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Procedia Chemistry 14 ( 2015 ) 176 – 185

2nd Humboldt Kolleg in conjunction with International Conference on Natural Sciences, HK-ICONS 2014 Biliproteins and Their Applications in Bioimaging

Hugo Scheera*, Xiaojing Yangb, Kai-Hong Zhaoc*

aDept. Biologie 1 – Botanik, Universität München, 80638 München, Germany bDepartment of Chemistry, The University of Illinois at Chicago, Chicago, IL 60607, USA cState Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, P.R. China

Abstract

The brightly fluorescent are light-harvesting pigments in and certain , covering almost the entire visible spectrum. Another type of biliproteins, the family, comprises regulatory photoswitches in plants, fungi and many bacteria; their spectra cover an even wider range extending into the near-infrared and near-ultraviolet. In both types, the chromophores can be tuned by modulating chromophore- interactions, including the transition between and photoswitching. These properties render biliproteins useful for bioimaging. Applications require not only the introduction of genes coding for apoproteins, but also genes coding for biosynthesis of the chromophores. Strategies for tuning and heterologous biosynthesis will be discussed.

© 2015 The H. Scheer,Authors. X. Published Yang, K.H. by Elsevier Zhao. B.V.Published This is byan Elsevieropen access B.V. article under the CC BY-NC-ND license (Peerhttp://creativecommons.org/licenses/by-nc-nd/4.0/-review under responsibility of the Scientific). Committee of HK-ICONS 2014. Peer-review under responsibility of the Scientifi c Committee of HK-ICONS 2014 Keywords: Bioimaging; cyanobacteriochrome; fluorescence; biosynthesis; ; phytochrome; optogenetics

Nomenclature

BV IXα Ho1 oxygenase BVR Fd-dependent reductase PEB DHBV 15,16-dihydrobiliverdin PUB phycourobilin Fd ferredoxin PVB phycoviolobilin PCB P)B phytochromobilin

* Corresponding authors. HS: Tel.: +49 89 17861 295; fax: +49 89 81099 334, E-mail address: [email protected]; KHZ: Tel/Fax: +86 27 8754 1634, E-mail address: [email protected]

1876-6196 © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the Scientifi c Committee of HK-ICONS 2014 doi: 10.1016/j.proche.2015.03.026 Hugo Scheer et al. / Procedia Chemistry 14 ( 2015 ) 176 – 185 177

1. Introduction

Fluorescent have revolutionized bioimaging1. The most versatile ones are E-barrel proteins in which the chromophore is derived autocatalytically from the protein in the presence of oxygen. Therefore, transfer of a single gene allows for the heterologous generation of a fluorescent marker under aerobic conditions. Many of them are derived from the green-fluorescent protein (GFP) from Aequora v ictoria. Additional related proteins have been isolated from other, mostly marine organisms, with special emphasis on red fluorescence: biological tissue is most transparent in the near-infrared region, pigments are therefore desirable that fluoresce at wavelengths 650 nm to 850 nm. Biliproteins are potential candidates. Phycobiliproteins, the brightly fluorescing light-harvesting pigments of cyanobacteria and certain algae, have been used for external applications since the early 1980s2. Intercellular applications or production under genetic control require, however, transfer of several genes. Firstly, the bilin chromophore has to be generated from heme3 and, secondly, covalent attachment generally requires a lyase4. Both processes were only poorly understood until recently. An alternative approach to red-fluorescing biliproteins relies on . Some of them require only a single , , for chromophore generation. Furthermore, they bind their chromophore autocatalytically. However, they are photochromic pigments that fluoresce only poorly. This situation has been improved by engineering protein variants with reduced and enhanced fluorescence. In this short review, current approaches that render fluorescent biliproteins heterologously accessible under genetic control will be discussed.

