Available online at www.sciencedirect.com
ScienceDirect
The family of phytochrome-like photoreceptors: diverse,
complex and multi-colored, but very useful
Katrin Anders and Lars-Oliver Essen
Bilin-dependent GAF domain photoreceptors cover the whole
spectrum of light with their absorbance properties. They can be
divided into three groups according to the domain architecture
of their photosensory module. Group I and Group II harbor
phytochromes with PAS-GAF-PHY and GAF-PHY domain
architecture, respectively. Group III consists of stand-alone
GAF domain photoreceptors, the cyanobacteriochromes.
Crystal structures of all three groups are now available to shed
light on possible downstream signaling pathways. Structures of
Group I and III photoreceptors in both states display changes in
the secondary structures during photoconversion. The
knowledge about the photoconversion in phytochromes and
CBCRs make them promising targets for applications in life
science and synthetic biology.
Address
Department of Chemistry, Philipps-University, Hans-Meerwein-Str. 4,
D-35032 Marburg, Germany
Corresponding author: Essen, Lars-Oliver
(essen@chemie.uni-marburg.de)
Current Opinion in Structural Biology 2015, 35:7–16
chromophores into three subfamilies dependent on their
common domain architecture (Figure 1a). Group I
includes canonical phytochromes whose sensory region
adopts a PAS-GAF-PHY domain architecture. Firstly
discovered in plants this domain organization is characteristic of bacterial, algal and fungal phytochromes as well
(Figure 1a). One consequence of their complex topology
is a figure-of-eight knot structure, where the variable Nterminus is threaded through an elongated loop of the
GAF domain [3,4] (Figure 2a). Cyanobacteria harbor
diverse phytochrome-like photoreceptors of Groups II
and III (Figure 1a). Cyanobacterial phytochromes of
Group II (Cph2) lack only the N-terminal PAS domain,
whereas Group III, the cyanobacteriochromes (CBCRs),
form a diverse subfamily of bilin-binding stand-alone
GAF domains. Together with their effector domains most
of these phytochromes apparently exist as dimers. Accordingly, the crystal structures of their photosensory
modules are often found as parallel or antiparallel dimers.
Interestingly, the two photostates of Group III members
can cover any region of light from ultraviolet (UV) to near
infrared radiation (NIR).
This review comes from a themed issue on Catalysis and regulation
Edited by Judith P Klinman and Amy C Rozenzweig
http://dx.doi.org/10.1016/j.sbi.2015.07.005
0959-440/# 2015 Elsevier Ltd. All rights reserved.
Introduction
Light perception allows life to adapt to changing illumination, for example, for controlling morphogenesis, photosynthesis or avoidance of harmful radiation.
Phytochromes utilize bilins as chromophore and occur
in most kingdoms of life, for example, algae, plants and
cyanobacteria, as well as in non-photosynthetic organisms
such as bacteria and fungi, but interestingly not in animals
and archaea [1]. Their chromophore senses two different
light qualities by adopting two stable, photoconvertible
isomers: In plant, fungal and many bacterial phytochromes the Pr state absorbs red light to convert to the
Pfr state, whereas the latter converts back by far-red
light absorption. Furthermore, conversion can proceed
independently of light as dark reversion [1]. Rockwell
et al. [2] classified GAF domain photoreceptors with bilin
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As linear tetrapyrroles, bilins form long-lived excited
states by their highly conjugated ring system [5] without
being toxic to the cell while allowing enhancement and
fine-tuning of their absorption by the protein environment. Bacteriophytochromes (BphP) and fungal phytochromes (Fph) link biliverdin IXa (BV), a degradation
product of heme, autocatalytically to an N-terminal cysteine residue as chromophore. Bilin reductases catalyze
