K

b 38,977.8 39,014 36.2 19.9

20 BL21

N311M:C313W:V366K

a 40,190.8 40,194 3.2 4.8

21 BL21 N311M:C313W b 38,978.9 38,982 3.1 2.6

22 BL21 N311M:C313W b 38,998.9 38,998 0.9 3.6

23 BL21 N311Q:C313W b 39,014.9 39,012 2.9 4.6

24 BL21

N311Q:C313W:V366K

b 39,030.9 39,015 15.9 10.7

25 BL21 N311Q:C313W b 39,013 39,014 1 9.8

26 BL21

N311M:C313W:V366A

a 40,210.9 40,211 0.1 21

27 BL21

N311M:C313W:V366A

a3----

28 BL21

N311M:C313W:V366A:

W382N b 39,061 39,063 2 1.1

1all DE3, 2yield per liter of cell culture, 3was not purified, 4not the main peak

2.7. Analytics of Canonical/Non-Canonical Amino Acids Incorporation

Larger scale protein production was performed to confirm the results from the flu-

orescence assays in small-scale 96-well plates. The protein yields are in good agreement

with the trends observed in the fluorescence experiments. We acquired the mass spectra

of the intact reporter proteins via electrospray ionization mass spectrometry (ESI-MS). To

facilitate MS data evaluation for AAs with a low incorporation efficiency, a reporter protein

with a C-terminal His

-tag was used. Table 1shows the optimal setup of reporter protein

production, the ESI-MS data, and protein yields for each substrate. The corresponding de-

convoluted ESI-MS-spectra are shown in the supplementary information (

Figures S17–S42

The incorporation of all substrates but five (14, 17, 18, 19, and 24) was confirmed in the

MS analytics. For substrate 14, the molecular weight of incorporated AA is 146.3 g/mol

which is equivalent to that of glutamine (146.2 g/mol). It is known that near-cognate

suppressor tRNAs, like tRNA

Gln

, read amber codons to some extent [

]. This means

Gln is incorporated at amber sites when the OTS is not working and then frequently ob-

served in MS analytics. The high reporter protein yield for a non-functioning OTS is most

likely the result of the general higher background suppression observed in RF1 knock-out

strains (

Figures S13–S16

). Gln incorporation is also observed for the setup of substrate

16, 17. For substrates 18, 19, and 24, the molecular weight of the incorporated AA is

165.3/164.3 g/mol

, which is equivalent to phenylalanine (165.2 g/mol), indicating that this

PylRS leads to Phe incorporation when no or inefficient substrate is present. The observed

high protein yield for the setup of substrates 18 and 19 is probably caused by a combination

Int. J. Mol. Sci. 2021,22, 11194 11 of 17

of the Phe incorporation activity and the use of a RF1 knock-out strain, although further

analytics would be required here. The same is true for substrate 24, though on a lower level

since no RF1 knock-out strain was used. Nonetheless, the fluorescence data with different

MbPylRS constructs clearly show that the incorporation is possible (Figure S14).

In summary, in this study, we generated 28 MbPylRS variants which can incorporate

23 ncAAs and one cAA. To the best of our knowledge, 17 of these ncAAs (besides 1, 3, 4,

17, 27, and 28) were not ribosomally incorporated by amber suppression before and 20 of

them were not previously incorporated with the PylRS system [33,35,40,41].

3. Materials and Methods

3.1. Canonical and Non-Canonical Amino Acids

Canonical amino acids were purchased from Carl Roth. Non-canonical amino acids

were obtained from Fluorochem, Iris Biotech, Chempur, Sigma-Aldrich (Merck), Chiralix,

Toronto Research Chemicals, Carl Roth, Thermo Fisher Scientific and TCI Deutschland

(see Table S1).

3.2. Plasmid Vector Construction

All plasmids were assembled by Golden Gate cloning and confirmed by DNA se-

quencing. Plasmids harboring the OTS (aaRS/tRNA

Pyl

) were constructed by cloning the

target aaRS gene into the pTECH vector (Addgene plasmid #104073) [42].

3.3. Site-Directed and Site-Saturation Mutagenesis

Point mutations were introduced by non-overlapping inverse PCR [

]. Focused

MbPylRS gene libraries were also created with non-overlapping inverse PCR, but random-

ization was performed using mutagenic primers (with NNK, whereby N = A, T, G, or C;

K = G or T) at designated positions (A267, V366, F349, and W382).

3.4. Analysis of SUMO-sfGFP Expression by Intact Cell Fluorescence.

For the small-scale expression of reporter constructs, E. coli BL21(DE) cells were used.