5 4 6 19 O O 1 O O 3 7 18 2 2 AAB 8 Fe, CO 17 D 3 1 N N 9 16 NH HN 4 4 e-, 4 H+ H NH HN 20 Fe 10 15 5 19 N N 11 Ho1 14 NH N 6 PebS NH N C 12 18 D 13 C B 7 17 13 12 8 16 14 11 9 15 10

COOH COOH COOH Heme COOH BV COOH PEB COOH

4 e-, 4 H+ Apoproteins - + Apoproteins 2 e , 2 H Lyases Hy2 PcyA Bacterial Phytochromes

H H O O O O Apoproteins Apoproteins NH HN NH HN Phytochromes Allophycocyanins NH N NH N Lyases

COOH COOH COOH COOH P)B PCB Fig. 1. Biosynthesis of biliprotein chromophores from heme. Note the different nomenclature of heme and bilins; the numbering is indicated in the top row for heme and BV.

2. Phycobiliproteins

Biosynthesis of biliprotein chromophores (Fig. 1) starts from protoheme that is almost ubiquitous. The macrocylic ring is opened at the C-5 position by heme oxygenase (Ho1), with the loss of the central iron and carbon monoxide derived from the original C-5. The resulting biliverdin IXD (BV) is then reduced at one or more double 178 Hugo Scheer et al. / Procedia Chemistry 14 ( 2015 ) 176 – 185

bonds by a family of closely related ferredoxin (Fd) dependent biliverdin reductases. These reduce double- bonds by a stepwise transfer of 2 electrons and 2 protons. They carry out one (or sometimes two, see below) of the following reactions: (i) reduction of the 18-vinyl to an ethyl group resulting in only a small (10 nm to 20 nm) spectral blue shift; (ii) reduction of the '15,16 double bond, resulting in an interruption of conjugation between rings C and D, and a concomitant large blue-shift of ~100 nm; c) reduction of the '2,3/'31,32 diene system to a '3,31-ene. The resulting ethylidene group at C-3 is an essential structural element required for covalent chromophore binding by thiol addition in phycobiliproteins and many Phy and CBCR. A model has been proposed in which the site specificity of the reduction is governed by positioning of proton donating amino acids in the otherwise mostly hydrophobic binding pocket5. A third enzyme is required for covalent attachment of the chromophores to the apoproteins. There are currently three types of lyases known that are distinguished by their chromophore and binding site specificities4. T-type lyases are single proteins, the most versatile S-type lyases are either single proteins, or require a 2nd subunit, and the E/F-type lyases always require two subunits. The latter lyase type is also capable of modifying the chromophore during the attachment reaction, thereby generating two additional chromophores, phycoviolobilin (PVB) and phycourobilin (PUB) (Fig. 3). Many apoproteins can bind chromophores in the absence of lyases, but the reaction is slow, prone to oxidative side reactions, and the fluorescence yield of the resulting is lowered compared to the correct, lyase-catalyzed attachment products. The lyases are, therefore, considered to have chaperone-like function, although they do not require ATP. This has been supported by the recent x-ray analysis of a T-type lyase6: the chemically labile chromophore is bound in the pocket of a calycin, a goblet-like E-barrel protein, and protected by dimerization of the lyase by which access to the chromophore is hindered. The pocket becomes accessible by displacement of one of the monomers by the apoprotein, and the chromophore is pulled out by a fishing-rod action of one of its loops that is only stiffened after chromophore binding (Fig. 2).

a b

Fig. 2. (a) X-ray structure of the dimer of the lyase, CpcT, with the bound chromophore (red arrow) largely shielded from the aqueous phase. (b) Model of the transfer of the chromophore from the lyase to the apoprotein, after displacement of one subunit of the dimer by the apoprotein. Note the conformational change of the chromophore, from a cyclic (ZZZsss) to an extended (ZZZasa) conformation. Adapted from ref.6. Taken together, heterologous generation of phycobiliproteins then requires the expression of at least two or more genes coding for enzymes for chromophore biosynthesis, one or two genes coding for a lyase, and the gene coding for the apoprotein. Several ways have been used to simplify the process. The first is to omit the lyase and rely on the capacity of certain apoproteins for spontaneous chromophore binding. (APC) is an example, where the D-subunit binds PCB in the absence of a lyase7,8. Even if the properties of these products are not fully identical to the native chromoproteins, there high fluorescence yield suffices for potential applications. The second approach is to simplify chromophore biosynthesis. In many organisms, each of the three elementary reaction steps (see above) is carried out by individual enzymes9,10. The two most common chromophores in phycobiliproteins, phycocyanobilin (PCB) and phycoerythrobilin (PEB) each have two double-bonds less than BV, they would then each require two reductases. Single enzymes are, however, available for both chromophores that that catalyze the respective two reduction steps (Fig. 1). PcyA reduces the '2,3/'31,32 diene system as well as the Hugo Scheer et al. / Procedia Chemistry 14 ( 2015 ) 176 – 185 179