BV reduction to the other phytobilins (Figure 1b), phytochromobilin (PFB) and phycocyanobilin (PCB) from
plant and cyanobacterial phytochromes that are attached
to a conserved cysteine in the GAF domain [3,4,6]. The
photocycle of these bilin chromophores depends on the
Z ! E and E ! Z photoisomerization of their C15 = C16
double bond (Figure 1b). BV-binding phytochromes
absorb NIR by their 15E-state and are thus the most
red-shifted GAF-containing photoreceptors. So far, all
bathyphytochromes (bathy-BphPs) that form Pfr as
ground state belong to this group of BV-binding phytochromes. Compared to bathy-BphPs PFB-binding plant
phytochromes are blue-shifted for their 15Z-states and
15E-states (Figure 1c). This is further pronounced in
PCB-binding phytochromes of Groups I and II with
the latter displaying the most blue-shifted absorbance
maxima, especially in the Pr state [7]. In contrast, CBCRs
undergo very diverse photocycles [8–15] with currently
four major types: Two types have opposite photocycles
Current Opinion in Structural Biology 2015, 35:7–16
8 Catalysis and regulation
Figure 1
Group II
(a)
PAS
(Cph2-like)
GAF
PHY
HKR
Group I
(canonical)
PhyA
PhyB
R
Cys
R
Cys
N
SynCph1
MCP
HK
GAF
PAS
PHY
TePixJ
R
Cys
MCP
AnPixJ
N
HK
RpBphP2
DrBphP
NcFph1
SynCph2
HK RR
EAL
(b)
O
O
O
BV
O
(15Z)
C
B
NH
HN
H
N
15 16
H
N
D
O
A
Cys
O
Group III
(CBCR)
O
O
O
PCB
B
O
R
Cys
R
Cys
R
C
NH
HN
H
N
15 16
H
N
D
O
Cys
R
A
O
(c)
Cys
760
R
O
O
O
PΦB
B
C
NH
HN
H
N
15 16
O
H
N
15 16
D
N
H
O
O
O
Cys
C
B
NH
O
D
HN
H
N
A
15 16
A
O
Cys
O
PVB
O
O
Cys
H
N
D
O
Absorbance maximum 15E-state (nm)
BV Pfr (15E)
Phytochromes
720
Pg/Pr CBCRs
680
640
Insert-Cys and
DxCF CBCRs
600
560
520
Pr/Pg CBCRs
480
400
440
480
520
560
600
640
680
720
Absorbance maximum 15Z-state (nm)
Current Opinion in Structural Biology
Domain architecture, chromophores and spectral diversity of members of the phytochrome family. (a) Sequence-similarity network of phytochrome-like
GAF domains (INTERPRO entry 16032; edges correspond to pair-wise BLAST E-values of <1075; each node to sequences of >70% sequence
identity) as calculated by the EFI-enzyme similarity web tool (efi.igb.illinois.edu/efi-est). Group I phytochromes are clustered in plant (light-red), bacterial
(dark-red) and fungal (brown) subfamilies. PAS and PHY domains are depicted in red and green, respectively. Blue stars indicate the bilin chromophore
and the location of the cysteine as chromophore attachment site that is either part of the GAF domain (light red) or the N-terminus (GAF domain: dark
red). Red lines depict the knot regions, dark-green loops the tongue-region of the PHY domain. Effector domains are abbreviated by R; CBCRs (gray)
may also have N-terminal domains. Domain organizations of Group I, II (orange) and III (gray, black) members including the effector domains are
shown as ellipses and rectangles (PAS: red; effector domains: white, gray or black; HKR domain: histidine kinase related domain; Syn: Synechocystis
sp. PCC 6803; Nc: Neurospora crassa; An: Anabaena sp. PCC 7120; Te: Thermosynechococcus elongates; Rp: Rhodopseudomonas palustris).
Current Opinion in Structural Biology 2015, 35:7–16
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Phytochromes: diverse, complex and multi-colored Anders and Essen 9
with green/red (G/R) CBCRs adopting a green-absorbing
(15Z) ground state and a red-absorbing (15E) photoproduct
[16], whereas red/green (R/G) CBCRs convert from a redabsorbing (15Z) state to a green-absorbing (15E) photoproduct [8,15,17,18]. The latter shows that the 15E-isomer
is not necessarily the long-wavelength absorbing state. The
insert-Cys and DxCF-types of CBCRs employ a second
cysteine residue for additional chromophore attachment
[14,19]. Their 15Z ground state absorbs at shorter wavelengths from near-UV to blue, whereas the 15E-state
absorbs at longer wavelengths, that is, from blue to orange.
