Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino Acids

Article abstract of DOI:10.1016/j.chembiol.2019.08.006

Imidazole glycerol phosphate synthase (ImGPS) is an allosteric bienzyme complex in which substrate binding to the synthase subunit HisF stimulates the glutaminase subunit HisH. To control this stimulation with light, we have incorporated the photo-respons

Full text of DOI:10.1016/j.chembiol.2019.08.006

Article
Light Regulation of Enzyme Allostery through Photo-
responsive Unnatural Amino Acids
Graphical Abstract
Authors
Andrea C. Kneuttinger, Kristina Straub,
¨
Philipp Bittner, ..., Burkhard Konig,
Rainer Merkl, Reinhard Sterner
Correspondence
reinhard.sterner@ur.de
In Brief
Within the bienzyme complex imidazole
glycerol phosphate synthase, the activity
of the subunit HisH was light controlled by
photo-responsive unnatural amino acids
located in HisF. Our findings demonstrate
that light regulation of allostery can be
used as a powerful tool for the
spatiotemporal control of metabolic
enzymes.
Highlights
d
d
d
Allostery in the HisH-HisF bienzyme complex was regulated
by light
Photo-sensitive unnatural amino acids in HisF were used to
control HisH activity
HisH activity depends on the conformational organization of
the catalytic H178
Kneuttinger et al., 2019, Cell Chemical Biology 26, 1–14
November 21, 2019 ª 2019 Elsevier Ltd.
https://doi.org/10.1016/j.chembiol.2019.08.006

Please cite this article in press as: Kneuttinger et al., Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino Acids, Cell
Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.08.006
Cell Chemical Biology
Article
Light Regulation of Enzyme
Allostery through Photo-responsive
Unnatural Amino Acids
Andrea C. Kneuttinger,1 Kristina Straub,1 Philipp Bittner,2 Nadja A. Simeth,2,3 Astrid Bruckmann,4 Florian Busch,5
1
1
5
6
2
1
¨
Chitra Rajendran, Enrico Hupfeld, Vicki H. Wysocki, Dominik Horinek, Burkhard Konig, Rainer Merkl,
and Reinhard Sterner1,7,
*
Institute of Biophysics and Physical Biochemistry, University of Regensburg, Universitatsstrasse 31, 93053 Regensburg, Germany
1
€
2
€
Institute of Organic Chemistry, University of Regensburg, Universitatsstrasse 31, 93053 Regensburg, Germany
3Centre for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands
4
€
Institute of Biochemistry, Genetics and Microbiology, University of Regensburg, Universitatsstrasse 31, 93053 Regensburg, Germany
5Department of Chemistry and Biochemistry and Resource for Native Mass Spectrometry Guided Structural Biology, The Ohio State
University, Columbus, OH 43210, USA
6
€
Institute of Physical and Theoretical Chemistry, University of Regensburg, Universitatsstrasse 31, 93053 Regensburg, Germany
7Lead Contact
*Correspondence: reinhard.sterner@ur.de
https://doi.org/10.1016/j.chembiol.2019.08.006
SUMMARY
2019). Less consideration has been dedicated to the photo-con-
trol of allosteric interactions, which are crucial regulatory features
Imidazole glycerol phosphate synthase (ImGPS) is an of enzymes in practically all metabolic pathways (Kastritis and
Gavin, 2018). Allostery in enzyme complexes describes the bind-
ing of a ligand to one subunit by which the activity of another,
allosteric bienzyme complex in which substrate
binding to the synthase subunit HisF stimulates the
glutaminase subunit HisH. To control this stimulation
with light, we have incorporated the photo-respon-
associated subunit is influenced (Makhlynets et al., 2015). Thus,
light regulation of allostery becomes most interesting for the tem-
poral control of synthetic processes in industrial biocatalysis,
sive unnatural amino acids phenylalanine-40-azoben-
such as in enzyme cascades that generally mimic metabolic
pathways (Schmidt-Dannert and Lopez-Gallego, 2016).
zene (AzoF), o-nitropiperonyl-O-tyrosine (NPY), and
methyl-o-nitropiperonyllysine (mNPK) at strategic
We have recently described photo-control of allostery within the
bienzyme complex imidazole glycerol phosphate synthase
positions of HisF. The light-mediated isomerization
of AzoF at position 55 (fS55AzoFE 4 fS55AzoFZ) re-
(ImGPS) from Thermotoga maritima (Figure 1A) (Kneuttinger
et al., 2018). ImGPS consists of the glutaminase subunit HisH,
sulted in a reversible 10-fold regulation of HisH activ-
ity. The light-mediated decaging of NPY at position which lacks measurable activity as a monomer, and the cyclase
subunit HisF, which poorly activates HisH by complexation (List
et al., 2012). Although the active sites of the two subunits are
˚
25 A apart (Douangamath et al., 2002), significant allosteric
39 (fY39NPY / fY39) and of mNPK at position 99
(fK99mNPK / fK99) led to a 4- to 6-fold increase
of HisH activity. Molecular dynamics simulations ex-
plained how the unnatural amino acids interfere with
the allosteric machinery of ImGPS and revealed addi-
tional aspects of HisH stimulation in wild-type
ImGPS. Our findings show that unnatural amino
acids can be used as a powerful tool for the spatio-
temporal control of a central metabolic enzyme com-
plex by light.
stimulation of HisH is initiated by binding of the HisF substrate
N’-[(50-phosphoribulosyl)formimino]-5-aminoimidazole-4-carbox-
amide ribonucleotide (PrFAR) (Lisi et al., 2017) or its analog
N’-[(50-phosphoribosyl)formimino]-5-aminoimidazole-4-carbox-
amide ribonucleotide (ProFAR) (List et al., 2012). Several compu-
tational studies have described an allosteric network that
connects the active sites of HisF and HisH and might transmit
the stimulation signal (Rivalta et al., 2012; VanWart et al., 2012;
Lisi et al., 2016; Negre et al., 2018). At the stimulation endpoint,
chemical activation of glutamine catalysis in HisH has been
postulated to occur through a backbone flip in the substrate
binding site that leads to the stabilization of the oxyanion reac-
INTRODUCTION
In the last decade, regulation of enzyme activity by light has tion intermediate (Chaudhuri et al., 2001, 2003; Lipchock and
received increasing attention in the field of synthetic biology. Loria, 2010; Rivalta et al., 2012). Activated HisH then hydrolyzes
Various approaches have been presented, which range from glutamine to glutamate and ammonia, which subsequently
the binding of light-responsive ligands at the active site to the travels through an intermolecular channel to the active site of
fusion of an enzyme with a light-oxygen-voltage sensing domain HisF (Douangamath et al., 2002). Ammonia there reacts
ꢀ
€
(Szymanski et al., 2013; Baker and Deiters, 2014; Hull et al., 2018; with PrFAR to produce imidazole glycerol phosphate (ImGP)
Losi et al., 2018; Lachmann et al., 2019; Schmermund et al., and 5-aminoimidazol-4-carboxamidribotide (AICAR), which are
Cell Chemical Biology 26, 1–14, November 21, 2019 ª 2019 Elsevier Ltd.
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A
B
C
Figure 1. Implementation Strategy of Allosteric Light Regulation of ImGPS by UAAs
(A) Activity cycle of the bienzyme complex ImGPS. PrFAR (barbell) binding to HisF (blue) stimulates HisH (purple) (activation). Glutamine is then turned over to
glutamate producing ammonia. After ammonia channeling through the interior of the central b barrel of HisF (dark blue), the active site of HisF cleaves PrFAR into
5-aminoimidazol-4-carboxamidribotide (AICAR) and ImGP (barbell fragments). Dissociation of the HisF products renders HisH inactive (deactivation).
(B) Four light-responsive unnatural amino acids (UAAs) were incorporated into HisF. AzoF isomerizes at the azo-bond (bold) from its E state (shown) to its Z state.
NBY, NPY, and mNPK cleave off their caging group (bold) upon irradiation, which sets free tyrosine or lysine. (In the case of mNPK, carbon dioxide (gray) is also
released.)
(C) To regulate the HisH reaction with light, a UAA is incorporated into HisF disrupting the activating allosteric machinery. Upon irradiation, the UAA isomerizes
(AzoF) or loses its caging group (NBY, NPY, or mNPK), restoring allosteric activation of HisH activity. This light-induced activation step is irreversible using the
caged UAAs, but can be reversed with AzoF (dashed arrow).
used for histidine and de novo purine biosynthesis, respectively.
The most commonly used light-responsive UAAs bear a natu-
By associating a photo-responsive ligand to the active site of ral amino acid as a scaffold caged with a photolabile protecting
HisF, we inhibited proper binding of PrFAR and were, hence, group (Figure 1B) (Curley and Lawrence, 1999; Courtney and
able to photo-control the allosterically stimulated activity of Deiters, 2018; Bardhan and Deiters, 2019). Irradiation with UV
HisH by a factor of 2 (Kneuttinger et al., 2018).
light induces a decaging reaction, setting free the natural amino
´
We have now established a more efficient and versatile light acid (Klan et al., 2013). One of the first caged UAA that has been
regulation of allostery within the ImGPS complex using transla- incorporated into proteins was o-nitrobenzyl-O-tyrosine (NBY)
tional incorporation of unnatural amino acids (UAAs). By (Deiters et al., 2006). Disadvantages of this UAA, however,
means of orthogonal tRNACUA/aminoacyl-tRNA-synthetase include slow and incomplete decaging, which could be improved
pairs, UAAs can be site-specifically incorporated into proteins, by the addition of a methylenedioxy moiety to form o-nitropiper-
providing new opportunities for synthetic biology (Liu and onyl-O-tyrosine (NPY) and by an additional methyl substituent
Schultz, 2010). Although light-responsive UAAs have been im- (Berroy et al., 2001; Luo et al., 2017). Selective incorporation
plemented by targeting positions that are directly associated has also been accomplished for other UAAs using, for example,
with the active site of an enzyme either as a substrate (Lemke serine (Lemke et al., 2007), cysteine (Uprety et al., 2014), or lysine
et al., 2007), a catalytic residue (Luo et al., 2017), or a residue (methyl-o-nitropiperonyllysine [mNPK]) (Gautier et al., 2010) as a
close to substrate binding (Luo et al., 2017; Schlesinger et al., scaffold. A general drawback of caged UAAs from the perspec-
2018; Wang et al., 2019), light-responsive UAAs have hitherto tive of light regulation is the irreversibility of the decaging reac-
only rarely been used to control allostery (Luo et al., 2018).
tion. For reversible photo-control, the azobenzene moiety has
2
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been successfully integrated into many biologically relevant mol- gate (*fR5 and *fK99), and the interface hinge (fD74) (Figure 2A).
ecules (Morstein et al., 2019; Schmermund et al., 2019) and spe- NBY was used to replace tyrosine *fY39, phenylalanine *fF23,
cifically incorporated into proteins as a derivative of phenylala- and aspartate fD74 (a phenylalanine in yeast ImGPS), whereas
nine (AzoF) (Bose et al., 2006). Irradiation with UV light mNPK was used to replace lysines fK13, *fK19, and *fK99. For
converts its E isomer into a Z isomer, which is thermodynamically the design of AzoF replacements, we first calculated its rotamer
less stable and reverts upon irradiation with light above 400 nm library and incorporated AzoF into each of the ten positions
(Zimmerman et al., 1958). The switching occurs rapidly, with in silico. The nine residues where the rotamers minimally overlap-
ꢀ
high quantum yields and little photobleaching (Szymanski ped with van der Waals radii of neighboring residues were re-
et al., 2013). placed by AzoF (fR5, fK13, fK19, fF23, fS29, fL35, fY39, fS55,
Our strategy to photo-control allosteric stimulation of HisH in and fK99). Fifteen UAA-HisFs were then expressed in Escheri-
ImGPS via the incorporation of UAAs in HisF (AzoF, NBY, NPY, chia coli cells using previously designed tRNACUA/aminoacyl-
and mNPK) is schematically outlined in Figure 1C. With the inten- tRNA-synthetase pairs; these pairs have been shown to scarcely
tion to disturb activation of HisH by HisF, we chose positions close incorporate endogenous, natural amino acids, while each UAA
to three known sites that are part of the previously described allo- was incorporated with high efficiency (Bose et al., 2006; Deiters
steric network (Rivalta et al., 2012; VanWart et al., 2012; Lisi et al., et al., 2006; Gautier et al., 2010). Purification resulted in yields of
2016; Negre et al., 2018). Furthest away from the HisH active site, 1–87 mg/L expression medium. UAA incorporation was analyzed
the highly flexible loop 1 of HisF is thought to be involved in by tryptic digest coupled to mass spectrometry (MS) analysis
coupling both catalytic activities of HisF and HisH (Douangamath and for fK13AzoF with UV-visible spectroscopy (Figure S1).
et al., 2002). Mutational studies (Beismann-Driemeyer and The two variants fK13mNPK and fK19mNPK were discarded af-
Sterner, 2001) and NMR experiments (Lisi et al., 2016) have sup- ter this step, because we could not confirm their identity. For the
ported the significance of loop 1 and suggested that its dynamics sake of brevity, we use the term fposUAA to name a HisF protein
(Lisi et al., 2016) and interactions (Rivalta et al., 2012) with hydro- containing a UAA at position pos, e.g., fS55AzoF designates
phobic residues in the core of HisF change upon PrFAR binding. the HisF protein with AzoF at position S55; likewise Im-
The other two sites are associated with PrFAR-stimulated motions GPS(fS55AzoF) designates the ImGPS complex containing
at the HisH:HisF interface. First, for ammonia to pass into the inter- AzoF at position S55 of HisF.