Electrocompetent cells were transformed with the orthogonal translation system and re-

porter plasmids. LB agar plates for plating contained 1% glucose and corresponding antibi-

otics. Single colonies of clones were used for inoculation of 2 mL LB (in

14 mL

tubes) with

1% glucose and appropriate antibiotics and were grown to saturation overnight. Assays

were conducted in 96-well plate format. Cultures were added to each well at

1:100 dilution

in ZYP-5052 auto induction medium to a final volume of 100

L supplemented with an-

tibiotics and ncAAs. Cells were grown in black

-plates (Greiner Bio-One, Kremsmünster,

Austria) covered with a gas permeable foil (Breathe-Easy

, Diversified Biotech, Dedham,

MA, USA) with orbital shaking for 24 h at 37

◦

C. For endpoint measurements (Tecan M200,

Männedorf, Switzerland), the plate foil was removed and fluorescence was measured

with an 85 gain setting. For OD

600

measurements, 50

L of ZYP-5052 medium was pipet-

ted into clear 96-well

-plates and 50

L of culture was added. Excitation and emission

wavelengths for fluorescence measurements were set to 481 nm and 511 nm, respectively.

Fluorescence values were normalized to the corresponding OD

600

. Biological triplicates

were used for measurements of each aaRS construct. Relative fluorescence was normalized

to the highest value. The data including standard deviation represent the mean of three

biological replicates.

3.5. Library Screening

After library creation and transformation, 96 clones were picked and grown overnight

in a 96-well plate in 100

L LB with 1% glucose and appropriate antibiotics. The next day a

96-well plate with 100

L ZYP-5052, appropriate antibiotics, and ncAAs was inoculated

with 1

L culture, grown for 24 h and measured afterwards as stated above. The 96-plate

which was used for inoculation was sealed with aluminum foil and stored at 4

◦

C. From

this plate, desired clones were analyzed via PCR gene amplification and sequencing of

Int. J. Mol. Sci. 2021,22, 11194 12 of 17

this PCR product afterwards. Calculations with the Toplib tool estimate the probability of

finding the best performing variant to be 96% (using a yield of 85%, which is the lower limit

of primer purity and therefore also the lower yield limit of created DNA constructs) [44].

3.6. Protein Expression

For expression of the SUMO-sfGFP variants, E.coli strains were used in 10 mL ZYP-

5052 medium supplemented with 10 mM ncAA and appropriate antibiotics. The expression

medium was inoculated with a starter culture (1:100). Shake flasks were incubated for 24 h

at 37

◦

C while shaking at 200 rpm. Cells were harvested by centrifugation and stored at

–80 ◦C or directly used for protein purification.

3.7. Protein Purification

Harvested cell pellets were resuspended (50 mM sodium phosphate, 300 mM NaCl,

20 mM imidazole, pH 8.0) and lysed with B PER

Bacterial Protein Extraction Reagent

(Thermo Scientific, Waltham, MA, USA) according to their protocol, with addition of

phenylmethanesulfonyl fluoride (PMSF, 1 mM final concentration), DNAse, and RNAse.

Cleared lysates were loaded onto a equilibrated Ni-NTA column and purified via the

P-1 peristaltic pump (Pharmacia Biotech, now Cytiva, Marlborough, MA, USA ). After

washing with 10 column volumes of resuspension buffer, elution buffer (50 mM sodium

phosphate, 300 mM NaCl, 500 mM imidazole, pH 8.0) was applied to elute the his-tagged

target proteins. The first 2 mL covering the void volume was discarded. Afterwards,

the eluate (1 mL) was collected and dialyzed in cellulose film tubings against 1 L buffer

(

50 mM

sodium phosphate, 300 mM NaCl, pH 8.0) for at least 2 h with three buffer changes.

Concentrations of purified reporter proteins were determined by measuring the sfGFP

chromophore absorption at 488 nm.

3.8. ESI-MS

Intact protein mass measurements of purified SUMO-sfGFP variants were performed

by electrospray LC-MS on a Waters H-class instrument with a Waters Acquity UPLC

protein BEH C4 column (300 Å, 1.7

m, 2.1 mm

50 mm). The following gradient used

a flow rate of 0.3 mL/min: A: 0.01% formic acid in H

O; B: 0.01% formic acid in MeCN.

5–95% B 0–6 min. Mass analysis was conducted with a Waters Xevo G2-XS QTof analyzer.

Proteins were ionized in positive ion mode applying a cone voltage of 40 kV. Raw data

were analyzed employing the maximum entropy deconvolution algorithm. The data were

exported and plotted with QtiPlot (version 0.9.9.7).