All CBCRs attach initially PCB as chromophore, but
DXCF-type CBCRs autocatalytically isomerize PCB into
PVB, thus shifting the absorbance of the 15E-state between teal and orange [12,13,20]. Interestingly, algae harbor Group I phytochromes, which clearly deviate from Pr/
Pfr-phytochromes of plant and fungal origin by exerting a
similar capability of spectral tuning as the CBCRs [21,22].
The structural repertoire of phytochrome-like
photoreceptors
Group I phytochromes show common features for binding
of BV, PFB or PVB in their GAF domain (Figure 2a).
Besides being involved in knot formation the N-terminus
clings to the chromophore-binding site and additionally
shields the chromophore from solvent access. The GAF
domain is connected via a long helical spine to the PHY
domain. The latter protrudes a tongue-like region that
reaches towards the GAF domain and covers the chromophore-binding pocket. The Group II phytochrome structure [23] closely resembles structurally Group I, but
lacks the N-terminal PAS domain and therefore the knot
in the structure. Both, in Group I and II structures the
tongue region shows common features depending on
the photochemical state of the protein. In the Pr-state
the stem of the tongue region is built by a two-stranded
b-hairpin-like structure. The length of the b-strands and
the region at the tip of the tongue vary but the positions
of the conserved WG/AG, PRxSF and WxE motifs are
invariant [3,23] (Figure 2c, left). The Pfr structures of
bathy-BphPs show different features [4,24] (Figure 2c,
right). Here, loop and a-helical sections build the stem
region. The tip of the tongue does not vary in length but
in the orientation of the loop region. The WG/AG, PRxSF
and WxE motifs are conserved in their positions but not
as strictly as in the Pr structures.
The photosensory GAF domain of CBCRs is structurally
highly related to Groups I and II apart from a truncated
loop region that is part of the knot structure in Group I
phytochromes and forms an additional b-strand in knotless Group II GAF domains (Figure 2b, red box). Another
difference is the a-helix facing the chromophore in nonBphPs that is distinctly oriented in Group I, II and III
photoreceptors. In addition, the loop region above the
chromophore-binding site differs (Figure 2b, inlet). Here,
group I and II phytochromes form a short helix that
includes the conserved DIP motif. The aspartate of this
motif is part of a salt bridge in Group I and II phytochromes that connects the tongue region of the PHY
domain with the GAF domain, the course of the following
loop/b-strand region is similar in both groups. In CBCRs
the DIP motif helix is missing, the subsequent loop
regions are more shifted towards the PCB-binding a-helix
thus narrowing the cleft that leads to the bilin-binding
site, which is otherwise blocked by the missing PHY
domain. In the TePixJ (15E) structure [25] (Figure 2b
inlet, blue) this loop region is shifted to an extent that it
nearly closes the cleft.
Photoconversion and down-stream signaling
in phytochromes and CBCRs
Structural changes upon photoconversion have been intensely studied to elucidate intramolecular pathways for
downstream signaling. 15Z-structures and 15E-structures
are now available both for phytochromes and CBCRs.
