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De novo synthetic biliprotein design, assembly and excitation energy transfer rsif.royalsocietypublishing.org Joshua A. Mancini1, Molly Sheehan2, Goutham Kodali1, Brian Y. Chow2, Donald A. Bryant3, P. Leslie Dutton1 and Christopher C. Moser1

Research 1Department of Biochemistry and Biophysics, and 2Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA 3Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA Cite this article: Mancini JA, Sheehan M, JAM, 0000-0001-5151-5643; GK, 0000-0001-6356-9016; DAB, 0000-0003-2782-5988; Kodali G, Chow BY, Bryant DA, Dutton PL, PLD, 0000-0002-3063-3154; CCM, 0000-0003-4814-8568 Moser CC. 2018 De novo synthetic biliprotein Bilins are linear with a wide range of visible and design, assembly and excitation energy near-visible light absorption and emission properties. These properties are transfer. J. R. Soc. Interface 15: 20180021. tuned upon binding to natural proteins and exploited in photosynthetic http://dx.doi.org/10.1098/rsif.2018.0021 light-harvesting and non-photosynthetic light-sensitive signalling. These pigmented proteins are now being manipulated to develop fluorescent experimental tools. To engineer the optical properties of bound bilins for specific applications more flexibly, we have used first principles of protein Received: 9 January 2018 folding to design novel, stable and highly adaptable -binding four- Accepted: 13 March 2018 a-helix bundle protein frames, called maquettes, and explored the minimal requirements underlying covalent bilin ligation and conformational restric- tion responsible for the strong and variable absorption, fluorescence and excitation energy transfer of these proteins. , and bind covalently to maquette Cys in vitro. A blue- Subject Category: shifted tripyrrole formed from maquette-bound phycocyanobilin displays Life Sciences–Chemistry interface a quantum yield of 26%. Although unrelated in fold and sequence to natural phycobiliproteins, bilin lyases nevertheless interact with maquettes during Subject Areas: co-expression in Escherichia coli to improve the efficiency of bilin binding bioenergetics, biophysics, biomimetics and influence bilin structure. Bilins bind in vitro and in vivo to Cys residues placed in loops, towards the amino end or in the middle of helices but bind poorly at the carboxyl end of helices. Bilin-binding efficiency and fluor- Keywords: escence yield are improved by Arg and Asp residues adjacent to the synthetic protein, maquette, biliprotein, ligating Cys on the same helix and by His residues on adjacent helices. light-harvesting, excitation energy transfer 1. Introduction Author for correspondence: Phycobiliproteins (PBPs) are a superfamily of proteins that bind linear tetra- Christopher C. Moser chromophores, i.e. bilins, and that assemble into supramolecular e-mail: [email protected] complexes known as phycobilisomes in cyanobacteria and red algae [1,2]. PBPs have intense visible absorption and exhibit high fluorescence quantum yields, consistent with their roles as light-harvesting proteins. PBPs have been extensively studied for decades, and details concerning their biogenesis, structure, assembly and function are well established (e.g. [1–8]). A recent cryoelectron microscopic study of the approximately 17 MDa phycobilisomes of the red alga Griffithsia pacifica establishes the organization of approximately 860 protein subunits and approximately 2048 individual bilin chromophores in each light-harvesting complex at a resolution of approximately 3.5 A˚ [9]. In spite of the very large numbers of chromophores (approx. 250 to greater than 2000) found in phycobilisomes, energy transfer efficiencies of approxi- mately 95% have typically been observed. PBPs generally absorb visible light, but recently PBPs absorbing light in the far-red wavelength range between 700 and 750 nm have been identified [10–12]. Each subunit of these brilliantly Electronic supplementary material is available coloured proteins carries one or more linear tetrapyrrole (i.e. bilin) chromo- online at https://dx.doi.org/10.6084/m9. phores that are usually bound to the protein through a single cysteinyl thioether linkage to a vinyl side chain on the bilin [1]. Over the figshare.c.4037237. past 20 years, the bilin lyases responsible for the specific attachment of bilin