molecular channel, the gate at the entrance, composed of resi-
The remaining 13 UAA-HisFs were screened for their ability to
dues fR5, fE46, fK99, and fE167 (f for HisF), needs to open activate HisH in the presence of ProFAR in their ‘‘as isolated’’
(Chaudhuri et al., 2001; Douangamath et al., 2002). Similarly, states (caged or E), after irradiation with UV light at a wavelength
PrFAR promotes a so-called ‘‘breathing motion’’ hinged at the of 365 nm (decaged or Z) and, for AzoF-HisFs, after additional irra-
cation-p interaction fR249-hW123 (h for HisH) that opens the diation with visible light at a wavelength of 420 nm (E) (Figure 2B).
interface above the glutamine binding site, possibly to allow for Suitable candidates for in-depth characterization were selected
glutamine binding (Amaro et al., 2007; Rivalta et al., 2012).
according to the following criteria: (1) At least 20% wild-type
Based on these considerations, we have incorporated the (WT) HisH activity had to be retained in ImGPS complexes con-
photo-responsive UAAs AzoF, NBY, NPY, and mNPK into posi- taining the irradiated caged UAA-HisFs or the more active isomer
tions close to loop 1, the ammonia channel, and the hinge region, of AzoF; (2) HisH activity was altered at least 1.5-fold upon
and identified three UAA-HisFs that showed the potential to irradiation (light regulation factor [LRF]). HisH activities in Im-
regulate HisH activity by light. We then characterized these pro- GPS(fK19AzoF), ImGPS(fF23AzoF), ImGPS(fY39AzoF), Im-
teins with respect to their structure, stability, and function, and GPS(fF23NBY), and ImGPS(fY39NBY) did not reach 20% WT ac-
optimized the isomerization and decaging processes. In the tivity, though irradiation of fY39NBY should in principle restore the
following, we could directly light regulate HisH activity 10-fold WT situation. Native MS suggests that considerable quantities
by our AzoF-HisF and 4- to 6-fold by our caged UAA-HisFs. (20%–60%) of reduced NBY existed in both fF23NBY and
Finally, molecular dynamics (MD) simulations revealed how fY39NBY, rationalizing missing decaging. Moreover, also the re-
each UAA affects HisH activity.
maining intact NBY-HisF proteins could not be decaged (Fig-
ure S2). However, during the course of these studies and as an
alternative to NBY, the UAA NPY was introduced and shown to
possess higher decaging efficiencies (Luo et al., 2017). Indeed,
HisH activities in ImGPS(fF23NPY) and ImGPS(fY39NPY) were
>20% WT after irradiation (Figure 2B). In the second selection
RESULTS
Identification of Three UAA-HisFs Leading to Light-
Dependent HisH Activity
We applied three criteria to identify suitable positions in HisF for step, LRFs of HisH in ImGPS(fF23NPY), ImGPS(fY39NPY), and
the incorporation of AzoF, NBY, and mNPK. First, the position in ImGPS containing the remaining eight UAA-HisFs were measured
˚
HisF should at least be 10 A away from the HisH active site avoid- and the results were displayed in a histogram (Figure 2C). Only
ing direct interactions of the UAA with catalytic residues. More- four UAA-HisFs led to LRFs >1.5, of which fF23NPY was the
over, UAAs should not hamper substrate binding in either the only caged UAA-HisF whose decaging did not restore WT-HisH
HisF or HisH active sites, nor impair the overall structure of the activity. This result was rationalized by a control experiment with
ImGPS complex. Based on these criteria, we tested ten positions fF23Y, which showed that a tyrosine residue at position 23 leads
in T. maritima HisF for the incorporation of light-responsive to a drastic reduction to ꢀ30% of WT-HisH activity. We therefore
UAAs. The positions include conserved (*) residues and excluded fF23NPY from our further studies and finally selected
are localized in or close to the allosteric sites of loop 1 fS55AzoF, fY39NPY, and fK99mNPK (Figure 2D) as most prom-
(fK13, *fK19, *fF23, fS29, fL35, *fY39, and *fS55), the ammonia ising UAA-HisFs for light regulation of ImGPS allostery.
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UAAs Weaken HisF Stability and Activity but Do Not
A
Impact Pr(o)FAR or Glutamine Binding
Before analyzing light regulation in detail, we characterized the
non-irradiated UAA-HisFs fS55AzoF, fY39NPY, and fK99mNPK
with respect to structure, stability, and function. Circular dichroism
spectroscopy and analytical size-exclusion chromatography
demonstrated that the three UAA-HisFs are properly folded mono-
mers and form stoichiometric complexes with WT-HisH (Figures
3A–3C). Crystal structure analysis of fY39NPY (PDB: 6rtz) and
fS55AzoF (PDB: 6ru0) in complex with WT-HisH showed that
only the tyrosine and phenylalanine moieties could be resolved
(Figures S3A–S3B), confirming the high flexibility of the UAA side
chains, as observed in MD simulations (Figures S3C–S3F). Never-
theless, in fY39NPY, an additional electron density cloud indicated
the approximate position of the o-nitropiperonyl moiety. This moi-
ety shoved aside fH228, causing a destabilization of helix a80 in
B
HisF (Figure S3G). As a consequence, certain residues of this helix
took on unallowed conformations in the Ramachandran plot
(Lovell et al., 2003) (Figure S3H). Similarly, the AzoF moiety led to
unallowed conformations of residues in loop 2 of fS55AzoF (Fig-
ure S3I). These structural effects translated into thermal destabili-
zation: fS55AzoF and fY39NPY, as well as fK99mNPK, which con-
tains a disturbed a4 helix in MD simulations (Figure S3J), showed
decreased denaturation midpoints compared with WT-HisF that
could be reversed upon irradiation for both caged UAA-HisFs (Fig-
ure 3D). Using steady-state enzyme kinetics, we observed that
fS55AzoF and fY39NPY, which are located relatively close to the
active site of HisF, display reduced (3- to 9-fold for fS55AzoF
and 15-fold for fY39NPY) turnover numbers (kcat), while
fK99mNPK, which is far remote from the active site has a kcat value
similar to that of WT-HisF (ꢀ173 minꢁ1) (Table 1; Figure S4). More-
C
over, we found that the Michaelis constants (KMPrFAR) of all UAA-
HisFs were identical to WT-HisF (ꢀ2.5 mM), indicating that sub-
strate binding to the active site was not compromised.
Before we started with the analysis of the allosteric control of
HisH activity by the three selected UAA-HisFs, we first
measured HisH activities in ImGPS complexes containing
these UAA-HisFs. The results showed that, albeit the kcat
values were decreased by 7- to 16-fold in ImGPS(fS55AzoF),
12-fold in ImGPS(fY39NPY), and 6-fold in ImGPS(fK99mNPK)
compared with WT-HisH (ꢀ17 minꢁ1), the KM
Gln
values were
unaltered (ꢀ0.4 mM) indicating that the affinity for the substrate
D
(PDB: 1gpw; Douangamath et al., 2002). The distances of the three finally
selected positions to the substrate glutamine, bound at the active site of HisH
(super-positioned from PDB: 3zr4; List et al., 2012) are shown.
(B) HisH activities (pH 8.5) in ImGPS complexes containing WT-HisF and UAA-
HisFs in the ‘‘as isolated’’ (ai) state, after irradiation with UV light (hnUV, 365 nm)
and, for AzoF-HisFs, after additional irradiation with visible light (hnVIS
,
420 nm). Mean SEM for three technical replicates are shown. Incorporation
of the UAA at each position was confirmed by tryptic digest coupled to liquid
mass spectrometry (MS) analysis (see also Figure S1). The two NBY proteins
fF23NBY and fY39NBY showed low decaging efficiencies (see also Figure S2).
(C) By comparison of HisH activities before and after irradiation, light-regula-
tion factors (LRFs) for each of the ten UAA-complexes with >20% WT-HisH
activity were calculated and plotted in a histogram. Four proteins stood out
with LRFs higher than 1.5.
Figure 2. Identification of Three UAA-HisFs Leading to Light-Depen-
dent HisH Activity
(D) 12.5% SDS-PAGE of 3 mg protein confirming >90% purity of fS55AzoF,
fY39NPY, and fK99mNPK and expression yield per liter TB medium. An
additional protein in fS55AzoF, identified as truncated HisF in LC-MS analysis,
was removed by a heat step of 75ꢂC for 30 min.
(A) Selected positions for incorporation of light-responsive UAAs are shown
in beige in the ImGPS overall structure, with HisF in blue and HisH in purple
4
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A
wt-HisF
B
C
− wt-HisH
+ wt-HisH
fS55AzoF
fY39NPY
fK99mNPK
40
20
15
10
5
wt-HisF
wt-HisF
12
8
0
0
40
15
10
5
fS55AzoF
fY39NPY
fS55AzoF
fY39NPY
20
0
0
4
40
15
10
5
20
0
0
0
40
15
10
5
fK99mNPK
fK99mNPK
−4
20
0
−8
0
190 210 230 250
Wavelength [nm]
8
9
10 11 12 13 14
8
9
10 11 12 13 14
Retention volume [mL]
Retention volume [mL]
D
0.9
0.8
0.7
0.6
wt-HisF: Tm = 93 °C
fS55AzoF ai: Tm = 88 °C
fY39NPY ai: Tm = 88 °C
fY39NPY hꢀ: Tm = 93 °C
fK99mNPK ai: Tm = 83 °C
fK99mNPK hꢀ: Tm = 93 °C
0.5
0.05
0.04
0.03
0.02
0.01
0.00
60 65 70 75 80 85 90 95
Temperature [°C]
Figure 3. UAAs Weaken HisF Stability but Not the Overall Fold of HisF
(A) Far-UV circular dichroism spectra indicate an intact overall fold of each UAA-HisF.
(B) Analytical gel filtration indicates that each UAA-HisF is monomeric and rules out soluble aggregates that might lower HisF activity.
(C) Analytical gel filtration indicates that each UAA-HisF forms a stoichiometric complex with HisH. Protein absorbance during chromatography was monitored at
280 nm (black). AzoF, NPY, and mNPK signals were detected at their characteristic absorption maxima of 334 nm (Bose et al., 2006) (green) and 360 nm (blue).
(D) Thermal stability measurements with Nano-DSF identified reduced Tm values for all three UAA-HisFs in their "as isolated" (ai) states. Upon irradiation (hn), WT-HisF
stability was recovered for fY39NPY and fK99mNPK. Stability of fS55AzoF could only be measured in its E state (ai) because the Z state is not thermally stable. Tm
values were mathematically determined at the transition midpoint (peak maximum of the first derivative). For further structural evaluation see also Figure S3.
glutamine was not compromised (Table 1; Figure S4). More- Table 1 and Figure 2C indicates that the LRFs of fS55AzoF,
over, the ProFAR concentrations that were required for the fY39NPY, and fK99mNPK increased slightly (ꢀ1.5-fold) from
UAA-HisFs to induce 50% HisH activity (Kac) were also not pH 8.5 to 7.0. Moreover, up to now, we irradiated caged UAA-
changed (ꢀ29 mM) (Table 1; Figure S4).
HisFs for 20 min with a conventional light bulb, to achieve
maximum decaging. Using a high-power LED (365 nm) instead,
we were able to accelerate this process by ꢀ10-fold. In addition,
the new light source improved the switching efficiency of
UAA-HisFs Allow for Direct Photo-control of HisH
Activity
Next, we identified optimum reaction conditions with respect to fS55AzoFE to fS55AzoFZ (Figure 4A). Whereas we only reached
pH and mode of irradiation. Comparison of the data shown in a photostationary state composed of 52% E and 48% Z with
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Table 1. Steady-State Kinetics of WT-ImGPS in Comparison with ImGPS(fS55AzoF), ImGPS(fY39NPY), and ImGPS(fK99mNPK)
PrFAR-Dependent HisF Activitya
Protein
State
kcat (minꢁ1
)
KmPrFAR (mM)
2.5 0.1
4.8 0.1
2.4 0.2
3.4 0.5
3.2 0.5
kcat/Km (Mꢁ1 sꢁ1
)
WT-HisF
fS55AzoF
fS55AzoF
fY39NPY
fK99mNPK
172.6 1.6
48.6 0.9
18.6 0.