Structures of the Group I phytochrome DrBphP from
Deinococcus radiodurans (Figure 3a–c) were solved in its Pr
(15Z) and Pfr (15E) state [26] and confirmed an earlier
Trp-switch model [23]. Here, Z ! E isomerization by
red light triggers structural changes, where an aspartatearginine salt bridge is broken with an accompanying swap
of tryptophans from the WG/AG and WxE motifs within
the GAF/tongue interface. The GAF-PHY domain linking helical spine is straightened during photoconversion
(Figure 3b), whereas the tongue undergoes further refolding from a b-hairpin-like to an a-helical structure, a b/atransition inferred before from differences between Pr
and bathy-BphP Pfr tongue structures (Figure 2c)
[23,27]. The PRxSF motif alters its conformation such
that new interactions are formed between the tongue and
the GAF domain [23] (Figure 4a). These changes also
affect the C-terminal a-helix that connects the photosensory with the effector module [26]. Single particle
electron microscopy (SPEM) of DrBphP full length
dimers in the Pr and Pfr conformation indicate a quaternary structure for both states [28], which corresponds to a
parallel head-to-head and intimately twisted arrangement
( Figure 1 Legend Continued ) The central part of the panel comprises isolated clusters, which are related to other Group I-III phytochromes with
E values of >1075. (b) Chromophores of phytochrome-like photoreceptors; BV: biliverdin; PCB: phycocyanobilin, PFB: phytochromobilin; PVB:
phycoviolobilin in the Pr (15Z) conformation. Differences relative to BV as well as the Pfr (15E) conformation of the C15-C16 double bond are highlighted
in red. (c) Absorbance maxima of the 15Z-states and 15E-states of photosensory modules from photoreceptors of Groups I-III. Black squares
represent Group I phytochromes; cyan colored triangles show bathy-BphPs with a Pfr ground state that also belong to this group. Red dots indicate
members of Group II, green triangles members of Group III, the CBCRs. The black line is a diagonal in the wavelength scale indicating either red shift
(above) or blue shift (below) upon Z ! E photoisomerisation. Black frames show the assignment of phytochrome-like proteins to subfamilies, red
circles unusual CBCRs or phytochromes.
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Current Opinion in Structural Biology 2015, 35:7–16
10 Catalysis and regulation
Figure 2
(a)
Group I
tongue
(b)
AtPhyB
C
GAF
PAS
PHY
N
SynCph1
AnPixJ
TePixJ_15E
90º
C
N
PaBphP
Group
SynCph1
I
SynCph2
II
TePixJ_15Z III
C
Group II
SynCph2
C
N
Group III: CBCRs
(c)
Tongue SynCph2
Tongue SynCph1
AnPixJ
GAF S
W
Tongue PaBphP
Tongue RpBphP1
W
F
R
x
C
W
GAF
P
R
F
W
P
x
N
S
PCB
TePixJ_15E
PAS
N
Pr
BV
Pfr
C
Current Opinion in Structural Biology
Phytochrome and CBCR structures. (a) Structure of the photosensory modules of Group I–III photoreceptors. PAS, GAF and PHY domains are
shown in red, orange/violet/gray and green, respectively. The chromophore is displayed in cyan. PDB-codes: AtPhyB: 4OUR, SynCph1: 2VEA,
PaBphP: 3NHQ, SynCph2: 4BWI, AnPixJ: 3W2Z, TePixJ (15Z): 4GLQ, 4FOF, TePixJ (15E): 3VV4. (b) Superimposition of the SynCph1 (Group I),
SynCph2 (Group II) and TePixJ (15Z) (Group III) GAF domains; the inlet and boxes illustrate differences between the GAF domains. The inset
displays a magnification of the chromophore-binding site regarded from the top. In addition, also AnPixJ and TePixJ (15E) are shown in this inlet.
The lower red box shows the knot-forming loop region in Group I phytochromes at the end of the b-sheet in the GAF domain. (c) Comparison
of the tongue region in the Pr conformation of SynCph1 and SynCph2 and the Pfr conformation of PaBphP and RpBphP1 (PDB code: 4GW9).
Conserved amino acids in the PRxSF motif as well as the tryptophan residues of the WG/AG and WxE motif are shown in stick representation.
Ca-atoms of the b-sheets in the tongue of the Pr state are indicated by red and blue dots so that their position in the Pfr state can be located.