& 2018 The Author(s) Published by the Royal Society. All rights reserved. Downloaded from http://rsif.royalsocietypublishing.org/ on June 13, 2018

chromophores to PBP apoproteins have been identified [8,13]. residues are favoured by de novo protein engineers because 2 Functionally, it is self-evident that PBPs are remarkably of their designability, i.e. many different sequences fold rsif.royalsocietypublishing.org versatile proteins that are exquisitely evolved to perform into the same structure [28,29]. Structural stability is built a specific task, harvesting light in the wavelength range in, resisting temperatures up to boiling [30]. These frames 400–750 nm for oxygenic photosynthesis [2]. are readily adapted to accommodate many natural and A second family of bilin-containing proteins are the phy- non-natural cofactors to achieve diverse functionality through tochromes and cyanobacteriochromes that are associated informed, iterative design [31–37]. They readily lend them- with light perception in cyanobacteria, algae and plants selves to progressive engineering directed towards further [14–17]. In these proteins, the bilin chromophore of a GAF synthetic applications [30,36,37]. This approach allows the domain may be singly or doubly bound to cysteine residues exploitation of design and sequence space undiscovered by to modify the number of conjugated double bonds and thus natural selection. the light-wavelength response of the chromophore. Light- The work reported here seeks to define some initial prin- Interface Soc. R. J. induced isomerization of the chromophore can trigger ciples to determine whether bilins can be effectively and conformational changes in the GAF domain that may be efficiently bound to completely artificial proteins with no transmitted to an output domain, often a histidine kinase sequence similarity to natural proteins, and if so, how the domain that can lead to downstream signalling to a response optical properties of the resulting proteins can be manipu-

regulator that controls the response to light [15]. They have lated and optimized. Here we report initial designs to 15 been recently engineered as opto-genetic infrared reporters artificial biliproteins to optimize three factors that control 20180021 : for deep-tissue and low-background fluorescence imaging the key optical properties of absorption, emission and fluor- [18–20]. escence yield: (i) the type of bilin; (ii) Cys localization for In both types of biliproteins, protein environment pro- bilin binding; and (iii) the bilin binding environment. This foundly influences bilin optical properties. By constraining work initiates the development of basic design rules for the flexibility and orientation of the pyrrole rings, the pi- these choices and tests the performance of initial de novo bili- conjugation of the highest occupied and lowest unoccupied protein light-harvesting maquettes in excitation energy molecular orbitals can be extended or shortened, shifting transfer (EET) from different bilins to cyclic tetrapyrrole absorption and emission to longer or shorter wavelengths acceptors bound within the same maquette. [3,21,22]. Restricted bilin mobility is associated with greater fluorescence yield [3]. Moreover, the binding pocket for the phycocyanobilin (PCB) chromophore of ApcD rigidly holds the PCB chromophore in a planar configuration, which 2. Results and discussion increases the effective conjugation and leads to the red-shifted absorption spectrum of AP-B compared to AP [22]. The protein 2.1. De novo biliprotein design choices may also provide hydrogen bonds, provide aromatic residues There are three principal choices in designing de novo bili- for pi–pi stacking with the bilin, alter the local dielectric proteins to control the key optical properties of absorption, environment and change pyrrole nitrogen pKa values with cor- emission and fluorescence yield. The most important choice responding effects on the absorption properties [3]. As a result is bilin type. The different conjugation lengths of the various of knowledge gained over many years of study, structure- bilins set the expected absorption and emission range, with focused, targeted mutational studies have recently succeeded the longest absorption and emission wavelengths of red to in increasing fluorescence yield substantially [23–27]. near IR for biliverdin (BV), shorter wavelengths for PCB In this report, we have tested the feasibility of drawing on and phycoerythrobilin (PEB), and the shortest wavelengths the advances made with natural biliproteins to reconstruct for tripyrroles (figure 1). Although not discussed here, and engineer biliproteins in minimal, compact protein other bilin choices are available [39,40] for in vivo ligation frames supporting selected functions that are expressible in to the de novo bundle scaffold. living cells with minimal metabolic load on cellular metab- The second choice is the location of the ligating Cys resi- olism. The challenge of dramatically redesigning natural due within the 4-helix bundle framework and the identity of biliproteins along these lines is daunting. Most PBPs require adjacent amino acids that control the yield of in vivo bilin specific interactions with lyase proteins that must be pre- binding during expression. These selections modulate the served to attach the bilin(s) covalently to the polypeptide accessibility of the Cys residue for interacting with free bilin [8]. Furthermore, natural PBPs only bind bilin chromophores, or bilin lyase and the ability of the region of the bundle to which constrains the design of multifunctional proteins. They unfold and permit access to the bundle hydrophobic core exhibit complex quaternary interactions that are difficult to during the ligation between the Cys sulfhydryl group and overcome without affecting the properties of the globin bilin vinyl moiety. domain that binds the chromophore(s) [9]. Changes to the The third choice is to select residues that control the bilin-binding environment to adjust absorption and fluor- environment of the bound bilin to modulate the bilin escence properties often have unintended consequences on conformation—specifically, the extension of the tetrapyrrole the folding of the protein. De novo protein design offers an chain, the angle of rotation between the pyrrole rings [22] alternative means to enhance biliprotein designability to and the bilin rigidity. These factors critically influence the achieve compactness and adaptable function. effective degree of pi-electron conjugation in the molecular De novo protein design uses first principles of protein orbitals of the bilin and thereby its optical properties [3]. folding to intentionally build minimal, structurally transpar- This is a special design challenge presented by bilin cofactors ent protein frameworks that have been proved robust to that is not a concern for previously studied rigid maquette extensive amino acid sequence variation. a-Helical bundles cofactors, including , , iron–sulfur clusters with a binary patterning of hydrophobic and hydrophilic and flavins [31]. Downloaded from http://rsif.royalsocietypublishing.org/ on June 13, 2018