11.5 0.5 3 105
1.7 0.0 3 105
1.3 0.1 3 105
0.6 0.1 3 105
5.9 0.9 3 105
E
Z
caged
caged
11.3 0.4
112.4 4.7
Glutamine-Dependent ImGPS Activitya
Protein
State
kcat (minꢁ1
41.4 1.1
6.0 0.2
2.6 0.2
3.5 0.2
7.3 0.4
)
KmGln (mM)
0.78 0.05
0.23 0.03
0.33 0.11
0.25 0.05
0.41 0.09
kcat/Km (Mꢁ1 sꢁ1
8.6 1.1 3 102
4.3 0.6 3 102
1.3 0.4 3 102
2.3 0.5 3 102
3.0 0.7 3 102
)
WT-ImGPS
ImGPS(fS55AzoF)
ImGPS(fS55AzoF)
ImGPS(fY39NPY)
ImGPS(fK99mNPK)
E
Z
caged
caged
ProFAR-Dependent HisH Activity (pH 7.0)b
Protein
State
kcat (minꢁ1
16.8 0.3
5.4 0.4
2.3 0.2
1.2 0.1
7.0 0.5
3.5 0.3
14.0 0.9
)
KacProFAR (mM)
32.8 1.2
19.5 3.8
35.1 5.0
32.3 4.2
28.7 4.2
19.6 3.7
32.1 3.2
LRF
2.3
5.9
4.0
WT-ImGPS
ImGPS(fS55AzoF)
E
Z
ImGPS(fY39NPY)
caged
decaged
caged
decaged
ImGPS(fK99mNPK)
See also Figures S4 and S6.
aValues SE for kcat and Km were determined by Michaelis-Menten fitting of the mean SEM for at least two technical replicates. Values SE for kcat
/
K
m were calculated according to the Gaussian law of error propagation.
bValues SE for kcat and KacProFAR were determined by fitting the mean SEM for at least two technical replicates to a hyperbolic function. LRF = kcat
/
E
kcatZ or kcatdecaged/kcat
.
caged
the conventional light bulb, the high-power LED led to the forma- Decaging Depends on the Protein Environment
tion of 11% E and 89% Z. Although HisH can be light regulated with the help of fY39NPY and
These results laid the foundation for direct light regulation of fK99mNPK, irradiation did not lead to complete decaging of these
HisH activity. To this end, the glutaminase reaction of HisH was two UAA-HisFs. Following irradiation of ImGPS complexes con-
stimulated by ProFAR-binding to UAA-HisFs at pH 7.0 and 8.5. taining the two UAA-HisFs, kcat values of HisH reached only
The reaction samples were then irradiated with our high-power ꢀ40% andꢀ80% oftheWTkcat value (ꢀ17minꢁ1)(Table1). Native
LED (365 nm) while product formation was still in the linear range MS was used to check whether incomplete decaging was caused
(Figure 4B). ImGPS(fS55AzoF) was irradiated a second time with by reduction of the nitropiperonyl groups of NPY and mNPK (Fig-
visible light (420 nm), to switch AzoF back into its E-enriched state. ure S6). Compared with NBY proteins (Figure S2), reduction of
In parallel, reactions were followed with as isolated complexes NPY and mNPK decreased from 20% and 60% to %10%, and
and pre-irradiated complexes as negative and positive controls, therefore did not explain incomplete recovery of WT-HisH activity.
respectively. ImGPS(fY39NPY) and ImGPS(fK99mNPK) showed However, native MS showed that decaging yields were lower for
only minor pH dependency of their LRFs and allowed us to control fY39NPY (27%) than for fK99mNPK (70%), consistent with HisH
HisH activity by ꢀ6- and ꢀ3-fold, respectively, consistent with activities following irradiation. The amount of decaging seems to
LRFs of ꢀ6 and ꢀ4 as determined in Kac measurements (Table 1). depend on the structural freedom of the UAA side chain: fK99 is
Activities before and after irradiation matched well with the nega- part of the gate at the ammonia channel entrance, whereas fY39
tive and positive controls. ImGPS(fS55AzoF), on the other hand, is buried by secondary structure elements. The potential for light
showed a stronger pH dependency and allowed us to reversibly activation by our caged UAA-HisFs was hence limited by the pro-
control HisH activity by ꢀ10-fold at pH 7.0 and by ꢀ2-fold at pH tein conformation, and neither longer irradiation times nor a stron-
8.5. We furthermore tested WT-HisF and as additional control ger light source led to complete decaging (Figure 4A).
fF23NPY and fF23AzoF; however, neither of the activities were
affected >1.5-fold by light (Figure S5). HisH activity was hence The Two fS55AzoF Light Isomers Change Various
unambiguously activated by light with fY39NPY and fK99mNPK, Structural Characteristics of ImGPS to Different Extents
and even more efficiently switched off and on in a pH-dependent In the following, we performed MD simulations with PrFAR-bound
manner by fS55AzoF.
ImGPS(fS55AzoF), ImGPS(fY39NPY), and ImGPS(fK99mNPK) to
6
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A
0.3
0.2
0.1
0.0
0.5
0.4
0.3
0.2
0.1
0.0
0.2
0.1
0.0
fS55AzoF
fY39NPY
fK99mNPK
ai
ai
ai
365nm40 s
365nm30 min
365nm30 min
365nm10–30 s
420nm10–30 s
365nm1–5 min
365nm1–5 min
250 300 350 400 450 500 550
Wavelength [nm]
250 300 350 400 450 500 550
Wavelength [nm]
250 300 350 400 450 500 550
Wavelength [nm]
ImGPS(fS55AzoF)
ImGPS(fY39NPY)
ImGPS(fK99mNPK)
B
negative control: 4.5 min−1
irradiated: 4.2 → 0.4 → 3.3 min−1
positive control: 0.7 min−1
positive control: 4.4 min−1
irradiated: 0.8 → 4.8 min−1
negative control: 0.7 min−1
positive control: 9.2 min−1
irradiated: 2.9 → 9.1 min−1
negative control: 3.0 min−1
0.20
0.15
0.10
0.05
0.00
0.15
0.10
0.05
0.00
0.4
0.3
0.2
0.1
0.0
+ complex hꢀUV
hꢀVIS
+ complex
hꢀUV
+ complex
hꢀUV
0
20
40
60
80
100 120
0
20
40
60
80
100 120
0
20
40
60
80
100 120
Time [min]
10x
Time [min]
6x
Time [min]
3x
pH 7.0
pH 8.5
pH 7.0
pH 8.5
pH 7.0
pH 8.5
2x
5x
2x
negative control: 1.5 min−1
positive control: 1.4 min−1
irradiated: 0.4 → 2.0 min−1
negative control: 0.2 min−1
positive control: 4.4 min−1
irradiated: 1.3 → 3.1 min−1
negative control: 1.5 min−1
irradiated: 0.9 → 0.5 → 0.7 min−1
positive control: 0.8 min−1
0.20
0.15
0.10
0.05
0.00
0.15
0.10
0.05
0.00
0.4
0.3
0.2
0.1
0.0
+ complex hꢀUV
hꢀVIS
+ complex
hꢀUV
+ complex
hꢀUV
0
20
40
60
80
100 120
0
20
40
60
80
100 120
0
20
40
60
80
100 120
Time [min]
Time [min]
Time [min]
Figure 4. UAA-HisFs Allow for Direct Photo-control of HisH Activity
(A) As described in Bose et al. (2006), fS55AzoF showed two signals in addition to the protein peak at 280 nm in the as isolated (ai) state (black). The signal at
~330 nm corresponds to the population of its E state. Upon irradiation with UV light (365 nm), the maximum loses intensity as the E/Z equilibrium is shifted toward
a Z-enriched photostationary state (blue arrows). The photostationary composition at this state, achieved with a high-power LED, was approximately 11% E to
89% Z (blue); with a conventional UV lamp (2 3 8 W), we only achieved 52% E and 48% Z (brown). Subsequent irradiation with visible light (420 nm) could reverse
the equilibrium toward an E-enriched state (green arrows). Caged UAA-HisFs fY39NPY and fK99mNPK exhibited a clear peak at ~360 nm next to the protein peak
at 280 nm. Upon irradiation with UV light, decaging could be followed due to a loss of intensity at this wavelength (downward blue arrows). In addition, two new
peaks were formed (upward arrows), which were caused by the cleaved-off nitrobenzylic species in their monomeric and dimeric forms, respectively (Zuman and
Shah, 1994). Formation of these species occurs simultaneously for NPY, but successively for mNPK (light blue arrows). The intensity at 360 nm reached a final
minimum after 2 min (blue); with a conventional UV lamp (2 3 8 W), the final minimum was reached after 20–30 min (brown). The final minimum does not represent
the fully decaged protein (see also Figure S6).
(B) Light-dependent HisH activities of ImGPS(fS55AzoF), ImGPS(fY39NPY), and ImGPS(fK99mNPK) were followed at pH 7.0 (upper panels) and at pH 8.5 (lower
panels). Blue curves show the reaction course of the as isolated complexes that were irradiated with UV light (365 nm) for decaging (fY39NPY, fK99mNPK;
(legend continued on next page)
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understand the underpinning mechanism of light regulation Finally, we wanted to track the breathing motion at the HisH:
of HisH. HisF interface that was previously shown to occur upon PrFAR
We started with UAA-HisF, in which the UAA is positioned binding to WT-HisF and leads to an opening of the interface
˚
furthest away (34 A) from the HisH active site, fS55AzoF. We above the glutamine binding site (Amaro et al., 2007; Rivalta
searched for reasons why HisH activity is lower in ImGPS(fS55A- et al., 2012; Negre et al., 2018). We considered the distance be-
zoFE) than in WT-ImGPS and why HisH activity is lower in Im- tween hG52 of the glutamine binding site and the opposite fF120
GPS(fS55AzoFZ) than in ImGPS(fS55AzoFE) (Figure 5A). For as an indirect measure of this movement. The mean distances of
this purpose, we derived normalized frequency distributions for hG52-fF120 suggest that the interface is more open in WT-
E
˚
˚
the atom distances (d) between the central catalytic residue ImGPS (d ꢀ 10.7 A) and in ImGPS(fS55AzoF ) (d ꢀ 11.3 A)
Z
˚
`
hH178 (Massiere and Badet-Denisot, 1998; Raushel et al., than in the least active ImGPS(fS55AzoF ) (d ꢀ 8.9 A) (Figure 5D).
1999) and either hE180 or fD98, which are both essential for To elucidate implications of this motion for the structure of HisH,
glutaminase catalysis (Zalkin and Smith, 1998; List et al., we visually compared RMSF values of secondary structure ele-
2012), from MD simulations (Figure 6). We concentrated on these ments. We orientated the 3D structure such that the hinge region
distances, because the stabilization of the hydrogen bond be- was located in the ‘‘left’’ half of the ImGPS complexes (Figure 5E).
tween hH178 and hE180, and the shortening of the hH178- Apart from the active site loops of HisF, which are similarly flex-
fD98 spacing, was previously observed as a consequence of ible in all three structures, the ‘‘right’’ half of WT-ImGPS and Im-
PrFAR binding in WT-HisF and the formation of the glutamyl- GPS(fS55AzoFE) containing the opening at hG52–fF120 is more
thioester reaction intermediate in WT-HisH (Myers et al., 2005). flexible with higher RMSF values than the left half containing the
A 2D diagram of these frequency distributions revealed distinct hinge region. In contrast, right and left halves remained rigid in
clusters that represent conformational ensembles of hH178 (Fig- the least active ImGPS(fS55AzoFZ). The mean RMSF values
ure 5B); representative MD snapshots are shown in Figure S7. In are in full agreement with this postulate: overall flexibility in
E
WT-ImGPS, three ensembles (ensWT,1-ensWT,3) were observed. WT-ImGPS (0.98 A) and ImGPS(fS55AzoF ) (1.00 A) is higher
˚
˚
Z
In ensWT,1 (prevalence 24%), hH178 assumes a pose with than in ImGPS(fS55AzoF ) (0.49 A). All differences of the mean
˚
˚
relatively short distances to both hE180 (d ꢀ 3.6 A) and fD98 values we determined here were low, but within the range we ex-
˚
(d ꢀ 6.9 A). This orientation suggests the formation of a direct pected, due to a recent comprehensive study comparing apo
hydrogen bond between hH178 and hE180 and leaves the pos- and holo structures (Clark et al., 2019).
sibility of a water-mediated hydrogen bond between hH178 and
In summary, our MD simulations suggest that fS55AzoFZ com-
fD98. In ensWT,2 (prevalence 44%), the hydrogen-bond distance promises the overall structure of HisF most strongly, and affects
between hH178 and hE180 remains unaltered, but the mean dis- the orientation of the catalytic residue hH178 and of the oxyanion
˚
tance of hH178 to fD98 increases (d ꢀ 8.6 A), which makes the hole, which are both crucial for HisH function (Figure 5F). In addi-
formation of a water-mediated hydrogen bond less likely. In tion, the breathing motion and the concomitant rearrangement of
ensWT,3 (prevalence 32%) hH178 cannot interact with hE180 (d the ImGPS structure seem less pronounced than in WT-ImGPS.