4. Conclusions

We were motivated by previous works implying that the PylRS, naturally specialized

for large bulky substrates, could be redesigned for small substrates. Therefore, we elu-

cidated the structure–activity relationship of a specific MbPylRS (MbSacRS), designed to

incorporate the shortest non-bulky ncAA (S-allyl-L-Cysteine, 1) possible to date. Based on

this knowledge, we have designed MbPylRSs for the incorporation of aliphatic amino acids

and various derivatives thereof, which are useful in biochemistry and structural biology.

Some of the incorporated substrates (allylglycine, 8 and propargylglycine, 13) were recently

synthesized

in vivo

in E.coli and would, therefore, open up the possibility of coupling

metabolic engineering and ncAA incorporation [

]. This approach could eliminate the

need to add ncAAs to the cultivation medium, which would drastically decrease costs and

simplify associated applications.

In addition, valuable information was gathered about the role of specific residues in

the active site that are responsible for the orthogonality of PylRS. We were able to determine

key residues that maintain orthogonality upon engineering the MbPylRS enzyme to ac-

commodate smaller substrates. This achievement will facilitate future enzyme engineering

efforts for the incorporation of structural analogs. To this end, PylRS libraries can now

be created with a smaller size, which simultaneously increases the likelihood of finding

Int. J. Mol. Sci. 2021,22, 11194 13 of 17

the desired enzymes and reduces the amount of work required to do so by eliminating

negative selection.

Supplementary Materials:

The following tables and figures are available online at https://www.

mdpi.com/article/10.3390/ijms222011194/s1.

Author Contributions:

Conceived the project and conceptualization, N.B. and N.G.K.; methodology,

N.G.K.; software, N.G.K.; validation, N.G.K.; formal analysis, N.G.K.; investigation, N.G.K.; resources,

N.B.; data curation, N.G.K. and N.B.; writing—original draft preparation, N.G.K.; writing—review

and editing, N.G.K., N.B., P.G. and J.R.; visualization, N.G.K.; supervision, P.G., J.R. and N.B.; project

administration, P.G., J.R., N.B.; funding acquisition, P.G., J.R. and N.B. All authors have read and

agreed to the published version of the manuscript.

Funding:

N.G.K. acknowledges funding by the “IBÖM04: XenoGlue”-Consortium supported by

the Federal Ministry of Education and Research of Germany (Grant Number: FKZ 031B0584A).

This work was also performed as part of the “Site-directed cross-linking of KLK proteases from

prostate” Project funded by the Lead Agency FWF (I 3877-B21) with DFG-D-A-CH (BU1404/12-1)

(P. G., N.G.K). N.B. thanks Canada Research Chairs Program (Grant Nr. 950-231971) for support.

This work was partially supported by the Cluster of Excellence “Unifying Systems in Catalysis”

(UniSysCat), funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)

under Germany’s Excellence Strategy–EXC 2008/1–390540038 (N.B. and J. R.)”.

Acknowledgments:

We are very grateful to Philipp Ochtrop (Leibniz-Forschungsinstitut für Moleku-

lare Pharmakologie, Hackenberger Group) for his support in ESI-MS measurements. N.B. thanks to

Christian Thomsen, President of the Technical University of Berlin, for his continuous support.

Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

AA amino acid

AMP adenosine monophosphate

AMP-PNP adenosine-50-[(β,γ)-imido]triphosphate

aaRS aminoacyl-tRNA synthetase

cAA canonical amino acid

ESI-MS electrospray ionization mass spectrometry

FTIR Fourier-transform infrared spectroscopy

MjTyrRS Methanocaldococcus jannaschii tyrosyl-tRNA synthetase

MmPylRS/MbPylRS Methanosarcina mazei/barkeri pyrrolysyl-tRNA synthetase

ncAA non-canonical amino a

NMR nuclear magnetic resonance spectroscopy

OTS orthogonal translation system

Pyl pyrrolysine

PylRS pyrrolysyl-tRNA synthetase

Sac S-allyl-L-cysteine

sfGFP superfolder GFP

SmbP small metal-binding protein

RF1 release factor 1

Appendix A

Appendix A.1. General Features and Perspectives of ncAAs Used in This Study

As mentioned in the introduction, ncAAs with side chain functionality closer to the

protein backbone would be highly advantageous for various applications. We used an

entire library of small side chain containing ncAAs that can be structurally and functionally

categorized into five classes, as shown in Figure 1. (i) Aliphatic Sac (1) analogs for structure

activity elucidation (2, 3, 5, 7, 10). (ii) Site-specific bioorthogonal reaction handles that

can be used for a variety of bioconjugation reactions (4, 6, 8, 9, 11, 12, 13, 14, 15, 16, 17,

27). These reactions include metal-free (e.g., click-chemistry, Staudinger ligations and

strain-promoted cycloadditions) and transition metal-mediated (e.g., ruthenium-based