Current Opinion in Structural Biology 2015, 35:7–16
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Phytochromes: diverse, complex and multi-colored Anders and Essen 11
Figure 3
(a)
Group I
DrBphP
Tongue Pr
GAF
N
GAF
S
x
F
Y
P
F
x
W
R
N
R
Y
P
S
BV
C
Tongue Pfr
BV
C
GAF: Pr
GAF: Pfr
PAS: Pr
PAS: Pfr
(b)
Pr
PAS
PAS
PHY: Pr
PHY: Pfr
Pr
(c)
Pfr
Pfr
(d)
Pr
Pfr
PHY
PHY
9Å
PAS / GAF
CBCR
HK
35º
124 Å
PAS / GAF
85 Å
(e)
HK
HK
HK
143 Å
Pfr’
HK
HK
90 Å
35º
PHY
5Å
124 Å
PAS / GAF
86 Å
PVB 15Z
PVB 15E
PVB 15Z
Cys
A
TePixJ 15Z
TePixJ 15E
D
C
B
Cys
Cys
PVB 15E
A
D
N
C
B
C
C
Current Opinion in Structural Biology
Comparison of the 15Z and 15E structures in Group I phytochrome and Group III CBCR structures. (a) Overlay of the Pr and Pfr structures of
DrBphP (PDB codes: 4O0P, 4O01). PAS; GAF, PHY domains and BV are displayed in red, orange, green and cyan, respectively. The Pfr structure
is represented in pale colors. The inlets show the tongue region of DrBphP in the Pr (left) and Pfr (right) photostate. The amino acids of the PRxSF
motif as well as the tryptophan and tyrosine residue of the WG/AG and W/YxE motifs are presented. (b,c) Illustration of the PHY domain
movement upon Pfr formation in DrBphP, regarded from the top looking on the tongue region (b) or in the dimer with side view (c). (d) Single
particle electron microscopy maps of the DrBphP dimer in the Pr, as well as in the Pfr and P0fr conformation [28]; surface-rendered front and side
views with the included Pr crystal structure of DrBphP (PDB code: 4Q0J) docked as rigid body. In this structure PAS, GAF and PHY domains are
shown in blue, green and orange, respectively, and the other monomer is displayed in gray. The PHY domain is rotated by 358 clockwise and
moved outside by 9 Å in Pfr and by 5 Å in P0fr . (e) Comparison of the 15Z-structures and 15E-structures of TePixJ. Left: overall structure; middle:
view on the chromophore binding pocket from the top; right side: the PVB chromophore of TePixJ.
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Current Opinion in Structural Biology 2015, 35:7–16
12 Catalysis and regulation
Figure 4
(a)
(b)
GAF1
DrBphP: Pr
DrBphP: Pfr
GAF
Pr
TePixJ 15Z
TePixJ 15E
2
C
D
F
G
G
R
W
E
P
Wx
S
x
C
ole
ind
C
C
~45º
(c)
R
R
GAF1
Pfr
W
P
F
W
x
R
X
N
N
2
S
GAF
D
E
G
G
(d)
EAL
EAL
*
GGDEF
GGDE
F*
GGDEF*
GGDEF*
EAL
EAL
HK
P
HK
HK
HK
P
Current Opinion in Structural Biology
Signal transduction pathways in phytochromes and CBCRs. (a) Model for conformational changes within the tongue region during
photoconversion [23]. Upon red-light illumination the aspartate–arginine salt bridge between the GAF1 domain and the tongue (inlet) is broken.
The tip of the tongue refolds and induces a disordering in the b-sheet stalk region while the tryptophan motifs are switched and the new
aspartate–serine hydrogen bond (inlet) is formed. (b) C-terminal a-helices of the photosensory modules from DrBphP (left) and TePixJ (right). In
DrBphP the helix is shifted from the Pr to the Pfr state, in TePixJ only a minor shift takes place but the helix is rotated about 458. (c,d) Different
mechanisms for downstream signaling by phytochrome-like photoreceptors. PAS, GAF and PHY domains are shown in red, orange and green,
respectively. The chromophore is displayed as a blue star. Arrows indicate illumination with red or far red light to achieve the Pfr or Pr state,
respectively. The blue ellipse shows a possible binding partner, the gray flash indicates the active state. Red circles indicate substrate molecules.
(c) Intermolecular interaction of the phytochrome with a possible binding partner in the Pfr state. (d) Intramolecular interactions: the structural
changes upon photoconversion to Pfr lead to a rearrangement of the HK domains, allowing the trans-phosphorylation (left). Structural
rearrangements lead to a better access to the active site of the effector domain thus activating the domain (right).