ꢀ 5.1 A) and is still far from fD98 (d ꢀ 8.6 A). Obviously, these Compared with fS55AzoFZ, the disadvantageous effects of
˚
˚
three specific conformational ensembles of hH178 are optimal fS55AzoFE seem to be generally less severe, as indicated by a
for HisH activity. In accordance with this assumption, Im- more structured catalytic site,
a typical h49-PGVG-52
GPS(fS55AzoFE) adopts only one conformational ensemble orientation, and a HisH:HisF arrangement that resemble the
(ensE) resembling ensWT,2, whereas ImGPS(fS55AzoFZ) adopts WT-ImGPS.
four ensembles (ensZ,1-ensZ,4) where only ensZ,1 resembles
ensWT,1
.
fY39NPY Causes a More Rigid ImGPS Complex
We continued our MD analyses with the UAA-HisF fY39NPY, in
˚
which NPY is located 26 A away from the HisH active site. We
Next, we concentrated on the h49-PGVG-52 motif (Fig-
ure 5C). In MD simulations of WT-ImGPS, PrFAR binding
has been observed to increase the flexibility of the backbones
of hG50 and hV51, which supposedly leads to a backbone flip
of the hV51 amine, stabilizing the oxyanion reaction intermedi-
ates (Lipchock and Loria, 2010; Rivalta et al., 2012). Our root-
mean-square fluctuation (RMSF) analyses of MD simulations
albeit showed that these residues are rather rigid in all three
ImGPS complexes. However, the adjacent hG52 backbone
is more flexible in WT-ImGPS and ImGPS(fS55AzoFE) than in
the least active ImGPS(fS55AzoFZ), which might allow it to
act as oxyanion intermediate stabilizer. In addition, the
motif adopts a different configuration in ImGPS(fS55AzoFZ),
with the consequence that hG52 leaves the glutamine binding
site.
searched for reasons why HisH activities are lower in caged Im-
GPS(fY39NPY) than in decaged ImGPS(fY39NPY), which is iden-
tical to WT-ImGPS (Figure 5A). We observed three structural en-
sembles of hH178, ensY,1 (prevalence 10%), ensY,2 (prevalence
34%), and ensY,3 (prevalence 49%) that resemble those in WT-
ImGPS (Figure 5B). However, the h49-PGVG-52 motif was
more rigid than in WT-ImGPS (Figure 5C) and, similarly as
deduced for ImGPS(fS55AzoFZ), the breathing motion at the
HisH:HisF interface seems less extensive; the mean hG52-
˚
fF120 distance is ꢀ1.5 A shorter than in WT-ImGPS (Figure 5D).
Consequently, both right and left halves of ImGPS(fY39NPY)
remain rigid (Figure 5E) and the mean RMSF of the full complex
˚
(0.93 A) is lower than the WT-ImGPS reference value (0.98 A).
˚
1.5 min) or E to Z isomerization (fS55AzoF; 30 s). ImGPS(fS55AzoF) was irradiated a second time with visible light (420 nm; 1 min) for back-isomerization to
E. Dark gray curves (negative control) and light gray curves (positive control) show the reaction courses of complexes that were not irradiated or pre-
irradiated, respectively. Reaction rates for the individual curves are given above each chart, LRFs are given below (pH 7.0) or above (pH 8.5) the charts (see also
Figure S5).
8
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A
B
C
D
E
F
Figure 5. Comparative MD Simulation Analysis of WT-ImGPS, ImGPS(fS55AzoFE), ImGPS(fS55AzoFZ), ImGPS(fY39NPY), and
ImGPS(fK99mNPK)
(A) Comparison of turnover numbers of HisH (see Table 1) shown to facilitate side-by-side comparison of MD results presented in (B–E).
(B) 1D and 2D frequency distributions for the distances hH178Nd-hE180Cd and fD98Cg--hH178Ne. The orientation of the hH178 residue in the 2D contour plots
suggests for WT-ImGPS three ensembles (enswt,1-enswt,3), for ImGPS(fS55AzoFE) only one (ensE) resembling enswt,2, for ImGPS(fS55AzoFZ) four (ensZ,1-ensZ,4
)
with ensZ,1 resembling enswt,1, for ImGPS(fY39NPY) three (ensY,1-ensY,3) with all three resembling enswt,1-enswt,3, and for ImGPS(fK99mNPK) two ensembles
(ensK,1 and ensK,2) with ensK,2 resembling enswt,3. MD snapshots representative of each of the conformational ensembles are shown in Figure S7.
(legend continued on next page)
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In summary, these findings suggest that fY39NPY impairs horseradish peroxidase activity by 1.5-fold with AzoF (Muranaka
HisH activity by reducing the h49-PGVG-52 flexibility and the et al., 2002). Remarkably, a new computationally aided strategy
breathing motion (Figure 5F).
that cages a residue in close proximity to the active site of an
enzyme (CAGE-prox) reduced tumor growth in mice by ꢀ5-fold
upon light irradiation (Wang et al., 2019). Recently, AzoF and de-
fK99mNPK Causes a Rearrangement of the HisH:HisF
Interface
rivatives were also used to photo-control firefly luciferase from
˚
˚
In the UAA-HisF fK99mNPK, mNPK is 14 A away from the HisH an allosteric position ꢀ16 A from its active site; however, only
active site. Again using MD, we searched for reasons why HisH a 2-fold LRF was achieved (Luo et al., 2018). We have identified
activities are lower in caged ImGPS(fK99mNPK) than in dec- our candidates by choosing known positions close to allosteric
aged ImGPS(fK99mNPK), which is identical to WT-HisF (Fig- sites in ImGPS; however, such knowledge might not be available
ure 5A). Here, we observed only two conformational ensembles for other potential target enzymes. Alternatively, computationally
of hH178, ensK,1 (prevalence 56%) and ensK,2 (prevalence guided methods similar to CAGE-prox or the identification of
29%), with the latter resembling the ensWT,3 of WT-ImGPS (Fig- allosteric regulation hotspots from coevolution studies (Rey-
ure 5B). Again, the deviation of these two conformations from nolds et al., 2011) might be applicable.
the three specific conformations observed in WT-ImGPS
Whereas AzoF incorporation and switching occurred straight-
seems to impair HisH activity. In addition, a comparison of forwardly, a large fraction of the caged UAA NBY was reduced in
representative MD snapshots with WT-ImGPS showed a E. coli and its decaging efficiency was below 10%. Significant
drastic backbone shift for both ensK,1 and ensK,2 (Figure S7). improvement was accomplished by the addition of a methylene-
Moreover, residues of the h49-PGVG-52 motif were also dioxy moiety, yielding NPY (Luo et al., 2017). Distinct spectral
affected by a backbone shift (Figure 5C). Due to this observa- features of NPY allowed easy verification of incorporation, its
tion, we analyzed the fD74-hS183 distance close to the inter- reduction was minimized in E. coli, and decaging increased by
face hinge, in addition to the fF120-hG52 distance at the up to 80%. On the downside, high levels of decaged species
HisH:HisF interface (Figure 5D). While the breathing motion were obtained before irradiation, possibly due to unintentional
above the glutamine binding site is absent almost completely, light exposure during protein synthesis or due to decaging by ni-
˚
the interface opened as far as ꢀ3.7 A at fD74-hS183. As a troreductases (Valiauga et al., 2017; Xie et al., 2017) in E. coli.
consequence, the left half of ImGPS(fK99mNPK) is more flex- Another drawback was the incomplete recovery of WT-HisH ac-
ible than the left half of WT-ImGPS (Figure 5E). The right half tivity upon irradiation of ImGPS complexes containing fY39NPY
is similarly flexible as the right half of WT-ImGPS, likely due to and fK99mNPK. This might be caused by reaction of the cleav-
˚
the motion between the different closing states (d ꢀ 4.6 A 4 age products with the protein to form imines (compare Fig-
˚
d ꢀ 9.1 A) at fF120-hG52. The mean RMSF value of the full com- ure S2A); however, we could not detect any adducts in native
˚
˚
plex (1.22 A) exceeds the WT-ImGPS value (0.98 A) and is a MS (Figure S6A). Instead, decaging seems to depend on the pro-
further indicator for higher flexibility.
tein environment. Specifically, neighboring residues such as
In summary, these findings suggest that fK99mNPK causes a fK19, fH228 and fR230 in fY39NPY or fD98, fE46, fR5, and
drastic rearrangement of the HisH:HisF interface, whereupon the fE167 in fK99mNPK might interfere with the photo-induced,
opening of the interface above the glutamine binding site is most acid-base catalyzed fragmentation process (Figure S6B) (Il’ichev
´
and Wirz, 2000; Klan et al., 2013). Different orientations of the
prominently affected (Figure 5F).
UAA, as observed for both NPY and mNPK in MD simulations,
might interrupt fragmentation more or less strongly, so that
different ratios of decaged proteins result for each position.
We performed MD simulations to understand how the different
DISCUSSION
In search for HisF positions that enable photo-control of the ac-
tivity of its partner protein HisH with UAAs, we identified three UAAs, positioned strategically close to known allosteric ele-
˚
promising candidates. Each of the positions is at least 10 A apart ments in HisF, affect HisH activity. In doing so, we also gained
from the HisH active site and, after UAA incorporation, neither further insight into structural events at the stimulation endpoint
Pr(o)FAR nor glutamine binding was affected. ImGPS(fS55AzoF) in HisH. In PrFAR-bound WT-ImGPS, we identified three confor-
allowed us to reversibly and pH-dependently control HisH activ- mational ensembles of the central catalytic residue hH178. The
ity by 10-fold. ImGPS(fY39NPY) and ImGPS(fK99mNPK) combination of these three ensembles––with ꢀ70% of hH178
achieved LRFs of 6 and 4. In direct comparison with photo-con- in hydrogen bonding distance to hE180 and ꢀ25% in water-
trol at the active site of an enzyme, our approach of allosteric mediated hydrogen bonding distance to fD98––generates the
light regulation reached equivalent LRFs; e.g., TEV protease ac- most active state of HisH. This combination is not present in Im-
tivity was regulated by 10-fold with NPY (Luo et al., 2017), and GPS(fS55AzoFE), in which only one distinct ensemble is
(C) Simulation endpoint orientation of the h49-PGVG-52 motif and the catalytic HisH residue hC84 color-coded by the residue-specific RMSF value; compare the
color bar. The substrate glutamine (Gln) was super-positioned from PDB: 3zr4 (List et al., 2012) and is shown in gray. In ImGPS(fS55AzoFZ) (middle panel) and
ImGPS(fK99mNPK) (right panel), the WT localization of h49-PGVG-52 is indicated semi-transparently.
(D) Frequency distributions for the distance fF120Cg-hG52Ca, which is an indicator for the extent of the ‘‘breathing motion.’’ In addition, frequency distributions for
the control distance fD74Ca-hS183Ca of residues close to the WT hinge region are given for ImGPS(fK99mNPK).
(E) Flexibility of secondary structure elements as indicated by their RMSF values. Mean RMSF values deduced from all atoms of the ImGPS complexes are given
below their cartoon representation. For easier interpretation, the location of fF120 and hG52, and the hinge region are indicated.
(F) Graphical representation of the effects observed in (B)–(E). Changes of the interface opening, the flexibility of secondary structure elements, and the orga-
nization of active site residues in HisH (see Figure 6) are indicated schematically.
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Figure 6. Reaction Mechanism of the Gluta-
A
B
minase HisH
(A) The active site of HisH harbors the catalytic triad
consisting of hE180, hH178, and hC84 (Zalkin and
Smith, 1998). HisF contributes a fourth residue that
was shown to be essential for catalysis, fD98 (List
et al., 2012). The central catalytic residue hH178
accepts the hydrogen from hC84, whereupon hC84
starts
a nucleophilic attack of the substrate
glutamine.
(B) The resulting oxyanion intermediate is stabilized
by a residue of the oxyanion hole, while hH178
submits its hydrogen to the amino group of gluta-
mine, setting free ammonia.
(C) hH178 accepts a hydrogen from water, which
undergoes a nucleophilic attack with the glutamyl-
thioester intermediate.
C
D
(D) The resulting oxyanion intermediate is stabilized
by a residue of the oxyanion hole, while hH178
submits its hydrogen to hC84, restoring this cata-
lytic residue and setting free glutamate. The mech-
anism was adapted from Massie` re and Badet-De-
nisot (1998).
The activity of HisH in ImGPS(fY39NPY)
was impaired by a similar mechanism but
to a lower extent than in ImGPS(fS55A-
zoFZ). Again, the hG52 backbone was
adopted, or in ImGPS(fS55AzoFZ), in which four distinct ensem- significantly more rigid than in WT-ImGPS, and the ImGPS inter-
bles are observed. It is not entirely clear why this different com- face remained closed correlating with a rigid right half of the
bination of ensembles leads to reduced HisH activity in Im- ImGPS complex. The UAA closest to the HisH active site,
GPS(fS55AzoFE) and in particular in ImGPS(fS55AzoFZ). mNPK in fK99mNPK, disrupted the structural integrity of the
However, it is plausible to assume that a productive positioning complete interface including HisH catalytic residues, and espe-
of the catalytically central hH178, which acts as a proton shuffle cially blocked the breathing motion above the glutamine binding
during the reaction cycle (Figure 6) (Zalkin and Smith, 1998; site. It is interesting to note that the binding of the substrate
Raushel et al., 1999), is essential for efficient glutaminase activ- glutamine is not disturbed in ImGPS(fK99mNPK), Im-
ity. Previous studies proposed a different process of HisH activa- GPS(fY39NPY), or ImGPS(fS55AzoFZ), all of which compromise
tion, which is caused by a rotation of the hV51 amine into the the opening of the ImGPS interface. Hence, the breathing motion
glutamine binding site that results in the formation of the oxyan- appears not to correlate with the binding of glutamine; however,
ion hole (Chaudhuri et al., 2001, 2003; Lipchock and Loria, 2010; an open interface seems to be associated with high turnover
Rivalta et al., 2012). With this in mind, we analyzed the conserved rates of HisH.
h49-PGVG-52 motif and observed significant conformational
As a conclusion, we found evidence that, in contrast to what
changes. However, in our MD simulations the backbone of has been postulated, glutamine turnover by HisH is not induced
hV51 remained rigid in WT-ImGPS and ImGPS(fS55AzoFE). by the proper formation of the oxyanion hole. Instead, our MD
Only in ImGPS(fS55AzoFZ), the backbone of the h49-PGVG-52 simulations indicate that the conformational organization of the
motif rotated, but this orientation was associated with the disrup- catalytic residue hH178 is essential for HisH activity. This obser-
tion of the glutamine binding site and an almost complete loss of vation will be the basis for further combined biochemical and
HisH activity. These findings are in agreement with our previous structural studies to uncover the changes that are induced in
assumption that, alternatively, hG52 can stabilize the oxyanion the HisH active site upon binding of PrFAR to HisF and transmit-
intermediate (List et al., 2012). This residue is located close to tance of the signal through an allosteric network.
the bound glutamine, its NH group points into the glutamine
Most importantly, we have demonstrated that UAAs can be
binding pocket, and it is highly flexible in PrFAR-bound WT- used to regulate allostery within a sophisticated metabolic
ImGPS. In contrast, in the least active ImGPS(fS55AzoFZ), enzyme complex such as ImGPS. Our results indicate that caged
hG52 was highly rigid and pointed away from the glutamine bind- UAAs are not optimal light switches in such complexes, because
ing site. In addition to the different conformational ensembles of the effect of irradiation is irreversible and complete decaging and
hH178 and the different rigidity of the h49-PGVG-52 motif, the recovery of WT activity is limited by the protein environment.
lower HisH activity of ImGPS(fS55AzoFZ) compared with Im- Photo-switchable proteins containing AzoF, on the other hand,
GPS(fS55AzoFE) correlates with the closure of the subunit inter- generally hold greater promise for allosteric photo-control of en-
face, consistent with previous studies in which low activity corre- zymes, especially because the effect of irradiation is reversible.
lated with a closed complex (Rivalta et al., 2016).