18-vinyl group3. PebS also reduces the '2,3/'31,32 diene system, but in addition the ' 15,16 double bond5. Thus, both chromophores are available from heme by two enzymes. Lyases do not discriminate the presence or absence of an 18-vinyl substituent: PCB (= 8-ethyl) can, for example, be replaced by phytochromobilin (P)B, 18-vinyl) by substituting the double function reductase, PcyA, by the single-function enzyme, HY211. An added bonus is the red- shift of about 10 nm as a result of the presence of the conjugated vinyl group.

Cys Cys S S

H

O H O H N N O N N O H N N H H N N H

COO- COO- COO- COO- BV (18-vinyl), MBV (18-ethyl) DBV

Cys Cys S S H H 18 H H 17 H H H 19 16 3 2 D 15 5 4 O H A 1 O H N 6 N O N N O H 14 N N H H N N H C B 13 7 11 9 12 10 8

COO- COO- COO- COO- PCB(18-ethyl), P)B (18-vinyl) PEB

Cys Cys S S

H H H H H

O H O H N O H O N H N N N H N N H H

- - COO COO - COO- COO PVB (18-ethyl), PtVB (18-vinyl) PUB

Cys Cys S S

H H

H O O N N O N N O H NH HN H H NH HN H

H H S S - - COO - COO - Cys COO Cys COO

PVR PCR

Fig. 3. Native biliprotein chromophores and their approximate colors. For full names, see list of abbreviation. The atom numbering and ring labeling is shown for PCB. PEB and PUB are sometimes linked to a 2nd cysteine via the C-18 vinyl group. Arrows mark the '15,16 double bond that photoisomerizes in phytochromes and cyanobacteriochromes. 180 Hugo Scheer et al. / Procedia Chemistry 14 ( 2015 ) 176 – 185

Fig. 4. E. coli cells modified by the dual plasmid approach for generation of differently colored fluorophores14

A third approach is the use of plasmids carrying more than one gene, thereby reducing the number of necessary transformations. This approach has been pioneered by Tooley et al.12,13 using one plasmid coding for chromophore biosynthesis, and the second coding for the lyase and the apoprotein. Multifunctional fusion proteins generated by this strategy have been used to express red-fluorescing biliproteins in E. coli14,15. A particular advantage of this approach is the versatility that can be gained by a modular approach: by combining a plasmid for biosynthesis and binding of a particular chromophore with one coding for the apoprotein, a toolbox can be generated for producing fluorescing biliproteins tailored to the specific need. A fourth approach relies on engineering of bilin binding proteins that improves their fluorescence. Free bilins have only low fluorescence: their excited state lifetimes are short due to efficient internal conversion (IC) of the excitation energy into vibrational energy and heat. This radiationless relaxation process is affected by many factors, among them prominently the rigidity of the chromophore16. Binding to a protein increases the rigidity of the chromophore, thereby reducing IC. In phycobiliproteins, this results in the intense fluorescence that is essential for their light-harvesting function. There are, however, other bilin-binding proteins where the mobility is only incompletely reduced. One is the phytochrome family (see below), the other are bilin-binding calycins including insect biliproteins17 as well as the S- and T-type phycobiliprotein lyases6,18,19. Engineering of calycins has been used to modify the substrate specificity and its biophysical properties, including the fluorescence enhancement of bound fluorescein20. An advantage of these systems is the possibility to use BV as chromophore, requiring only heme oxygenase for its synthesis.