Int. J. Mol. Sci. 2021,22, 11194 14 of 17

olefin cross-metathesis or palladium based oxidative HECK and SONOGASHIRA cross-

coupling reactions) approaches [

]. (iii) ncAAs are/could be used as biophysical probes,

e.g., in vibrational Stark, IR, and NMR spectroscopy (18, 19, 26) and as genetically encoded

photo-crosslinker (20) [

]. (iv) Methionine analogs (23, 24, 25) as tools to elucidate

the role of methionine oxidation in proteins, enzymes, and cells [

]. (v) Substrates with a

terminal alkene as site-specific chemical cleavage sites (4, 6, 8, 21) [

–

]. For example, the

cleavage reaction with substrate 8 proceeds presumably via iodolactonization, suggesting

that this reaction could also proceed with substrates 6 and 8. The transition state would

change from a five-membered iodolactone to a 6/7 membered one [

–

]. In contrast to

the classical site-specific peptide cleavage with cyanogen bromide at a methionine position,

these ncAAs could be cleaved with non-toxic iodine under mild conditions [57].

In yeast, substrates 3 and 4 have already been incorporated with an orthogonal E. coli

leucyl-tRNA synthetase [

]. Although we used the five aliphatic ncAAs (2, 3, 5, 7, 10)

to estimate the size of the narrowed active site, some of these ncAAs could potentially

help to address certain questions regarding post-translational lipidation of proteins [

–

Lipidation is also a common strategy to improve the pharmacokinetic properties of biophar-

maceuticals, especially to prolong the systemic half-life in patients with a corresponding

increase in bioavailability [

]. This approach opens a potentially interesting biomedical

application area for theses ncAAs. Notably, ncAA substrates 5, 9, 12, 15, 17, 20, and 25 have

been incorporated in a residue specific manner (using auxotrophy-based methods) but

never in a site-specific mode, besides 17 [

–

]. Substrate 9 contains a cyclopropane

ring with properties closely resembling to those of an olefinic double bond, which could be

exploited for a wide range of site-specific bioorthogonal protein conjugation reactions [

These reactions include enzymatic halogenation with a haloperoxidase [

], enzymatic oxi-

dation with a mono-oxygenase [

], nucleophilic substitutions, electrophilic ring opening

reactions, and a plethora of other reactions [

]. Lastly, allylglycine (8) and propargyl-

glycine (13) have recently been synthesized

in vivo

in E. coli and would, therefore, open up

the possibility of coupling metabolic engineering and the incorporation of ncAA [

]. This

procedure could eliminate the need to add ncAAs to the cultivation medium, which would

drastically decrease costs and simplify associated applications.

References

Pagar, A.D.; Patil, M.D.; Flood, D.T.; Yoo, T.H.; Dawson, P.E.; Yun, H. Recent Advances in Biocatalysis with Chemical Modification

and Expanded Amino Acid Alphabet. Chem. Rev. 2021,121, 6173–6245. [CrossRef] [PubMed]

Groff, D.; Thielges, M.C.; Cellitti, S.; Schultz, P.G.; Romesberg, F.E. Efforts toward the direct experimental characterization of

enzyme microenvironments: Tyrosine 100 in dihydrofolate reductase. Angew. Chem. Int. Ed.

2009

,48, 3478–3481. [CrossRef]

[PubMed]

Baumann, T.; Hauf, M.; Schildhauer, F.; Eberl, K.B.; Durkin, P.M.; Deniz, E.; Löffler, J.G.; Acevedo-Rocha, C.G.; Jaric, J.; Martins,

B.M.; et al. Site-Resolved Observation of Vibrational Energy Transfer Using a Genetically Encoded Ultrafast Heater. Angew. Chem.

Int. Ed. 2019. [CrossRef] [PubMed]

Minnihan, E.C.; Young, D.D.; Schultz, P.G.; Stubbe, J. Incorporation of fluorotyrosines into ribonucleotide reductase using an

evolved, polyspecific aminoacyl-tRNA synthetase. J. Am. Chem. Soc. 2011,133, 15942–15945. [CrossRef] [PubMed]

Li, J.C.; Nastertorabi, F.; Xuan, W.; Han, G.W.; Stevens, R.C.; Schultz, P.G. A Single Reactive Noncanonical Amino Acid Is Able to

Dramatically Stabilize Protein Structure. ACS Chem. Biol. 2019,14, 1150–1153. [CrossRef]

Agostini, F.; Völler, J.S.; Koksch, B.; Acevedo-Rocha, C.G.; Kubyshkin, V.; Budisa, N. Biocatalysis with Unnatural Amino Acids:

Enzymology Meets Xenobiology. Angew. Chem. Int. Ed. 2017,56, 9680–9703. [CrossRef]