(a) 3 rsif.royalsocietypublishing.org

(b) (c) 1.0 TPB 0.2 free protein bound

visible Interface Soc. R. J. 0.5 0.1 absorbance

PEB PCB BV 15 20180021 :

0 0 300 400 500 600 700 800 300 400 500 600 700 800 wavelength (nm) wavelength (nm) Figure 1. Spectral diversity of bilins and increase in the visible absorption band upon binding to protein. (a) Structure of free bilins. (b) Absorbance of free bilins in phosphate-buffered saline, pH 7. (c) Absorbance of bilins bound to protein. TPB and BV are bound to a maquette, while PCB and PEB are bound to the CpcA subunit fused to a maquette [38]. The shorter wavelength major absorption bands are referred to as Soret or UV and the longer wavelength major bands as Q or Vis.

polar 3 4 b f e view down c helix axis a g d 2 non polar 1

heptad position: helix 1 loop 1 helix 2 loop 2 helix 3 loop 3 helix 4

net charge: negative helix: –4 positive helix: +1 neutral loop: positive loop: short loop: Figure 2. a-Helical-bundle designs of maquettes based on binary patterning of heptad-based sequences (upper left) connected by loops. Non-polar residues (purple) line the interior face of the helix, while polar (black), positive (blue) or negative (red) residues cover the exterior face. An end-on view of the bundle based on a maquette NMR structure [41] is shown at the upper middle, showing non-polar residues (purple) buried in the bundle core. A side view (upper right) colours helices 1, 2, 3 and 4 in red, orange, yellow and green, respectively. A flattened amino acid sequence map (middle) shows non-polar heptad positions a, d and e as purple stripes. The designs of this work use either four net negative helices or alternate negative and positive helices connected by either long or short loops (bottom).