However, also with AzoF as the UAA, the identification of
Cell Chemical Biology 26, 1–14, November 21, 2019 11

Please cite this article in press as: Kneuttinger et al., Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino Acids, Cell
Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.08.006
B Biochemical Synthesis of ProFAR
B Auxiliary Enzymes
positions leading to high LRFs is not trivial and might remain a
cumbersome trial-and-error process in the future. Nevertheless,
we envision that the transfer of the approach presented here for
ImGPS, to other enzymatic systems, will allow for interesting
synthetic or medicinal applications.
B Subcloning of the hisF and hisH Genes
B Subcloning of the aaRS/tRNACUA Pairs
B Site-Directed Mutagenesis of hisF
B Building a Rotamer Library
B Expression and Purification of ImGPS
B Tryptic Digest and MS Analysis
B Screening of HisF Positions
SIGNIFICANCE
The artificial spatiotemporal control of biological macro-
molecules by light is an exciting and rapidly emerging
sub-discipline of synthetic biology. Within this framework,
one central research goal is the photo-control of mono-
meric enzymes by reversible obstruction of the active
site. The next level of sophistication is the light regulation
of allosteric interactions in enzyme complexes, which
might pave the way for the regulation of enzyme cascades
in industrial biocatalysis. We have made a first step toward
this goal and developed a versatile strategy for the light
regulation of allostery, namely the manipulation of signal
propagation by photo-sensitive unnatural amino acids
(UAAs). To implement our new approach, we used the
enzyme complex imidazole glycerol phosphate synthase
(ImGPS), which consists of the synthase subunit HisF and
the glutaminase subunit HisH. Substrate binding to HisF
stimulates the glutaminase activity of HisH over a distance
B Native MS Analysis
B CD Analysis
B UV/Vis Analysis
B Analytical Size-Exclusion Chromatography
B Steady-State Kinetics
B Direct Photo-control of HisH Activity
B Crystallization
B Data Collection, Structure Solution and Refinement
B Molecular Dynamics (MD) Simulations
B Nano Differential Scanning Fluorimetry
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND CODE AVAILABILITY
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.
chembiol.2019.08.006.
˚
of more than 25 A. To put this long-range allosteric
stimulation under the control of light, we have incorporated
the light-responsive UAAs phenylalanine-40-azobenzene
(AzoF), o-nitropiperonyl-O-tyrosine, and (NPY) methyl-o-
nitropiperonyllysine (mNPK) at ten strategic positions of
HisF. The three most promising candidates for the light-
dependent regulation of HisH activity were purified and
analyzed by various biochemical and biophysical methods.
Kinetic measurements showed that HisH activity was light
regulated as much as 10-fold by AzoF-HisF and 4- to 6-fold
by NPY-HisF and mNPK-HisF. Crystal structure analysis
and MD simulations revealed the different mechanisms by
which each UAA affects the allosteric machinery in ImGPS
and provided additional insight into HisH activation. Taken
together, we present an innovative approach for the light
regulation of long-range enzyme allostery by the use of
photo-sensitive UAAs. Allostery is a crucial regulatory
feature of many central metabolic enzymes. Our work dem-
onstrates a general strategy how this feature can be put un-
der the control of light.
ACKNOWLEDGMENTS
We thank Sabine Laberer and Jeannette Ueckert for excellent technical assis-
tance and would like to further express our gratitude to Peter Schultz for sup-
€
plying us with the pEVOL-vectors as well as to Gernot Langst for providing the
Nano-DSF equipment. This work was supported by a grant of the Deutsche
Forschungsgemeinschaft to R.S. (STE 891/12-1) and a grant from the NIH to
V.H.W. (P41 GM128577). N.A.S. thanks the Studienstiftung des Deutschen
Volkes for a doctoral scholarship.
AUTHOR CONTRIBUTIONS
A.C.K. and R.S. equally conceptualized the idea. A.C.K. was responsible for
the acquisition of all data, delegating experiments where necessary, final anal-
ysis and interpretation, as well as writing of the original draft. K.S. performed all
computational methods and bioinformatical analyses. P.B. (lead) and E.H.
(supporting) delivered data for mNPK proteins. P.B. synthesized mNPK and
N.S. synthesized NPY and AzoF. A.B. performed tryptic digest and liquid chro-
matography-MS analysis. F.B. (lead) and V.H.W. (supporting) performed
native MS analysis. C.R. supervised X-ray crystallography, as well as collect-
ing and refining structural data. K.S. (lead) and D.H. (supporting) created ro-
tamer libraries for the unnatural amino acids. R.S., R.M., and B.K. supervised
the project and acquired funding. A.C.K., K.S., E.H., R.M., and R.S. collabora-
tively evaluated the data and, together with P.B., N.A.S., F.B., V.H.W., D.H.,
and B.K. revised and edited the paper.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d LEAD CONTACT AND MATERIALS AVAILABILITY
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
d METHOD DETAILS
DECLARATION OF INTERESTS
The authors declare no competing interests.
B Chemistry, Materials and Instrumentation
B Synthesis of AzoF
Received: June 18, 2019
Revised: July 31, 2019
B Synthesis of NPY
Accepted: August 19, 2019
Published: September 5, 2019
B Synthesis of mNPK
12 Cell Chemical Biology 26, 1–14, November 21, 2019

Please cite this article in press as: Kneuttinger et al., Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino Acids, Cell
Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.08.006
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tem for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395, 361–374.
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Standardized cloning vectors for protein production and generation of large
gene libraries in Escherichia coli. BioTechniques 64, 24–26.
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donor. Adv. Enzymol. Relat. Areas Mol. Biol. 72, 87–144.
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Alfonta, L. (2018). Photo-switchable microbial fuel-cells. Biotechnol. Bioeng.
115, 1355–1360.
Zimmerman, G., Chow, L.-Y., and Paik, U.-J. (1958). The photochemical isom-
erization of azobenzene. J. Am. Chem. Soc. 80, 3528–3531.
¨
Schmermund, L., Jurkas, V., Ozgen, F.F., Barone, G.D., Buchsenschutz, H.C.,
ꢁ
€
€
Zuman, P., and Shah, B. (1994). Addition, reduction, and oxidation reactions of
Winkler, C.K., Schmidt, S., Kourist, R., and Kroutil, W. (2019). Photo-bio-
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14 Cell Chemical Biology 26, 1–14, November 21, 2019

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Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.08.006
STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Chemicals, Peptides, and Recombinant Proteins
AzoF
This paper
N/A
NBY
Accela ChemBio
This paper
Cat#SY008208
N/A
NPY
mNPK
This paper
N/A
ProFAR
This paper
N/A
HisA
This paper
N/A
HisG/IE
This paper
N/A
wt/UAA-HisF
This paper
N/A
HisH
This paper
N/A
Glutamate dehydrogenase (GDH)
NAD+, free acid
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Merck
CAT#10197734001
CAT#124542
CAT#G8540
CAT#G1924
CAT#P8125
CAT#100206
CAT#06800
Glutamine
Glutamate-oxidase (GOX)
Hydrogen peroxidase (HRP)
Phenol
2-aminoantipyrine
Deposited Data
Sigma-Aldrich
ImGPS structure from Thermotoga maritima
Douangamath et al., 2002
Chaudhuri et al., 2003
PDB-ID 1gpw
PDB-ID 1ox5
ImGPS structure from Saccharomyces
cerevisiae
ImGPS(fS55AzoF) structure
ImGPS(fY39NPY) structure
Oligonucleotides
This paper
This paper
PDB-ID 6ru0
PDB-ID 6rtz
Primers: see Table S1
Recombinant DNA
pET28aTEV
This paper
N/A
This paper
This paper
This paper
This paper
This paper
This paper
N/A
N/A
N/A
N/A
N/A
N/A
pET28a_HisFwt
pET28aTEV_BsaI
pET28a_HisHwt
pEVOL_mNPK
pET28a vectors containing stop codon
mutations: see Table S1
pEVOL_AzoF
Peter Schultz (Scripps Research Institute,
La Jolla, USA) (Young et al., 2010; Bose
et al., 2006)
N/A
N/A
pEVOL_NBY
Peter Schultz (Scripps Research Institute,
La Jolla, USA) (Young et al., 2010; Deiters
et al., 2006)
Software and Algorithms
Gromacs version 5.1.2
Acpype
Berendsen et al., 1995
Sousa da Silva and Vranken, 2012
Wang et al., 2006
https://www.gromacs.org
https://www.pypi.org/project/acpype/
https://www.ambermd.org/AmberTools.php
https://www.originlab.com
AmberTools14
Origin 2018
OriginLab
Data Analysis 4.2
Bruker Daltonics
https://www.bruker.com
(Continued on next page)
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Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.08.006
Continued
REAGENT or RESOURCE
Yasara version 17.4.17
Pymol 2.0
SOURCE
IDENTIFIER
Krieger et al., 2004
https://www.yasara.org
https://www.pymol.org/2/
https://plot.ly
¨
Schrodinger, 2015
plotly
Plotly
Python 2.7 with packages numpy,
scipy, yasara, plotly
Python
https://www.python.org
R version 3.6.0
UniDec version 2.6.5.
MolProbity
The R Foundation
Marty et al., 2015
Davis et al., 2007
https://www.r-project.org
http://unidec.chem.ox.ac.uk/
http://molprobity.biochem.duke.edu/
Other
‘‘Conventional UV lamp’’: two F8 T5 BLB
black light bulbs (8 W)
Sylvania
EAN#5410288000244
High Power LED, UV Z5 series, 420 nm
High Power LED, LZ4 series, 365 nm
Seoul Viosys
LED Engin
CAT#CUN26A1B
CAT#LZ4-04UV00-0000
LEAD CONTACT AND MATERIALS AVAILABILITY
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Reinhard
Sterner (reinhard.sterner@ur.de).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
E. coli BL21 Gold (DE3) cells were originally purchased from Agilent Technologies and E. coli NEB Turbo from New England Biolabs.
The strains were further maintained following the manufacturer’s guidelines for the preparation of chemically competent cells. Cells
were grown in lysogenic broth (LB) medium at 37ꢂC, and stored in 80% glycerol at ꢁ80ꢂC.
METHOD DETAILS
Chemistry, Materials and Instrumentation
All reagents and solvents other than AzoF, NPY, mNPK and ProFAR were purchased in analytical grade or higher from commercial
sources and were used without further purification, if not otherwise stated. AzoF, NPY, mNPK and ProFAR were synthesized and
purity confirmed as described below. NBY was purchased from Accela ChemBio (96% pure).
All reactions were performed under magnetic stirring at room temperature (r.t.) unless otherwise specified. Light-sensitive com-
pounds were kept in the dark using aluminum foil to cover reaction vessels or were worked-up under red light (LEDs > 650 nm).
˚
For TLC, silica coated aluminum plates (Macherey-Nagel GmbH & Co. KG 60 A silica gel, 0.25 mm) were utilized. Visualization
was carried out with a UV lamp at 254 or 366 nm or through suitable staining. For MPLC, a Biotage Isolera One Flash Purification
System with manually packed columns using Macherey-Nagel GmbH & Co. KG 60M (0.04–0.063 mm, 230–400 grain diameter).
Nuclear magnetic resonance spectroscopy (NMR) was carried out using a Bruker Avance 300 MHz (1H: 300 MHz, 13C: 75 MHz, T =
300 K) or Bruker Avance 400 MHz spectrometer (1H: 400 MHz, 13C: 101 MHz, T = 300 K). Chemical shifts are reported in d [ppm]
relative to an internal standard (solvent residual peak). The solvents used are indicated for each spectrum. Coupling constants
are reported in Hertz [Hz]. Characterization of the signals: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, bs = broad
singlet, dd = doublet of doublet, dt = doublet of triplet. Integration is directly proportional to the number of the protons.
Synthesis of AzoF
AzoF was synthesized following a protocol developed by (Bose et al., 2006). The identity and purity of products was determined
through 1H-NMR.