3. Phytochromes and cyanobacteriochromes

Most engineering emphasis has been placed on Phy and, more recently, CBCR21-25. Both Phys and CBCRs are sensory photoreceptors where one or more photosensory domains of the GAF (or the related PAS) type are coupled to one of a number of output domains (see below). The GAF domains carry bilin chromophores that can exist in two states that are interconvertible by light. In all cases, the primary reaction is a Z/E isomerization of the chromophores at the '15,16 double bond between rings C and D (Fig. 4), that is followed by rearrangements of the protein that is transmitted to the output domain where it eventually results in a physiological signal. In some cases, the initial photoreaction is also followed by chemical modifications of the chromophore that are reversible as well. One of the states is thermostable, the other is metastable, reverting to the thermostable state over times from minutes to hours. The phytochromes proper have red-absorbing (650 nm to 690 nm) 15Z-configured chromophores (PCB, P)B) in the thermostable state. The far-red (700 nm to 750 nm) absorbing metastable state has a chromophores with 15E configuration (see Fig. 1 for nomenclature). The red-shift results mainly from changed pigment-protein interactions that are currently only partly understood26-28. These canonical Phys were originally identified as photoreceptors in plants, but the Phy family has greatly expanded. Members are found in many bacteria, fungi and algae, and their photochemistries are much more divers. There are, firstly, non-canonical phytochromes from bacteria where the stabilities are inverted29. A much larger diversity is found in the cyanobacteriochromes21,22,24,25,30. They absorb over Hugo Scheer et al. / Procedia Chemistry 14 ( 2015 ) 176 – 185 181 most of the visible spectrum extending even into the near UV, and they show red or blue shifts of the metastable compared to the thermostable states (Fig. 5). Amazingly, this diversity is brought about by only two chromophores, PCB and PVB. Absorption shifts up to 100 nm result from protein-chromophore interactions. Pigments absorbing in the blue and near-UV result from thiol (cysteine) addition to the central C-10 by which rubinoid chromophores are generated (Fig. 2). Most Phy or CBCR are far too large for labeling applications. Moreover, the wt chromoproteins also tend to dimerize, but this can be inhibited by mutagenesis of the interaction surface31. The chromophore photochemistry is, however, retained in isolated GAF domains. Moreover, these domains bind the chromophores autocatalytically, thereby omitting the need for a lyase. The photochemically competent chromophore-binding domains of phytochromes are in the 30 kDa to 40 kDa range, and the GAF domains of CBCR are even smaller, of comparable size to the phycobiliproteins. The large drawback of the chromophorylated GAF domains from Phy and CBCR is, however, their low fluorescence. This situation has been successfully changed by mutagenesis31-34.

En e r g y

S1 Energy transfer heat

n

e

o

i c

s

n

heat n

r

o Photochemistry

e

e

i

c t

v

s

p

n

r

heat e

o

r o

c

o

s

l

u b heat

l

a

A

F

n

r

e light t heat light

n

I heat S 0

Jablonsky Diagram Fig. 5. Simplified Jablonsky diagram showing the ground and first excited states, and transitions between them. Absorption of light results in excitation to S1, which can relax back to the ground state by fluorescence (emission of light) or internal conversion (release of heat). The excited state energy can also be transferred to another chromophore (e.g. by FRET), or initiate photochemistry (e.g. the 15 Z/E isomerisation in Phy and CBCR). Fluorescence yield is 100% when internal conversion, energy transfer and photochemistry are completely blocked. Opening of any of these channels results in reduced fluorescence.