2.2. Binary patterning in helical bundle design these heptads places non-polar residues along one face of Maquettes exploit first principles of protein folding by using the helix with polar residues on the other. Ends of helices repeating amino acid heptads (figure 2) [42]. In an a-helical are connected with simple glycine (Gly)-rich loops. In aqu- fold, seven amino acids form complementary hydrogen eous solution, this single polypeptide chain spontaneously bonds to complete two helical turns. Binary patterning of sequesters the non-polar residues to form the core of a Downloaded from http://rsif.royalsocietypublishing.org/ on June 13, 2018

(a) (b) 4 rsif.royalsocietypublishing.org

4 0.3 5 8 5 9 7 6 4 C Interface Soc. R. J. XXX 6

0.2 loop CXXX helix CLRD amino terminus absorbance 2CGRI 15

0.1 20180021 : 7 8 acid 3 CGRD terminus 9 1 CGEI

400 500 600 700 400 500 600 700 wavelength (nm) wavelength (nm) Figure 3. (a) Effect of introducing downstream Arg near the loop Cys anchor. PCB binding yield increases and the 650 nm band is enhanced. (b) Effect of placing a CLRD motif near amino (yellow) or acid (brown) terminals of helix 2. Protein concentrations, assayed by tryptophan absorbance at 280 nm, are held constant. Variants are grown in parallel to the same cell densities and purified in parallel with comparable protein yields. Protein is in excess of bound bilin, thus larger absorbances correspond to larger ligation efficiency. Binding yields are given in table 1.

4-helix bundle. Maquette frames are highly tolerant to in vitro (figure 6). We selected the PCB plasmid for the most changes in sequences that maintain the general binary pat- extensive analysis. terning. We have the design freedom to move the bilin ligating Cys around the helical and loop regions of the maquette, make additional sequence changes around the 2.4. Modulating bilin binding through Cys exposure To test the hypothesis that surface exposure of the ligating Cys, change the net charge of individual helices, alter lengths Cys residue may modulate bilin binding, Cys residues were of loops and make changes in the hydrophobic core, all with- inserted at different positions in the helical heptad. Even out compromising the highly tolerant folding of the protein. though maquettes are expressed in surplus over bilin, we can compare the relative yields of bound bilin to determine which binding sites are most favourable. When individual 2.3. Bilin lyase co-expression Cys residues are inserted in the middle of a helix, both hydro- We exploited the expression system developed previously phobic core sites (heptad positions a or d) and surface [40,43,44], which imports bilin synthesis and attachment exposed sites (heptad positions b or c) lead to similar bilin- machinery for ligating diverse to PBP apopro- binding yields (see the electronic supplementary material, teins expressed into E. coli. The plasmids code for the figure S2 and table S1). On the other hand, Cys residues following: haem oxygenase-1 to catalyse BV synthesis from inserted in a conformationally flexible loop position dis- haem; a reductase to reduce BV to either PCB or PEB; and played greater absorbance and enhanced binding yield. a bilin lyase. We added plasmid PJ414 encoding a maquette Spontaneous PCB binding without lyase is negligible. These to this system. Despite the completely unnatural sequence observations support the hypothesis that local unfolding of of the designed bundle, successful coupling between the the protein sequence is more important than Cys exposure bilin lyase and the maquette is clear. The higher copy in interfacing with bilin lyases in vivo. Local unfolding has number of the maquette PJ414 expression vector tends to out- been proposed in a structural model that docks the lyase strip the bilin cofactor biosynthetic machinery on the low CpcT with the natural biliprotein CpcB; in this model the copy number pACYCDuet vector. Nevertheless, expression Cys-containing sequence is inserted into the bilin-carrying of the bilin S-type lyase CpcS from Thermosynechococcus elon- cleft of the lyase, which brings the Cys-sulfhydryl and the gatus [44] greatly increases the amount of bilin bound. bilin vinyl into apposition for catalytic ligation [45]. Without PCB lyase, purified maquettes carried minimal amounts of bound PCB along with small amounts of 2.5. Modulating bilin binding through nearby Arg bound BV and haem (see the electronic supplementary material, figure S1, for spectra). and Asp In this study, we show the bilins BV, PCB and PEB all Cys-containing sequences in natural biliproteins are highly bind covalently to Cys residues in maquettes in vivo (figures 1 diverse and different sequence classes are associated with and 3; and electronic supplementary material, figure S20) and specific bilin lyase families [8,13]. Restricting a biliprotein Downloaded from http://rsif.royalsocietypublishing.org/ on June 13, 2018