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(S,E)-2-((tert-butoxycarbonyl)amino)-3-(4-(phenyldiazenyl)phenyl)propanoic acid (3)
Compound 3 was synthesized adapting a previously reported procedure (Bose et al., 2006). Nitrosobenzene (1.91 g, 17.84 mmol)
was added to butoxycarbonyl-p-aminophenylalanine (5.00 g, 17.84 mmol) dissolved in glacial acetic acid (1.0 L). The flask was
covered with aluminum foil and the reaction mixture was stirred at ambient temperature for 18 h. The reaction mixture was poured
onto crushed ice/water (ꢀ 1.5 L) and extracted with EtOAc (2 x 300 mL). The combined organic phases were washed with water
(2 x 200 mL) and brine (1 x 200 mL) and dried over Na2SO4. The volatiles were removed in vacuo and the crude product was purified
by MPLC (10% MeOH in DCM). Compound 3 was isolated as orange solid (4.09 g, 11.06 mmol, 62% yield). 1H-NMR (300 MHz,
Chloroform-d): d = 7.95 – 7.83 (m, 4H), 7.59 – 7.45 (m, 3H), 7.34 (d, J = 8.1 Hz, 2H), 4.75 – 4.57 (m, 1H), 3.35 – 3.08 (m, 2H),
1.42 (s, 9H).
(S,E)-2-Amino-3-(4-(phenyldiazenyl)phenyl)propanoic acid (AzoF)
AzoF was synthesized following the previously reported protocol (Bose et al., 2006) employing compound 3 (4.09 g, 11.06 mmol).
AzoF3HCl was dried via lyophilization to yield an orange solid (3.38 g, 11.06 mmol, quantitative; purity > 98% by NMR). 1H-NMR
(300 MHz, DMSO-d6): d = 7.97 – 7.83 (m, 4H), 7.72 – 7.55 (m, 3H), 7.54 – 7.43 (m, 2H), 4.17 (t, J = 6.4 Hz, 1H), 3.21 (t, J = 6.8 Hz, 2H).
Synthesis of NPY
NPY was synthesized following a protocol developed by (Luo et al., 2017). The identity and purity of products was determined
through 1H-NMR.
5-(Bromomethyl)-6-nitrobenzo[d][1,3]dioxole (6)
Compound 6 was synthesized following the previously reported protocol (Luo et al., 2017) employing 5 (1.00 g, 5.07 mmol). After
purification by MPLC (20% EtOAc in petroleum ether), the product was obtained as yellow powder (685 mg, 2.64 mmol, 52% yield).
1H NMR (400 MHz, Chloroform-d): d = 7.57 (s, 1H), 6.95 (s, 1H), 6.14 (s, 2H), 4.79 (s, 2H) (Tietze et al., 2013).
(2S)-2-Amino-3-(4-(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)phenyl)propanoic acid x HCl (NPY)
NPY was synthesized following the previously reported protocol (Luo et al., 2017) employing L-tyrosine (490 mg, 2.70 mmol) and
compound 6 (537 mg, 2.08 mmol). NPY3HCl was isolated as yellow powder (361 mg, 0.87 mmol, 42% yield; purity ꢀ 85% by
NMR). 1H-NMR (400 MHz, DMSO-d6): d = 7.74 (s, 1H), 7.25 (s, 1H), 7.19 (d, J = 8.3 Hz, 2H), 6.96 (d, J = 8.3 Hz, 2H), 6.24 (s, 2H),
3.92–3.87 (m, 1H), 3.10–2.92 (m, 2H).
Synthesis of mNPK
mNPK synthesis was adopted from a protocol developed by (Gautier et al., 2010). The identity and purity of products was determined
through 1H-NMR.
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N2-(tert-butoxycarbonyl)-N6-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)-L-lysine (9)
1-(6-Nitro-1,3-benzodioxol-5-yl)ethanol 7 (1.00 g, 4.7 mmol) was dissolved in dry THF (10 mL) and supplemented with K2CO3 (0.66 g,
4.7 mmol) at 0ꢂC under nitrogen atmosphere. Diphosgene (0.6 mL, 4.7 mmol) was added dropwise and stirring continued for 12 h
allowing the reaction mixture to warm to r.t. After filtration the quantitatively converted chloroformate 8 (1.30 g, 4.7 mmol) was dis-
solved in THF and added dropwise to a solution of Na-Boc-lysine (1.28 g, 5.2 mmol) in NaOH (13 mL, 1 M, aq) under stirring at 0ꢂC.
Stirring was continued for 12 h allowing the mixture to warm to r.t. The deep red aqueous phase was washed with Et2O (10 ml), cooled
to 0ꢂC and acidified with ice-cold HCl (1 M, aq) to pH 1 producing a bright yellow suspension. The crude product was extracted with
EtOAc (3 x 30 mL). The organic phases were combined, dried over Mg2SO4 and filtered. The volatiles were evaporated to afford 9
(1.94 g, 4.1 mmol, 85% yield) as a yellow foam. 1H-NMR (400 MHz, CDCl3): d, [ppm] = 7.47 (s, 1H), 7.00 (s, 1H), 6.28-6.21
(s, 1H.), 6.20-6.10 (m, 2H), 5.04-5.24 (m, 1H), 4.28 (bs, 1H), 3.12 (bs, 2H), 1.28-1.80 (m, 18H).
N6-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)-L-lysine TFA salt (mNPK)
The Boc-protected lysine 9 (500 mg, 1.0 mmol) was dissolved in DCM (6 mL), supplemented with triethylsilane (0.3 mL, 2.1 mmol) and
the mixture was added dropwise to a 0ꢂC solution of TFA (6 mL) under nitrogen atmosphere. After the reaction mixture was stirred for
2 h at r.t., the volatiles were evaporated under a constant N2-stream without heating. The residue was dissolved in MeOH (2 mL) and
added dropwise to ice-cold Et2O (200 mL) under vigorous stirring. The supernatant was decanted, the precipitate was washed with
ice-cold Et2O (100 mL) and decanted again. The volatiles were evaporated to afford the caged lysine TFA salt mNPK (360 mg,
0.8 mmol, 73 % yield; purity ꢀ 85% by NMR) as a light-yellow, hygroscopic solid. 1H-NMR (400 MHz, D2O): d [ppm] = 7.38
(s, 1H), 6.98 (s, 1H), 6.00 (s, 2H), 5.97-5.92 (m, 1H), 3.55-3.52 (m, 1H), 2.91 (m, 2H), 1.85-1.73 (m, 2H), 1.42-1.24 (m, 7H).
Biochemical Synthesis of ProFAR
ProFAR was synthesized following the standard protocol developed by (Davisson et al., 1994) from 5-phospho-D-ribosyl a-1-py-
rophosphate 10 and adenosine triphosphate 11 in 50 mM NH4COOCH3 pH 7.8. Reaction progress was tracked spectrophotomet-
rically as described (Davisson et al., 1994). Products were purified with ion-exchange chromatography using a POROS column (HQ
20, 10 mL, Applied Biosystems) and a linear gradient of NH4COOCH3 (50 mM / 1 M). ProFAR concentration was determined at
300 nm (e300 = 6069 Mꢁ1cmꢁ1) (Klem and Davisson, 1993) and ProFAR purity through the absorbance ratio A290/A260 for each frac-
tion. Highly concentrated and > 90% pure (A290/A260 = 1.1–1.2 accounts for > 95% purity) (Smith and Ames, 1964) fractions were
lyophilized and stored at ꢁ80ꢂC. ProFAR identity was confirmed by total turnover measurements to ImGP and AICAR, with HisA
and HisF, using the HisF assay described in the methods section (in ammonia saturation). Concentrations determined by total turn-
over were compared to spectrophotometrically measured concentrations. For steady-state kinetics, ProFAR-concentrations were
always determined by total turnover.
Auxiliary Enzymes
HisA from T. maritima was produced by heterologous gene expression in E. coli BL21 Gold (DE3) (Agilent Technologies) from
pET21_HisA (List et al., 2012). The recombinant protein was purified from the soluble fraction of the crude extract by heat precipi-
tation (15 min at 73ꢂC) of the host proteins, followed by nickel-affinity chromatography. To this end, the extract was loaded onto
a column (HisTrap FF crude 5 ml; GE Healthcare) that had been equilibrated with 50 mM potassium phosphate, 300 mM NaCl,
1 mM imidazole (pH 7.5). The column was washed with the equilibration buffer, and the bound protein was eluted by applying a linear
gradient of 1–300 mM imidazole. Fractions with pure protein were pooled and dialyzed extensively against 50 mM potassium phos-
phate (pH 7.5). Based on SDS-PAGE analysis, the purity of all samples was at least 90%. The proteins were dripped into liquid
nitrogen and stored at ꢁ80ꢂC
HisG/IE was produced by heterologous gene expression in E. coli BL21 Gold (DE3) (Agilent Technologies) from p_hisGIE_tac
(Davisson et al., 1994). The strains containing the respective expression vectors were grown in lysogeny broth medium (2 L) at
37ꢂC to an OD600 of 0.7. Protein expression was induced by isopropyl-b-D-thiogalactopyranoside (IPTG; 1 mM) and incubated over-
night. Bacterial pellets were harvested and resuspended in 10 mM KP pH 7.5, 2.5 mM EDTA, and 1 mM DTT. Sonication and repeated
centrifugation yielded the protein-containing supernatant, which was subjected to ion-exchange chromatography with a MonoQ col-
umn (HR 16/10, 20 mL, Pharmacia). Proteins were eluted with a linear gradient of KP (10 / 500 mM), and fractions containing either
e4 Cell Chemical Biology 26, 1–14.e1–e9, November 21, 2019

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HisG or HisIE or both were pooled and dialyzed against 50 mM KP pH 7.5, 2.5 mM EDTA, and 1 mM DTT. Proteins were identified
based on their molecular weight using SDS-PAGE analysis and simultaneously checked for > 90% purity. Concentrated proteins
were dripped into liquid nitrogen and stored at ꢁ80ꢂC.
Subcloning of the hisF and hisH Genes
The hisF and hisH genes were cloned into vectors encoding an N-terminal His6-tag. For this purpose, the thrombin cleavage site of
pET28a(+) (Novagen) was substituted for a TEV-cleavage site. For hisF incorporation, the vector was amplified using primers flanking
the region encoding the thrombin cleavage site (P1 and P2, Table S1) using PWO Polymerase (Sigma Aldrich). Both primers con-
tained a part of the TEV cleavage site (bold), creating an 18 bp overlap facilitating circularization. pET28a(+) was amplified with
each primer separately and, after digestion with DpnI (NEB), a mixture of both reactions was transformed into E. coli NEB Turbo
(NEB) to yield the target plasmid pET28aTEV independent of ligation. The correct exchange was confirmed by Sanger Sequencing
(Microsynth Seqlab) starting from the T7 terminator.
The hisF gene was amplified from pET11c_HisF (Beismann-Driemeyer and Sterner, 2001) with primers P3 and P4 (Table S1) using
PWO polymerase (Sigma Aldrich). The primers carried an overhang containing an NdeI and a HindIII restriction site, respectively.
After digestion of both pET28aTEV and the PCR product with the appropriate enzymes (HF-enzymes, NEB), both fragments were
purified via agarose gel electrophoresis by means of a Cleanup Kit (Thermo Scientific). Afterwards, the fragments were mixed and
ligated with T4 Ligase (NEB). The correct sequence of the hisF gene was confirmed by Sanger Sequencing (Microsynth Seqlab) start-
ing from the T7 terminator.
For hisH incorporation, pET28a_BsaI (Rohweder et al., 2018) designed for golden-gate cloning, was modified to encode for a TEV-
cleavage site C-terminal of the N-terminal His6-tag. For this purpose, the plasmid was amplified in a standard PCR reaction using
primers P5 and P6 (Table S1). P5 contained the TEV cleavage site (bold).The purified (agarose gel Cleanup Kit, Thermo Scientific),
linear PCR fragment was re-cyclized in a ligation reaction with addition of T4 PNK (NEB), yielding pET28aTEV_BsaI.
The gene encoding hisH from T. maritima was optimized for E. coli codon usage and purchased as a linear gene string (GeneArt,
Thermo Scientific) carrying two terminal BsaI cleavage sites for golden-gate cloning. hisH was cloned into pET28aTEV_BsaI as
described previously (Rohweder et al., 2018). Because the N-terminus of HisH is shielded and therefore inaccessible for proteolytic
cleavage, an additional two amino acid linker (MG) was inserted between the TEV cleavage site and the hisH gene. To this end, the
plasmid carrying the gene was amplified in a standard PCR reaction using primers P7 and P8 (Table S1). After purification, the PCR
product was cyclized in a ligation reaction with addition of T4 PNK (NEB). The correct sequence of both pET28aTEV_BsaI and pE-
T28a_HisHwt was confirmed by Sanger Sequencing (Microsynth Seqlab) starting from the T7 terminator.
Subcloning of the aaRS/tRNACUA Pairs
Plasmids for incorporation of AzoF and NBY/NPY were provided by Prof. Peter Schultz (Scripps Research Institute, La Jolla, USA).