Modifications converting a fluorescent into a photoswitchable biliprotein, and vice versa, were first obtained by chance, but an increasingly rational design has become possible based on x-ray structures and detailed spectroscopic studies (see references 21 and 35 for leading references). The basis to this is that both efficient photochemistry and intense fluorescence require an extension of the excited state lifetime of the chromophore. Most free bilins, in particular the red absorbing ones, have very short excited stated lifetimes, in the range of few ps. This is due to very rapid internal conversion, a process by which the excited state energy is converted to molecular vibrations and, eventually, to heat. The so-called natural lifetime of bilins, in the absence of internal conversion (IC) (and other processes like intersystem crossing to triplets), is in the range of 3 ns to 5 ns. If competing de-excitation processes like IC are completely inhibited, the chromophore would have a fluorescence yield of 100 %, and a fluorescence lifetime equal to the natural lifetime. In free bilins, the 1 000 fold faster IC of free bilins results in a 1 000 fold reduction of the fluorescence yield. In the phycobiliproteins, IC is almost completely inhibited, mainly by restricting the mobility of the chromophore (see above). The same is true for Phy and the CBCRs. However, here the chromophore is not fully immobilized, but defined local movements are allowed in the ring D region. This allows for another deexcitation channel, the photochemical isomerization of the double bond to ring D. Thus, the main distinction between switchable and fluorescent biliproteins is whether or not such local movements are allowed, or not, respectively. This simple picture is supported by mutagenesis experiments where fluorescence is greatly increased, at the expense of photochemistry31-34. In some cases, single mutations suffice for this conversion. In the 182 Hugo Scheer et al. / Procedia Chemistry 14 ( 2015 ) 176 – 185

case of type II phytochromes that bind BV, it then suffices to introduce two genes only: one coding for heme oxygenase, the other for the GAF domain.

Fig. 6. Photoreversibly photochromic biliproteins of the extended phytochrome family. Spots indicate the position of the absorption maxima in the thermostable (abscissa) and light-induced states (ordinate). The CBCR, CcaS, is shown as an example, absorbing at 645 nm in the thermostable state, and at 545 nm in the light-induced state. Phy = phytochromes, the canonical ones shift to the red upon irradiation of the thermostable state and are, thus, located above the diagonal, the non-canonical ones shifting to the blue upon irradiation of the thermostable state lie below the diagonal. CcaS = receptor for complementary chromatic acclimation from Fremyella diplosiphon, TePixJ and SyCikA are examples with exceptionally large shifts absorbing in the blue in the thermostable state and in the green or orange, respectively, in the light-induced state. Adapted from ref. 43.

4. Superresolution imaging

In the foregoing discussion, fluorescence and photochemistry have been discussed as mutually exclusive all-or- none processes. In reality, this is only approximated: all Phy and CBCR show some fluorescence, usually with low quantum yield (< 1 %). Vice versa, many phycobiliproteins show residual photochemistry, in particular in isolated subunits or under conditions where the protein becomes softened; this can be by low pH, or the addition of non- denaturing amounts of urea (< 4 M). Since generally isolated subunits are preferred for their small size, their comparably low fluorescence in the range of 50 % or even less can be improved for conventional fluorescence labeling. There are, however, applications where both photochemistry and fluorescence are desirable. The possibility for balancing the photoconversion/fluorescence ratio renders, in particular, GAF domains interesting for novel microscopic applications that rely on single molecule imaging36-38. These techniques allow optical with resolutions far beyond the diffraction limit; acronyms for such superresolution techniques are PALM or STORM. A single fluorophore gives, under the microscope, a glowing spot whose size is determined by the diffraction limit: for visible light, this is in the order of 0.5 μm. If an area of 1 μm × 1 μm is labeled, for example, by 105 chromophores that fluoresce simultaneously, most of these spots will overlap, thereby limiting the resolution and blurring the picture. If, however, only a few of them fluoresce, then most of the individual spots will no longer overlap. The centers of each spot can now be determined with a precision in the nanometer range. By applying fluorophores that “blink” between fluorescent and non-fluorescent states, it is then possible to collect many images in which at any time only few are fluorescing at precisely defined positions. By superposition, this will result in a high-resolution picture to which all fluorophores contribute. It is desirable to use fluorophores with well defined blinking properties that can be actively controlled by, for example, a suitable background illumination. An example of such a controllable fluorophore is the photoactive yellow protein36. Suitably tailored of chromophorylated GAF domains could largely increase the wavelength range and, in particular, extend it to the red and even NIR spectral region. Hugo Scheer et al. / Procedia Chemistry 14 ( 2015 ) 176 – 185 183

Fig. 7. Dependence of absorption maximum of PCB in phyco-biliproteins on co-planarity of the four pyrrole-rings. a) Side view on several native chromophores from x-ray structures, b) correlation of deviation from co- planarity with absorption maximum. Structure of AP-B has pdb code 4PO538.