Current Opinion in Structural Biology 2015, 35:7–16
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Phytochromes: diverse, complex and multi-colored Anders and Essen 13
of the two monomers. For Pr EM maps reveal a central
hole and a C-terminal bulky density reflecting the paired
HKs (Figure 3d). The Pr crystal structure of DrBphP can
be docked into the map, although the helical spine is
placed in a central hole of the map without being covered
by density. Photoactivation apparently generates two
populations of particles, Pfr and P0fr (Figure 3d, middle,
right), with an increased diameter of the central hole and
altered arrangements of the PHY domains. Interestingly,
upon docking the Pfr DrBphP crystal structure in the
SPEM map, the PHY domain has to be rotated and
displaced to fit the Pfr and P0fr conformations [28]. The
change of the chromophore itself upon photoconversion is
limited to the Z ! E double bond isomerization between
C15 and C16 and minor relaxations of the molecule that
can be also observed in cryo-trapped intermediate states
[29]. Evidences for a possible A-ring instead of a D-ring
rotation in Group II phytochromes upon photoconversion
[30] could be disproved [31] and derived apparently from
the high solvent accessibility of the PCB chromophore
along rings A and B [23].
Recombinant CBCRs usually contain a mixture of PCB
and PVB [20]. Accordingly, the structure of the B/Gabsorbing DxCF-type CBCR TePixJ from Thermosynechococcus elongatus was analyzed in its PVB-bound form as Pb
(C10-Zs/C15-Za) [25] and Pg (C10-Zs/C15-Ea) state
[32], respectively, as well as in the PCB-bound form
as Pb state [25]. The Pb states of TePixJ exhibit a second
thioether linkage between the C10 carbon of the chromophore and the cysteine from the conserved DxCF
motif [25]. This causes a rubinoid bilin species with
shortened p-system, whereas in the Pg state this
thioether linkage is broken [32]. Conformational
changes occur mainly in regions of the chromophorebinding pocket (Figure 3e), where in Group I and II
phytochromes the tongue shields the chromophore. During Z ! E photoconversion, the terminal strand of the
b-sheet diminishes solvent access to the chromophore
by converting into a flexible region and a 310-helix that
moves towards the PVB-binding a-helix. Like in phytochromes [24,33] the C-terminal a-helix of the GAF
domain in AnPixJ and TePixJ continues as helix into
the downstream effector domain [32]. As these helical
spines apparently transmit signals over long distances [4]
this feature is probably not only crucial for signal transduction by TePixJ [34], but generally for all GAF-containing photoreceptors. Light-triggered release of the
terminal b-strand in TePixJ may be hence crucial for
downstream signaling, as its N-terminal and C-terminal
a-helices are coupled to the b-strands [34]. Additionally,
an axial rotation of the C-terminal helix by 458 during
15Z ! 15E photoconversion (Figure 4b) transmits to
an altered orientations of the flanking HAMP domains
[34], which are known to act to as rigid transmitters
by rotation of the helix bundles in their dimerization
interface [35].
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Synthetic biology applications for
phytochromes
Near-infrared is attractive for applications in life sciences,
because autofluorescence, scattering and absorption by
cellular components is mostly negligible thus allowing
photocontrol in thick tissues and even live animals [36].
As Group I phytochromes have fluorescence and action
spectra in this wavelength range, they are promising
templates for the engineering of biomarkers, biosensors
and optogenetic switches.
To engineer NIR-fluorescent phytochromes the stability
of their Pr conformation is optimized by (a) hindrance of
the C15 = C16 double bond isomerization after photoexcitation, (b) destabilization of the chromophore formed
after photoisomerization to an early intermediate called
Lumi-R, and (c), blockage of its deprotonation transiently
occurring during the further Pr ! Pfr photoconversion
[36]. Removal of the PHY domain also fosters fluorescence properties as in Wi-Phy, a monomeric PAS-GAF
variant of DrBphP, that harbors mutations in the dimerization interface as well as for the DIP motif’s aspartate
and Y263 in the D-ring environment [36]. At least one WiPhy variant is applicable as NIR-fluorescent biomarker in
mammalian cells and mice, where it incorporates endogenous BV [37]. Resonance-Raman and molecular dynamics studies on iRFP, a biomarker based on RpBphP2 from
Rhodopseudomonas palustris, indicate that the chromophore is distorted relative to wild type by an increased
tilt angle between rings C and D and a loss of hydrogen
bond interactions to the ring D carbonyl. Here, subsequent loss of water molecules in the binding pocket [38]
may cause rigidification of the chromophore-binding domain and thus a decrease of non-radiative decay processes. Likewise, the IFP1.4 biomarker derived from DrBphP
shows tight hydrophobic packing around ring D [39] and
very long life times for excited Pr states. Nevertheless,
current phytochrome-based NIR-biomarkers still have
a restricted potential due to quantum yields of <10%.