Drienovská, I.; Mayer, C.; Dulson, C.; Roelfes, G. A designer enzyme for hydrazone and oxime formation featuring an unnatural

catalytic aniline residue. Nat. Chem. 2018,10, 946–952. [CrossRef]

Burke, A.J.; Lovelock, S.L.; Frese, A.; Crawshaw, R.; Ortmayer, M.; Dunstan, M.; Levy, C.; Green, A.P. Design and evolution of an

enzyme with a non-canonical organocatalytic mechanism. Nature 2019,570, 219–223. [CrossRef]

9. Liu, C.C.; Schultz, P.G. Adding New Chemistries to the Genetic Code. Annu. Rev. Biochem. 2010,79, 413–444. [CrossRef]

10. Mukai, T.; Lajoie, M.J.; Englert, M.; Söll, D. Rewriting the Genetic Code. Annu. Rev. Microbiol. 2017,71, 557–577. [CrossRef]

11. Chin, J.W. Expanding and reprogramming the genetic code. Nature 2017,550, 53–60. [CrossRef] [PubMed]

12.

Wan, W.; Tharp, J.M.; Liu, W.R. Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion

tool. Biochim. Biophys. Acta Proteins Proteom. 2014,1844, 1059–1070. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2021,22, 11194 15 of 17

13.

Yanagisawa, T.; Ishii, R.; Fukunaga, R.; Kobayashi, T.; Sakamoto, K.; Yokoyama, S. Multistep Engineering of Pyrrolysyl-tRNA

Synthetase to Genetically Encode N

-(o-Azidobenzyloxycarbonyl) lysine for Site-Specific Protein Modification. Chem. Biol.

2008

15, 1187–1197. [CrossRef]

14.

Yanagisawa, T.; Kuratani, M.; Seki, E.; Hino, N.; Sakamoto, K.; Yokoyama, S. Structural Basis for Genetic-Code Expansion with

Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase. Cell Chem. Biol.

2019

,26, 936–949. [CrossRef] [PubMed]

15.

Guo, L.T.; Wang, Y.S.; Nakamura, A.; Eiler, D.; Kavran, J.M.; Wong, M.; Kiessling, L.L.; Steitz, T.A.; O’Donoghue, P.; Söll, D.

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution. Proc. Natl. Acad. Sci. USA

2014

,111, 16724–16729. [CrossRef]

16.

Wang, Y.S.; Fang, X.; Wallace, A.L.; Wu, B.; Liu, W.R. A rationally designed pyrrolysyl-tRNA synthetase mutant with a broad

substrate spectrum. J. Am. Chem. Soc. 2012,134, 2950–2953. [CrossRef]

17.

Tseng, H.; Baumann, T.; Sun, H.; Wang, Y.; Ignatova, Z.; Budisa, N. Expanding the Scope of Orthogonal Translation with

Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids. Molecules 2020,25, 4418. [CrossRef]

18.

Xiao, H.; Peters, F.B.; Yang, P.Y.; Reed, S.; Chittuluru, J.R.; Schultz, P.G. Genetic incorporation of histidine derivatives using an

engineered pyrrolysyl-tRNA synthetase. ACS Chem. Biol. 2014,9, 1092–1096. [CrossRef]

19.

Völler, J.S.; Biava, H.; Hildebrandt, P.; Budisa, N. An expanded genetic code for probing the role of electrostatics in enzyme

catalysis by vibrational Stark spectroscopy. Biochim. Biophys. Acta Gen. Subj. 2017,1861, 3053–3059. [CrossRef]

20.

Kavran, J.M.; Gundllapalli, S.; O’Donoghue, P.; Englert, M.; Söll, D.; Steitz, T.A. Structure of pyrrolysyl-tRNA synthetase, an

archaeal enzyme for genetic code innovation. Proc. Natl. Acad. Sci. USA 2007,104, 11268–11273. [CrossRef] [PubMed]

21.

Trudeau, D.L.; Tawfik, D.S. Protein engineers turned evolutionists—the quest for the optimal starting point. Curr. Opin. Biotechnol.

2019,60, 46–52. [CrossRef] [PubMed]

22.

Arnold, F.H. Directed Evolution: Bringing New Chemistry to Life. Angew. Chem. Int. Ed.

2018

,57, 4143–4148. [CrossRef]

[PubMed]

23.

Bloom, J.D.; Labthavikul, S.T.; Otey, C.R.; Arnold, F.H. Protein stability promotes evolvability. Proc. Natl. Acad. Sci. USA

2006

103, 5869–5874. [CrossRef] [PubMed]

24.

Tokuriki, N.; Tawfik, D.S. Stability effects of mutations and protein evolvability. Curr. Opin. Struct. Biol.