5. And beyond

Studies on Phys and CBCRs are further fuelled by their potential of using the output domains for controlling biological functions by light40,41. Current application of the technique, termed optoge netics, is focused on controlling nerve cell action by channelrhodopsins (chR). With these membrane-bound retinal proteins, ion fluxes (Ca++) over the cell membrane of neurons and, thereby, neuronal activity can be modulated by light. The method is fast, contactless, and spatially confined to the volume of the activating light spot. Biliproteins of the Phy family have again the potential of extending the useful wavelength range. They combine a photoreceptor domain with a variety of output domains that can be chosen according the targeted mechanism. A pioneering example is the recent use of a bacterial phytochrome that synthesizes cyclic di-GMD upon irradiation41. Another type of application makes use of energy transfer after excitation of a chromophore. The very function of phycobiliproteins is not only light absorption and preservation of excitation energy over relatively long times (nanoseconds), but also the efficient transfer of excitation energy within the large and on to the reaction centers, a transfer over distances exceeding dozens of nm. It is achieved mainly by stepwise hopping of the excitation between chromophores by the mechanism of fluorescence resonance energy transfer (FRET), also termed Förster transfer after the pioneer of this mechanism42. Each transfer step between a donor and an acceptor has to be much faster than the excited state lifetime. Its speed depends mainly on their distance, on their relative orientations, on the fluorescence yield of the donor, and on the spectral overlap between the emission of the donor and the absorption of the receptor. These factors are optimized in the phycobilisome for each of a dozen or more steps, such that the overall quantum yield is nearly 100 %. FRET can be used to enhance fluorescence intensity and, in particular, the Stokes shift of a fluorophore. The latter is the difference between the excitation and emission wavelength. It should be large enough to avoid an overlap, which is often not ensured with strongly fluorescing chromophores. By combining two biliproteins, one like with short-wavelength absorbing PEB and PUB chromophores, and one like APC with longer wavelength absorbing PCB chromophores, FRET from the former to the latter results in an exceptionally large Stokes shift2. FRET is also possible between phycobiliproteins and other types of chromophores FRET can also be used for interaction studies and is efficient only over distances less than about 5 nm. By labeling two proteins, or other biologically active compounds, it can then be used to study their interactions including, in favorable cases, even estimating their distances. Phycobiliproteins have been used to study the 184 Hugo Scheer et al. / Procedia Chemistry 14 ( 2015 ) 176 – 185

interaction of surface proteins. Heterologous generation of two suitable ones acting as donor and acceptor, respectively, could extend this to intracellular studies. Since phycobiliproteins tend to interact with each other mainly by their N-terminal helix-turn-helix domain, this should be removed in order to avoid false-positives. FRET; and many other applications depend critically on appropriately chosen absorption and fluorescence wavelengths. In natural biliproteins, the the absorption maximum of individual chromophores can shift by almost 100 nm: PCB at position E -155 in C- absorbs around 590 nm, while the same chromophore absorbs at 670 nm in the terminal emitters of the phycobilisome. The recent x-ray structure of one terminal emitter, allophycocyanin B, indicated that the degree of co-planarity of the four pyrrole rings correlates with the absorption maximum39 (Fig. 7). Exceptionally large red-shifts, beyond this correlation, are found in phytochromes; they may also relate to the different conformations ( syn/anti) of the A/B ring system. Massive mutagenesis should be useful for such spectral tuning.

6. Conclusion

This short review is meant as an introduction to some principles underlying the photophysics and photochemistry of biliproteins, and to the potential of this class of pigments in bioimaging. It is far from complete, and so are the references that are meant to guide further interest. While the eventual applications often involve genetic engineering, it should be emphasized that much of the recent progress relies on exploiting the biodiversity: biliproteins are much more widespread and involved in many more functions than thought only a dozen years ago, and it is likely that this will continue in the future.

Acknowledgements

This work is supported by the National Natural Science Foundation of China (grants 31110103912 to HS and KHZ, 31270893 and 21472055 to KHZ), and Key State Laboratory of Agricultural Microbiology. XY acknowledges supports from Dept. of Biochemistry and Molecular Biology, the University of Chicago and National Institute of Health grant R01EY024363 .

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