The pEVOL-vector system was especially designed for the incorporation of UAAs with two copies of the respective aaRS (Young
et al., 2010). pEVOL_NBY carries the Methanocaldococcus jannaschii tyrosyl-tRNA synthetase / tRNACUA (MjTyrRS-TyrT) pair
evolved for NBY incorporation with the mutations Y32G, L65G, F108E, D158S, and L162E (Deiters et al., 2006). The same construct
was used for NPY incorporation as previously confirmed (Luo et al., 2017). pEVOL_AzoF carries the MjTyrRS-TyrT pair evolved for
AzoF incorporation with the mutations Y32G, L65E, F108A, Q109E, D158G, and L162H (Bose et al., 2006). Both vectors were
checked by Sanger Sequencing (Microsynth Seqlab) starting from the araBAD promoter.
pEVOL_AzoF was further used as a template for the design of pEVOL_mNPK. The genes encoding the tyrosyl-tRNA synthetase
and its respective tRNACUA were substituted for the pyrollysyl-tRNA synthetase and its respective tRNACUA from Methanosarcina
barkeri (MbPylRS-PylT), which was evolved for caged-lysine derivatives with the mutations M241F, A267S, Y271C, and L274M
(Gautier et al., 2010). Initially, tyrT-tRNA was substituted by PCR-amplification of pEVOL_AzoF with primers P9 and P10 (Table
S1) containing pylT-tRNA (bold) and overlaps to the sequences flanking the original tyrT-tRNA. The PCR product was cyclized by
blunt end ligation using T4 polynucleotide kinase (NEB) and T4 Ligase (NEB) according to the manufacturer’s instructions to obtain
pEVOL_AzoF’_pylT.
Exchange of the first copy of MjtyrRS was prepared by amplifying pEVOL_AzoF’_pylT with primers P11 and P12 (Table S1) con-
taining overhangs with a BsaI restriction site and flanking the MjtyrRS gene. MbpylRS, synthesized (GeneArt) with BsaI restriction
sites complementary to those in the PCR product, was then joined with the linearized vector in a golden gate cloning procedure (Eng-
ler et al., 2008) to build pEVOL_mNPK’. The deletion 3’ of the open reading frame (ORF), resulting from deleting the BsaI restriction
site, does not include the transcription terminator and thus leaves the regulation of transcription intact. Analogous to the first copy,
the second MjtyrRS gene was substituted with MbpylRS by the same strategy using primers P13 and P14 (Table S1) leading to no
further deletions and resulting in the final pEVOL_mNPK vector. After each cloning step, the correct modification of the target site was
confirmed by Sanger Sequencing (Microsynth Seqlab) with the sequencing primers (Table S1) P15 for pEVOL_AzoF’_pylT, P16 for
pEVOL_mNPK’, and P17 for pEVOL_mNPK.
Site-Directed Mutagenesis of hisF
Stop codon point mutations (TAG, underlined) were introduced into pET28a_HisFwt according to the protocol of the Phusionꢀ site-
directed mutagenesis kit from Finnzymes with 5’ phosphorylated (PHO) and HPLC-purified primers (Metabion) P18–P37 (Table S1).
Correct mutagenesis was checked by Sanger Sequencing (Microsynth Seqlab) starting from the T7 terminator.
Cell Chemical Biology 26, 1–14.e1–e9, November 21, 2019 e5

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Building a Rotamer Library
In order to deduce a backbone-dependent rotamer library (Dunbrack and Karplus, 1993), MD simulations of a tri-peptide (Ala-UAA-
Ala) were analyzed to sample the conformational space of the respective side chains. MD simulations were performed using
Gromacs (version 5.1.2) (Berendsen et al., 1995) with GAFF (Wang et al., 2004) topologies generated by means of Acpype (Sousa
da Silva and Vranken, 2012), which is part of the AmberTools14 (Wang et al., 2006). The tripeptide has been solvated using tip4p/
˚
2005 water (Abascal and Vega, 2005) in a 3.5 A box, which was sufficiently large to accommodate the tri-peptide. After equilibration,
the temperature was kept at 300 K using a stochastic velocity-rescaling thermostat (Bussi et al., 2007) with a relaxation time of 1 ps,
and the pressure was set to 1 bar using a Berendsen barostat (Berendsen et al., 1984) with a relaxation time of 1 ps. The electrostatic
interactions were calculated by smooth particle-mesh Ewald summation, and the Lennard-Jones interactions were smoothly trun-
˚
cated between 0 and 10 A. The simulation time was 50 ns; during this period, all chi, phi, and psi angles were determined resulting in
the joint probability distributions of each angle combination. Binning the probabilities and calculating the median angle of each bin led
to several distinct angle combinations, their probability of occurrence, and their relative free energy. As described for the rotamer
library of SwissSidechain (Gfeller et al., 2013), the angle combinations were processed to enable their import into PyMOL. This import
provided the prerequisites for the interactive assessment of mutation experiments introducing UAAs. For MD simulations, conforma-
tions of minimal clashes with the adjacent residues were chosen for each UAA.
Expression and Purification of ImGPS
HisF and HisH proteins were produced by heterologous gene expression in E. coli BL21 Gold (DE3) (Agilent Technologies). Strains
containing the respective vector were grown in lysogeny broth (LB) medium (2–4 L) at 37ꢂC to an OD600 of 0.6. Protein expression was
induced after 30 min at 30ꢂC with 0.5 mM IPTG. Incubation overnight at 30ꢂC was followed by harvesting of bacterial pellets and
suspension in either 50 mM Tris,HCl pH 7.5, 100 mM NaCl, and 10 mM imidazole (HisF) or 50 mM KP pH 7.5, 100 mM NaCl, and
10 mM imidazole (HisH). Proteins were obtained from the supernatant after sonication and repeated centrifugation steps. The ma-
jority of E. coli proteins were precipitated in a heat step (15 min, 75ꢂC HisF and 15 min, 70ꢂC HisH) and subsequent centrifugation.
Proteins were subjected to nickel-affinity chromatography (HisTrapꢀ FF Crude column, 5 mL, GE Healthcare) and eluted with a linear
gradient of imidazole (10 / 750 mM). Fractions containing the proteins were identified by SDS-PAGE analysis, pooled and further
purified with a size-exclusion chromatography column (Superdex 75 HiLoad 26/600, GE Healthcare) by using 50 mM HEPES pH 7.5
and 100 mM NaCl as running buffer. Fractions were checked on SDS-PAGE analysis for > 90% purity, pooled, concentrated, and
dripped into liquid nitrogen for storage at ꢁ80ꢂC.
For incorporation of UAA into HisF, a slightly adjusted expression protocol was used. After cotransformation of the pET28a vector,
carrying the desired TAG codon, and pEVOL_NBY, pEVOL_AzoF or pEVOL_mNPK, respectively, into E. coli BL21 Gold (DE3), the
strains were grown in LB medium (6 L) at 37ꢂC to an OD600 of 0.6. Then, bacterial pellets were harvested by centrifugation at
room temperature and suspended in terrific broth (TB) medium (600 mL). Bacterial growth was resumed to an OD600 of approximately
10 at 37ꢂC and incorporation was induced by addition of 1 mM UAA and 0.02% L-arabinose. Omission of IPTG turned out to be favor-
able for expression of UAA-HisF proteins, as higher yields could be obtained in its absence. Cultures were incubated overnight at
30ꢂC and proteins were purified as described above.
Tryptic Digest and MS Analysis
Recombinant T. maritima HisF proteins were run on a 15% SDS-PAGE and stained with Coomassie G250 (SimplyBlue SafeStain,
Lifetech). Protein bands were cut out from the gel, washed with 50 mM NH4HCO3, 50 mM NH4HCO3/acetonitrile (3/1), 50 mM
NH4HCO3/acetonitrile (1/1) and lyophilized. After a reduction/alkylation treatment and additional washing steps, proteins were
in gel digested with trypsin (Trypsin Gold, mass spectrometry grade, Promega) overnight at 37ꢂC. The resulting peptides were
sequentially extracted with 50 mM NH4HCO3 and 50 mM NH4HCO3 in 50% acetonitrile. After lyophilization, peptides were reconsti-
tuted in 20 mL 1% TFA and separated by reversed-phase chromatography. An UltiMate 3000 RSLCnano System (Thermo Fisher Sci-
entific, Dreieich) equipped with a C18 Acclaim Pepmap100 preconcentration column (100 mm i.D.x20 mm, Thermo Fisher Scientific)
and an Acclaim Pepmap100 C18 nano column (75 mm i.d.x250 mm, Thermo Fisher Scientific) was operated at flow rate of 300 nL/min
and a 60 min linear gradient of 4% to 40% acetonitrile in 0.1% formic acid. The LC was online-coupled to a maXis plus UHR-QTOF
System (Bruker Daltonics) via a CaptiveSpray nanoflow electrospray source. Acquisition of MS/MS spectra after CID fragmentation
was performed in data-dependent mode at a resolution of 60,000. The precursor scan rate was 2 Hz processing a mass range be-
tween m/z=175 and m/z=2,000. A dynamic method with a fixed cycle time of 3 s was applied via the Compass 1.7 acquisition and
processing software (Bruker Daltonics). Prior to database searching with Protein Scape 3.1.3 (Bruker Daltonics) connected to Mascot
2.5.1 (Matrix Science), raw data were processed in Data Analysis 4.2 (Bruker Daltonics). A customized database comprising the
T. maritima entries from UniProt as well as manually added sequences of the mutated HisF proteins and common contaminants
was used for database search with the following parameters: enzyme specificity trypsin with two missed cleavages allowed, precur-
sor tolerance 10 ppm, MS/MS tolerance 0.04 Da. As general variable modifications, deamidation of asparagine and glutamine, oxida-
tion of methionine, carbamidomethylation or propionamide modification of cysteine were set. Specific variable modifications for
identification of unnatural amino acids were o-nitrobenzyltyrosine (NBY), o-nitropiperonyltyrosine (NPY), methyl-o-nitropiperonylly-
sine (mNPK), or phenylazophenylanine (AzoF). AzoF was detected as modification of phenylalanine, why each position of AzoF
e6 Cell Chemical Biology 26, 1–14.e1–e9, November 21, 2019

Please cite this article in press as: Kneuttinger et al., Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino Acids, Cell
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incorporation was changed to a phenylalanine in the query, respectively. NBY and NPY were detected as modification of tyrosine,
why each position of NBY and NPY incorporation was changed to a tyrosine in the query, respectively. Spectra of peptides contain-
ing unnatural amino acids were inspected manually.
Screening of HisF Positions
Caged HisF proteins were either used ‘‘as isolated’’, or irradiated for 20 min at 365 nm (conventional UV lamp, two fluorescent black
light bulbs with 8 W, Sylvania, settings: 250 mA and 220 V). The proteins were each diluted to 30 mM and cooled in a metal rack during
irradiation. HisF proteins containing AzoF were also used either ‘‘as isolated’’, irradiated for 40 s at 365 nm (conversion to the Z state)
or further irradiated for 40 s at 420 nm (back conversion to the E state) (High Power LED, Seoul Viosys, settings: 600 mA and 4.5 V).
Irradiation occurred in ꢀ 1 cm distance to the light source. UAA-HisFs were then mixed at equimolar concentrations with wt-HisH in
50 mM HEPES pH 7.5, and 100 mM NaCl to form the ImGPS complex and kept in the dark. HisH activity was determined in a coupled
enzymatic assay (Beismann-Driemeyer and Sterner, 2001) with glutamate-dehydrogenase (GDH, 99% pure, Roche) as auxiliary
enzyme and NAD+ as co-substrate. Reaction conditions included 50 mM Tris-acetate pH 8.5, glutamine in saturation (5 mM),
1 g/L GDH, 70 mM ProFAR, and 10 mM NAD+. All reactions were set up in a 96-well plate, started with 2 mM ImGPS and followed
continuously at l = 340 nm [De340(NADHꢁNAD+) = 6,300 Mꢁ1 cmꢁ1] using a plate reader (Tecan Infinite M200 Pro).
Native MS Analysis
Identity of fF23NBY, fY39NBY, fY39NPY, and fK99mNPK and decaging efficiencies were analyzed by online buffer exchange MS
using an UltiMateꢀ 3000 RSLC (Thermo Fisher Scientific) coupled to an Exactive Plus EMR Orbitrap instrument (Thermo Fisher Sci-
entific) modified to incorporate a quadrupole mass filter and allow for surface-induced dissociation (VanAernum et al., 2019). Proteins
were either analyzed in their ‘‘as isolated’’ state or after exposure to UV light (UVP BL-15; Analytik Jena US; CA 91786) for 20 min.
100 pmol protein were injected and online buffer exchanged to 200 mM ammonium acetate, pH 6.8 (AmAc) by a self-packed buffer
exchange column (Waitt et al., 2008) (P6 polyacrylamide gel, BioRad) at a flow-rate of 100 mL per min. Mass spectra were recorded for
1,000–8,000 m/z at 35,000 resolution as defined at 200 m/z. The injection time was set to 200 ms. Voltages applied to the ion optics
were optimized to allow for efficient ion transmission while minimizing unintentional ion activation. fY39NPY and fK99mNPK showed
a minor tendency to form higher oligomers under the given conditions. Only m/z corresponding to the monomer were considered for
deconvolution and subsequent relative quantitation. Mass spectra were deconvoluted with UniDec version 2.6.5 (Marty et al., 2015)
using the following processing parameters: sample mass every 0.3 Da; peak FWHM 0.3 Thompson, Gaussian peak shape function.
CD Analysis
Circular dichroism (CD) spectra in the far-UV range of 190–250 nm were recorded in a Jasco J-815 spectrophotometer with five ac-
cumulations. The spectra were measured with 5–12 mM protein in 50 mM potassium phosphate, pH 7.5 in a 0.1 cm cuvette at 25ꢂC.