2009

,19, 596–604.

[CrossRef]

25.

Grasso, K.T.; Yeo, M.J.R.; Hillenbrand, C.M.; Ficaretta, E.D.; Italia, J.S.; Huang, R.L.; Chatterjee, A. Structural Robustness Affects

the Engineerability of Aminoacyl-tRNA Synthetases for Genetic Code Expansion. Biochemistry 2021,60, 489–493. [CrossRef]

26.

Hu, L.; Qin, X.; Huang, Y.; Cao, W.; Wang, C.; Wang, Y.; Ling, X.; Chen, H.; Wu, D.; Lin, Y.; et al. Thermophilic Pyrrolysyl-tRNA

Synthetase Mutants for Enhanced Mammalian Genetic Code Expansion. ACS Synth. Biol. 2020,9, 2723–2736. [CrossRef]

27.

Yanagisawa, T.; Ishii, R.; Fukunaga, R.; Kobayashi, T.; Sakamoto, K.; Yokoyama, S. Crystallographic Studies on Multiple

Conformational States of Active-site Loops in Pyrrolysyl-tRNA Synthetase. J. Mol. Biol. 2008,378, 634–652. [CrossRef]

28.

Vargas-Cortez, T.; Morones-Ramirez, J.R.; Balderas-Renteria, I.; Zarate, X. Expression and purification of recombinant proteins in

Escherichia coli tagged with a small metal-binding protein from Nitrosomonas europaea. Protein Expr. Purif.

2016

,118, 49–54.

[CrossRef]

29.

Ko, J.H.; Wang, Y.S.; Nakamura, A.; Guo, L.T.; Söll, D.; Umehara, T. Pyrrolysyl-tRNA synthetase variants reveal ancestral

aminoacylation function. FEBS Lett. 2013,587, 3243–3248. [CrossRef]

30.

Baumann, T.; Hauf, M.; Richter, F.; Albers, S.; Möglich, A.; Ignatova, Z.; Budisa, N. Computational aminoacyl-tRNA synthetase

library design for photocaged tyrosine. Int. J. Mol. Sci. 2019,20, 2343. [CrossRef]

31.

Owens, A.E.; Grasso, K.T.; Ziegler, C.A.; Fasan, R. Two-Tier Screening Platform for Directed Evolution of Aminoacyl–tRNA

Synthetases with Enhanced Stop Codon Suppression Efficiency. ChemBioChem 2017,18, 1109–1116. [CrossRef] [PubMed]

32.

Takimoto, J.K.; Dellas, N.; Noel, J.P.; Wang, L. Stereochemical Basis for Engineered Pyrrolysyl-tRNA Synthetase and the Efficient

in Vivo Incorporation of Structurally Divergent Non-native Amino Acids. ACS Chem. Biol. 2011,6, 733–743. [CrossRef]

33.

Exner, M.P.; Kuenzl, T.; To, T.M.T.; Ouyang, Z.; Schwagerus, S.; Hoesl, M.G.; Hackenberger, C.P.R.; Lensen, M.C.; Panke, S.;

Budisa, N. Design of S-Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl-tRNA Synthetase Variant.

ChemBioChem 2017,18, 85–90. [CrossRef] [PubMed]

34.

Wang, Y.S.; Russell, W.K.; Wang, Z.; Wan, W.; Dodd, L.E.; Pai, P.J.; Russell, D.H.; Liu, W.R. The de novo engineering of pyrrolysyl-

tRNA synthetase for genetic incorporation of l-phenylalanine and its derivatives. Mol. Biosyst.

2011

,7, 714–717. [CrossRef]

[PubMed]

35.

Liu, J.; Cheng, R.; Wu, H.; Li, S.; Wang, P.G.; DeGrado, W.F.; Rozovsky, S.; Wang, L. Building and Breaking Bonds via a Compact

S-Propargyl-Cysteine to Chemically Control Enzymes and Modify Proteins. Angew. Chem. Int. Ed.

2018

,57, 12702–12706.

[CrossRef] [PubMed]

36.

Johnson, D.B.F.; Xu, J.; Shen, Z.; Takimoto, J.K.; Schultz, M.D.; Schmitz, R.J.; Xiang, Z.; Ecker, J.R.; Briggs, S.P.; Wang, L. RF1

knockout allows ribosomal incorporation of unnatural amino acids at multiple sites. Nat. Chem. Biol.

2011

,7, 779–786. [CrossRef]

[PubMed]

37.

Mukai, T.; Hoshi, H.; Ohtake, K.; Takahashi, M.; Yamaguchi, A.; Hayashi, A.; Yokoyama, S.; Sakamoto, K. Highly reproductive

Escherichia coli cells with no specific assignment to the UAG codon. Sci. Rep. 2015,5, 9699. [CrossRef]

38.