Phytochromes are proposed as fluorescent sensors for
redox potentials, metal ions or protein-protein interactions by exploiting their modular multidomain organization and potential for spectral fine-tuning by altering their
chromophore or 3D-structure. For example, a BphPbased biosensor for mercury uses the high affinity of
Hg2+ ions to cysteines for competing with BV-attachment
[40]. To detect protein-protein interactions the PAS and
GAF domains of RpBphP2 are employed as iSplit biosensors. Here, these domains are parts of separate polypeptide chains, each fused to a possible interaction
partner, respectively. Upon association the PAS and
GAF domains assemble so that BV gets covalently attached and the iSplit phytochrome starts to fluoresce.
This split NIR-biosensor functions in human cell lines
and whole mice [41]. The third direction of phytochrome
engineering is optogenetics with currently three types of
Current Opinion in Structural Biology 2015, 35:7–16
14 Catalysis and regulation
applications: The photosensory module of SynCph1 was
fused to the HK region of EnvZ to control HK activity by
red light for repressing the expression of OmpR-dependent genes [42,43]. Alternatively, the light-regulated
PhyB-PIF3 interaction can control gene expression,
e.g. by splitting the DNA-binding and transactivation
domains of GAL4 [44], or co-localize target proteins to
subcellular components [45–47]. Finally, a DrBphP-phosphodiesterase fusion steers cyclic nucleotide levels by
light in vivo [48].
Conclusions
Overall, the mechanistic understanding of phytochromelike photoreceptors faces two challenges: Firstly, how
do we use structural and spectroscopic data for a concise
understanding of color tuning? Given the difficulty to
rationalize color-tuning in microbial and animal rhodopsins, which depend on the ‘simpler’ chromophore retinal,
it is not surprising that theoretical approaches such as
full-scale quantum chemical and QM/MM calculations
on complex bilin chromophores in their protein environment are still in their infancy [49–51]. Here, progress is
not only impeded by radiation effects on the chromophore during structural analyses [23,52], but also by
intrinsic conformational heterogeneity of the Pr state and
[53–56] and to some extent also the Pfr state [57]. The
second issue is the chosen mode of downstream signaling, which apparently differs in the family of phytochrome-like photoreceptors (Figure 4c and d). So far,
we are missing structural views how plant phytochromes
form light-dependent complexes with the plethora of
known phytochrome-interacting factors such as COP1,
PIFs or FHY1. In terms of bacterial phytochromes we
can at least extrapolate the activation/inactivation of
effector domains by extrapolating from other photosensory systems [58].
One pressing issue for synthetic phytochromes to be
applicable in optogenetics is chromophore supply, as
animals provide only BV from heme breakdown. Accordingly, PCB production requires at least two additional
transgenes [59] and makes BphPs to more attractive
templates. Another challenge is the complexity of the
architecture of Group I and II phytochromes. Here,
CBCRs indeed excel by high fluorescence, e.g. GAF3
from the RGS protein from Synechocystis sp. displays a twofold higher fluorescence quantum yield than SynCph2
(1-2) [7,60]. Furthermore, CBCRs are now known, which
do not only incorporate PCB as chromophore, but also
the better bioavailable BV [17]. In this context, the small
size and high spectral diversity of CBCRs calls for their
future engineering and application.
Conflict of interest statement
No conflict of interest.
Current Opinion in Structural Biology 2015, 35:7–16
Acknowledgements
The authors are grateful for funding by the Deutsche
Forschungsgemeinschaft (ES152/9, ES152/10) and the LOEWE-Center for
Synthetic Microbiology.
References and recommended reading
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