The curves were smoothed in Origin 2018 (OriginLab). Data were normalized to obtain the mean residue ellipticity as described in
(Kelly et al., 2005).
UV/Vis Analysis
UV/Vis spectra in the range of 230–550 nm were recorded in a Jasco V650 spectrophotometer. The spectra were measured with
15–30 mM protein in 50 mM HEPES, pH 7.5 and 100 mM NaCl in a 1 cm cuvette at 25ꢂC. Irradiation of fS55AzoF took place by
removing the cuvette from the spectrophotometer and exposing it to 365 nm UV light (conventional UV lamp, two fluorescent black
light bulbs with 8 W, Sylvania, settings: 250 mA and 220 V) or subsequently to 420 nm visible light (High Power LED, Seoul Viosys,
settings: 600 mA and 4.5 V). Caged proteins were irradiated by removing the cuvette and exposing it to 365 nm UV light with the con-
ventional UV lamp. High-power irradiation was performed with a 365 nm LED (High Power LED, LED Engin, settings: 700 mA and 16
V) and a 420 nm LED (High Power LED, Seoul Viosys, settings: 300 mA and 4 V) installed perpendicular to the measurement beam.
After each irradiation step, spectra were recorded and baseline corrected. Estimation of photostationary state composition from
UV/Vis spectra was performed as described in (Calbo et al., 2017).
Analytical Size-Exclusion Chromatography
30 mM HisF monomer or 30 mM HisF mixed with 30 mM wt-HisH were subjected to a S75 10/300 GL (GE Healthcare) column pre-equil-
ibrated in 50 mM HEPES, pH 7.5 and 100 mM NaCl. Samples were eluted in the same buffer, and protein as well as UAA peaks were
detected at 280 nm, 334 nm (AzoF), and 360 nm (NPY, mNPK).
Steady-State Kinetics
UAA-HisFs were either used ‘‘as isolated’’, or irradiated with 365 nm light as described above (screening). Proteins were then used as
single subunit for the determination of steady-state constants of HisF activity or mixed at equimolar concentrations with HisH in
50 mM HEPES, pH 7.5, and 100 mM NaCl. The ammonia-dependent HisF activity and the ImGPS activity were measured continu-
ously following PrFAR turnover at 300 nm [De300(PrFARꢁAICAR) = 5,637 Mꢁ1 cmꢁ1] (Beismann-Driemeyer and Sterner, 2001). Re-
action conditions of HisF activity measurements were 50 mM Tris-acetate pH 8.5, 100 mM ammonium acetate (saturated), 0.6 mM
HisA (to convert ProFAR to PrFAR), varying concentrations of ProFAR (1–40 mM), and 0.1–0.3 mM HisF at 25ꢂC. Reaction conditions of
HisH activity measurements were 50 mM Tris-acetate pH 8.5, 0.6 mM HisA (to convert ProFAR to PrFAR), 40 mM ProFAR (saturated),
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Please cite this article in press as: Kneuttinger et al., Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino Acids, Cell
Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.08.006
varying concentrations of glutamine (0.1–10 mM), and 0.1–0.2 mM ImGPS complex at 25ꢂC. In order to circumvent the stimulating
effect of NAD+ on HisH activity as in the GDH-coupled assay used in the screening (Beismann-Driemeyer and Sterner, 2001), a
different continuous assay was performed for determination of ProFAR activation constants (Kac). For this, the HisH reaction was
coupled to glutamate-oxidase (GOX, 99% pure, Sigma-Aldrich) producing a-ketoglutarate and hydrogen peroxide out of
glutamate. In a second step, hydrogen peroxide was turned over by horse-radish peroxidase (HRP, 99% pure, Sigma-Aldrich)
together with colorless 2-aminoantipyrine and phenol to form red-colored quinoneimine. The reaction was followed at 505 nm
with e505(quinoneimine) = 6,400 Mꢁ1 cmꢁ1 (Green and Hill, 1984). Initial optimization of this coupled reaction identified pH 7.0 as
an optimum, hence, reaction conditions included 20 mM Tris,HCl pH 7.0, 5 mM glutamine (saturated), 20 mU/mL GOX, 0.15 g/L
HRP, 1 mM 4-aminoantipyrin, 1 mM phenol, varying concentrations of ProFAR (5–70 mM), and 0.05–1 mM ImGPS complex at
25ꢂC. All reactions were started by the addition of the ImGPS complex. Measurements were performed either with a Jasco V650-
UV/Vis spectrophotometer or for the GOX assay with a plate reader (Tecan Infinite M200 Pro). Activities were deduced from the initial
slopes of the transition curves and analyzed using the Michaelis-Menten equation.
Direct Photo-control of HisH Activity
ProFAR-stimulated HisH-activity was followed in the GOX-assay with increased concentrations of auxiliary enzymes to prevent irra-
diation effects on the light-sensitive cofactors of GOX and HRP. Reaction conditions included 20 mM Tris,HCl pH 7.0 or pH 8.5, 5 mM
glutamine, 100 mU/mL GOX, 70 mM ProFAR, 0.75 g/L HRP, 3 mM 2-aminoantipyrin, 3 mM phenol, and 0.2 mM ImGPS complex. All
reactions were started by the addition of the ImGPS complex and followed at l = 505 nm with e505(quinoneimine) = 6,400 Mꢁ1 cmꢁ1
(Green and Hill, 1984). Measurements were performed in a plate reader (Tecan Infinite M200 Pro) and activities were deduced from
the slopes of the transition curves. Each sample was measured simultaneously with three controls in a 96-well plate. Negative and
positive controls, started with non-irradiated and pre-irradiated ImGPS complex, respectively, were kept in the dark. Pre-irradiation
was performed with 365 nm for 1.5 min (High Power LED, LED Engin, settings: 700 mA and 16 V). In a fourth control, buffer was added
instead of the ImGPS complex, in order to monitor the remaining effect on the auxiliary enzymes. Irradiation of each sample and its
buffer control occurred by pausing the measurement in the plate reader, taking out the plate, and irradiating the two wells while keep-
ing the other two wells (positioned furthest away), containing both negative and positive control, covered. wt and caged proteins were
irradiated once after 60 min for 1.5 min with 365 nm (High Power LED, LED Engin, settings: 700 mA and 16 V). AzoF-proteins were
irradiated after 40 min for 30 s with the 365 nm High Power LED and after another 40 min for 1 min with a 420 nm High Power LED
(High Power LED, Seoul Viosys, settings: 300 mA and 4 V). The turnover curves of irradiated samples were baseline corrected by
subtraction of the buffer control. Negative and positive controls were baseline corrected by subtraction of a linear fit of the first
40–60 min of the buffer control.
Crystallization
ImGPS(fY39NPY) and ImGPS(fS55AzoF) were crystallized following a protocol described for PDB-ID 1gpw (Douangamath et al.,
2002). Prior to crystallization, His6-tags of HisF and HisH proteins were removed by cleavage with TEV protease (20 mg per 1 mg pro-
tein) at 20ꢂC overnight. Pure complex was obtained by size-exclusion chromatography (Superdex 75 HiLoad 26/600, GE Healthcare)
of equimolar amounts of HisH and HisF in 10 mM Tris,HCl pH 8.0. Fractions containing the complex were identified by SDS-PAGE
analysis, pooled and concentrated.
1 mL of ꢀ 20 mg/mL complex was then mixed with 1 mL of reservoir solution, containing 13–17% PEG 8000, 0.7–0.9 M ammonium
nitrate, 0.1 M HEPES,NaCl pH 8.5, 10 mM DTT, and 5% (v/v) MPD. Crystals grew within one week in a rod-like morphology similar to
wt-ImGPS using the hanging drop vapor diffusion method. Crystals were mounted onto a nylon loop and shock-frozen in liquid ni-
trogen without addition of further cryoprotectants.
Data Collection, Structure Solution and Refinement
˚
Data sets were collected to ꢀ 2.8 A resolution each, using synchrotron radiation from the Swiss Light Source (SLS), Switzerland at
beamline PXIII and PXI. Data collection for both ImGPS(fY39NPY) or ImGPS(fS55AzoF) was done at cryogenic temperature. Data
were processed using XDS (Kabsch, 1993), and the data quality was assessed using the program PHENIX (Adams et al., 2002). Struc-
tures were solved by molecular replacement with MOLREP within the CCP4i (Potterton et al., 2004) suite using PDB-ID 1gpw (Douan-
gamath et al., 2002) as search model. Initial refinement was performed using REFMAC (Murshudov et al., 1997). The model was
further improved in several refinement rounds using automated restrained refinement with PHENIX (Adams et al., 2002) and interac-
tive modelling with Coot (Emsley and Cowtan, 2004), in which the mutation D11N present in PDB-ID 1gpw was restored to wt. The
final model was analyzed using the program MolProbity (Davis et al., 2007). Structural and refinement statistics are listed in Tables S2
and S3.
Molecular Dynamics (MD) Simulations
MD simulations were conducted with Yasara (Krieger et al., 2004), version 17.4.17, force field AMBER03 on the basis of the ImGPS
complex (PDB-ID 1gpw, chains C, D). Residue 11 in HisF was mutated back to the wt aspartate, missing side chains in flexible loops
of HisF were replaced, and PrFAR was positioned as in the Saccharomyces cerevisiae ImGPS structure (PDB-ID 1ox5). UAAs were
incorporated into HisF and sterically oriented by using the previously generated rotamer library. A hexagonal simulation cell was
˚
created, which was 5 A larger than the protein along each axis, filled with water to a density of 0.997 g/mL and with counterions
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Please cite this article in press as: Kneuttinger et al., Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino Acids, Cell
Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.08.006
to a final concentration of 0.9% NaCl. Three independent simulation runs of 100 ns length were performed with slightly different tem-
peratures (25 0.01ꢂC) adjusted by the Berendsen thermostat to alter the starting conditions. Each minimization consisted of an
initial equilibration step of 14 ps length. During the next 100 ns of simulation, a snapshot was recorded every 20 ps. The root-
mean-square fluctuation (RMSF) value representing the deviation from the mean position for each atom of the ImGPS complexes
was deduced for each series of snapshots. For this calculation, the function R:rmsf (version 3.6.0) was used (Skjærven et al.,
¨
2014); corresponding values resulting from the three temperature-specific runs were averaged. By means of PyMol (Schrodinger,
2015), RMSF values were mapped onto the 3D structure and visualized. RMSF values were read as B factors, and a color gradient
˚
˚
˚
ranging from blue over white to red was used to indicate RMSF values in the range of [0 A, 1.5 A]; values R 1.5 A are shown in red.
Distances between specific atoms were calculated using the Yasara function Distance; corresponding values resulting from the three
˚
MD runs were pooled in histograms (bin size 0.05 A) and plotted in the form of probability distributions. Minima and maxima were
determined by utilizing a sliding window of size five. By means of the plotly (https://plot.ly) function Histogram2DContour, a 2D con-
tour density plot was generated for the pair of distances hH178Nd–hE180Cd and fD98Cg–hH178Ne. Scripts for automated processing
were written in Python 2.7 (Oliphant, 2007).
Nano Differential Scanning Fluorimetry
The Nano-DSF technique was used to analyze the thermal stability of proteins. All samples were prepared in 30 mM concentration,
filled into capillaries and scanned at 330 nm and 350 nm during a heating gradient of 35–95ꢂC (1ꢂC minꢁ1) in a NanoDSF Prometheus
NT.48 (Nanotemper technologies). For analysis of melting temperatures (Tm), the ratio 350 nm/330 nm was plotted against the tem-
perature. Tm was determined as the transition midpoint with Origin 2018 (OriginLab), also defined as the peak maximum in the first
derivative (f’) of the denaturing curve. Melting temperatures were measured for wt-HisF, fY39NPY, fS55AzoF, and fK99mNPK in their
‘‘as isolated’’ forms. fY39NPY and fK99mNPK were also analyzed after 30 min irradiation (365 nm, conventional UV lamp, two
fluorescent black light bulbs with 8 W, Sylvania, settings: 250 mA and 220 V). fS55AzoF, however, could only be measured in its
E-enriched state, since its Z-enriched state is thermally unstable.
QUANTIFICATION AND STATISTICAL ANALYSIS
All activity data points shown in the figures are the mean SEM (standard error of mean) of at least two technical replicates. Fitting of
data points using Origin 2018 (OriginLab) resulted in the fitting values SE (standard error; scaled with square root of reduced Chi-
square). Detailed description of the used fitting functions can be found in the figure legends, table footer, and the Method Details
section. For data calculated from two values SE(M.), the SE was calculated according to the Gaussian law of error propagation.
All analyses and data representations were performed and assembled in Origin 2018 (OriginLab) except for MS analysis (Data Anal-
ysis 4.2, Bruker Daltonics), native MS analysis (UniDec version 2.6.5 (Marty et al., 2015)), crystal structure analysis (MolProbity (Davis
et al., 2007)), and computational analysis (Yasara (Krieger et al., 2004), plotly (https://plot.ly), Gromacs (version 5.1.2) (Berendsen
et al., 1995)). Details can be found in the method details section and data collection and refinement statistics of crystal structure anal-
ysis can be found in the Supplemental Information.
DATA AND CODE AVAILABILITY
The crystal structures generated during this study are available at the Protein Data Bank (http://www.rcsb.org) with the following
accession numbers: ImGPS(fY39NPY) PDB: 6rtz and ImGPS(fS55AzoF) PDB: 6ru0.
Cell Chemical Biology 26, 1–14.e1–e9, November 21, 2019 e9