Lajoie, M.J.; Rovner, A.J.; Goodman, D.B.; Aerni, H.R.; Haimovich, A.D.; Kuznetsov, G.; Mercer, J.A.; Wang, H.H.; Carr, P.A.;

Mosberg, J.A.; et al. Genomically recoded organisms expand biological functions. Science 2013,342, 357–360. [CrossRef]

Int. J. Mol. Sci. 2021,22, 11194 16 of 17

39.

O’Donoghue, P.; Prat, L.; Heinemann, I.U.; Ling, J.; Odoi, K.; Liu, W.R.; Söll, D. Near-cognate suppression of amber, opal and

quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion. FEBS Lett. 2012,586, 3931–3937. [CrossRef]

40.

Ai, H.W.; Shen, W.; Brustad, E.; Schultz, P.G. Genetically encoded alkenes in yeast. Angew. Chem. Int. Ed.

2010

,49, 935–937.

[CrossRef]

41.

Wang, Y.; Chen, X.; Cai, W.; Tan, L.; Yu, Y.; Han, B.; Li, Y.; Xie, Y.; Su, Y.; Luo, X.; et al. Expanding the Structural Diversity

of Protein Building Blocks with Noncanonical Amino Acids Biosynthesized from Aromatic Thiols. Angew. Chem.

2021

,133,

10128–10136. [CrossRef]

42.

Bryson, D.I.; Fan, C.; Guo, L.T.; Miller, C.; Söll, D.; Liu, D.R. Continuous directed evolution of aminoacyl-tRNA synthetases. Nat.

Chem. Biol. 2017,13, 1253–1260. [CrossRef] [PubMed]

43.

Dominy, C.N.; Andrews, D.W. Site-Directed Mutagenesis by Inverse PCR. In E. coli Plasmid Vectors; Humana Press: Totowa, NJ,

USA, 2003; pp. 209–224.

44.

Nov, Y. When second best is good enough: Another probabilistic look at saturation mutagenesis. Appl. Environ. Microbiol.

2012

78, 258–262. [CrossRef] [PubMed]

45.

Marchand, J.A.; Neugebauer, M.E.; Ing, M.C.; Lin, C.-I.; Pelton, J.G.; Chang, M.C.Y. Discovery of a pathway for terminal-alkyne

amino acid biosynthesis. Nature 2019,567, 420–424. [CrossRef] [PubMed]

46.

Boutureira, O.; Bernardes, G.J.L. Advances in chemical protein modification. Chem. Rev.

2015

,115, 2174–2195. [CrossRef]

[PubMed]

47.

Chen, W.-N.; Kuppan, K.V.; Lee, M.D.; Jaudzems, K.; Huber, T.; Otting, G. O-tert -Butyltyrosine, an NMR Tag for High-Molecular-

Weight Systems and Measurements of Submicromolar Ligand Binding Affinities. J. Am. Chem. Soc.

2015

,137, 4581–4586.

[CrossRef]

48.

Mishra, P.K.; Yoo, C.; Hong, E.; Rhee, H.W. Photo-crosslinking: An Emerging Chemical Tool for Investigating Molecular Networks

in Live Cells. ChemBioChem 2020,21, 924–932. [CrossRef]

49.

Kim, G.; Weiss, S.J.; Levine, R.L. Methionine oxidation and reduction in proteins. Biochim. Biophys. Acta Gen. Subj.

2014

,1840,

901–905. [CrossRef]

50.

Wang, B.; Lodder, M.; Zhou, J.; Baird, T.T.; Brown, K.C.; Craik, C.S.; Hecht, S.M. Chemically Mediated Site-Specific Cleavage of

Proteins. J. Am. Chem. Soc. 2000,122, 7402–7403. [CrossRef]

51.

Baird, T.; Wang, B.; Lodder, M.; Hecht, S.M.; Craik, C.S. Generation of Active Trypsin by Chemical Cleavage. Tetrahedron

2000

,56,

9477–9485. [CrossRef]

52.

Wang, B.; Brown, K.C.; Lodder, M.; Craik, C.S.; Hecht, S.M. Chemically Mediated Site-Specific Proteolysis. Alteration of

Protein−Protein Interaction†. Biochemistry 2002,41, 2805–2813. [CrossRef] [PubMed]

53.

Liutkus, M.; Fraser, S.A.; Caron, K.; Stigers, D.J.; Easton, C.J. Peptide Synthesis through Cell-Free Expression of Fusion Proteins

Incorporating Modified Amino Acids as Latent Cleavage Sites for Peptide Release. ChemBioChem

2016

,17, 908–912. [CrossRef]

[PubMed]

54.

Nolsøe, J.M.J.; Hansen, T.V. Asymmetric Iodolactonization: An Evolutionary Account. Eur. J. Org. Chem.

2014

,2014, 3051–3065.

[CrossRef]

55.

Kristianslund, R.; Tungen, J.E.; Hansen, T.V. Catalytic enantioselective iodolactonization reactions. Org. Biomol. Chem.

2019

,17,

3079–3092. [CrossRef] [PubMed]

56.

Lodder, M.; Golovine, S.; Laikhter, A.L.; Karginov, V.A.; Hecht, S.M. Misacylated Transfer RNAs Having a Chemically Removable

Protecting Group. J. Org. Chem. 1998,63, 794–803. [CrossRef] [PubMed]

57.

Gross, E.; Witkop, B. Nonenzymatic cleavage of peptide bonds: The methionine residues in bovine pancreatic ribonuclease. J.

Biol. Chem. 1962,237, 1856–1860. [CrossRef]

58.

Jiang, H.; Zhang, X.; Chen, X.; Aramsangtienchai, P.; Tong, Z.; Lin, H. Protein Lipidation: Occurrence, Mechanisms, Biological

Functions, and Enabling Technologies. Chem. Rev. 2018,118, 919–988. [CrossRef]

59.

Chen, J.J.; Boehning, D. Protein lipidation as a regulator of apoptotic calcium release: Relevance to cancer. Front. Oncol.

2017

,7,

138. [CrossRef]

60.

Coleman, D.T.; Gray, A.L.; Kridel, S.J.; Cardelli, J.A. Palmitoylation regulates the intracellular trafficking and stability of c-Met.

Oncotarget 2016,7, 32664–32677. [CrossRef]

61. Hang, H.C.; Linder, M.E. Exploring Protein Lipidation with Chemical Biology. Chem. Rev. 2011,111, 6341–6358. [CrossRef]

62.

Bech, E.M.; Pedersen, S.L.; Jensen, K.J. Chemical Strategies for Half-Life Extension of Biopharmaceuticals: Lipidation and Its

Alternatives. ACS Med. Chem. Lett. 2018,9, 577–580. [CrossRef]

63.

Acevedo-Rocha, C.G.; Geiermann, A.-S.; Budisa, N.; Merkel, L. Design of protein congeners containing

-cyclopropylalanine.

Mol. Biosyst. 2012,8, 2719. [CrossRef]

64.

Budisa, N.; Steipe, B.; Demange, P.; Eckerskorn, C.; Kellermann, J.; Huber, R. High-level Biosynthetic Substitution of Methionine

in Proteins by its Analogs 2-Aminohexanoic Acid, Selenomethionine, Telluromethionine and Ethionine in Escherichia coli. Eur. J.

Biochem. 1995,230, 788–796. [CrossRef]

65.

Kiick, K.L.; Saxon, E.; Tirrell, D.A.; Bertozzi, C.R. Incorporation of azides into recombinant proteins for chemoselective modifica-

tion by the Staudinger ligation. Proc. Natl. Acad. Sci. USA 2002,99, 19–24. [CrossRef]

Int. J. Mol. Sci. 2021,22, 11194 17 of 17

66.

Tanrikulu, I.C.; Schmitt, E.; Mechulam, Y.; Goddard, W.A.; Tirrell, D.A. Discovery of Escherichia coli methionyl-tRNA synthetase

mutants for efficient labeling of proteins with azidonorleucine

in vivo

.Proc. Natl. Acad. Sci. USA

2009

,106, 15285–15290.

[CrossRef]

67.

Wang, Z.A.; Kurra, Y.; Wang, X.; Zeng, Y.; Lee, Y.; Sharma, V.; Lin, H.; Dai, S.Y.; Liu, W.R. A Versatile Approach for Site-Specific

Lysine Acylation in Proteins. Angew. Chem. Int. Ed. 2017,56, 1643–1647. [CrossRef]

68. Salaün, J. Cyclopropane Derivatives and their Diverse Biological Activities. Top. Curr. Chem. 2000,207, 1–67.

69.

Geigert, J.; Neidleman, S.L.; Dalietos, D.J. Novel haloperoxidase substrates. Alkynes and cyclopropanes. J. Biol. Chem.

1983

,258,

2273–2277. [CrossRef]

70.

Dalton, H.; Golding, B.T.; Waters, B.W.; Higgins, R.; Taylor, J.A. Oxidations of cyclopropane, methylcyclopropane, and arenes

with the mono-oxygenase system from Methylococcus capsulatus. J. Chem. Soc. Chem. Commun. 1981,28, 482. [CrossRef]