(a) (b) 0.2 5

10 CLRD rsif.royalsocietypublishing.org loop

0.1 11 CLRD helix DRLC

Abs/Abs 280 nm 13 helix

CLRK Interface Soc. R. J. C L RD helix DR L C L R D 12 0 400 500 600 700 (c) (d) 0.1

15 DRLC 15 add His 20180021 :

CLRD 13 DRLC 10 loop helix

14 DRAC helix Abs/Abs 280 nm A

C L RD H C X R D 0 400 500600 700 800 wavelength (nm) Figure 4. Comparable binding of CLRD and DRLC motifs in loop and helix 4. (a) Sequence position (black, red, blue). (b) Spectra: replacing D with K reduces yield dramatically (magenta). (c) Overlay of X-ray structure of natural allophycocyanin (green backbone ribbon, purple bilin) with molecular dynamics structure of the maquette (grey) by superimposing allophycocyanin CLRD onto maquette DRLC. Cys (yellow), Arg (blue), Asp (red). (d) Corresponding spectra.

BLAST search to those associated with the S-type lyase used 10.5 A˚ from the carboxyl terminus of a helix, bilin-binding here indicates that Arg and Asp residues are usually found yield decreased and the absorption broadens (brown spectra downstream from the Cys in a Cys-Xxx-Arg-Asp (CXRD) in figure 3b). Cys anchoring close to the carboxyl terminus pattern, where Xxx is typically a non-polar residue [46]. (less than the 13 A˚ of an extended bilin) may force a twisted Arginines can form charge-pairs with bilin propionates, geometry. while Asp can interact with pyrrole nitrogens [44]. Figure 3a shows that introducing an Arg two positions after 2.7. Modulating bilin binding with CXRD order a loop Cys boosts the bilin-binding yield by a factor of 2 When the Cys is positioned in the middle of a helix, introduc- and adds structure to the absorption spectrum. Longer wave- tion of a negatively charged Asp two residues downstream length absorption features in natural biliproteins are greatly improves bilin binding in maquettes relative to posi- associated with more extensive pi-orbital conjugations of tively charged lysine (Lys; figure 4a). This is evidence that the more extended bilin conformations and those with smal- in contrast to Asp in loops, the carboxylate of Asp in a ler dihedral angles between the pyrrole rings, especially helix is positioned for favourable interactions with the nitro- between the rings C and D. However, adding an Asp in a gens of pyrrole ring B/C. When the Asp carboxylate is loop CXRD motif did not improve binding yield. replaced with a Glu carboxylate, analogous to the CXRE motif found in some BV-binding bacteriophytochromes, 2.6. Modulating bilin-binding yield with CXRD position PCB binds less favourably, while comparable amounts of While cysteines in CXRD motifs are comparably effective in BV are bound (electronic supplementary material, figure S21). binding PCB at both surface-exposed or core buried helix Unexpectedly, the reverse motif, DRXC, appears equally heptad positions (figure 3b), there is a conspicuous asymme- effective in driving bilin ligation in maquettes (figure 4a). try in bilin-binding efficiency of the amino end of the helix While the amount of bilin bound to the maquette varies compared to the carboxyl end of the helix. Bilin-binding depending upon details of growth conditions and the relative yield increases by nearly fivefold when CXRD is placed rate of cellular production of both the bilin chromophore and near the amino rather than the carboxyl terminal of helices. the maquette protein frame, under controlled conditions of Helical unwinding for insertion of Cys into the lyase for lig- parallel growth and purification, there is comparable PCB ation is expected to be easier near either end of the helix. binding yield for the DRLC and CLRD motifs (table 2). However, when the Cys was placed within two turns or These results indicate that within a helix, proximity of the Downloaded from http://rsif.royalsocietypublishing.org/ on June 13, 2018

Table 1. Relative binding yields and optical qualities of maquette designs shown in figure 3. Designs vary residues immediately downstream from the bilin- 6 ligating Cys and Cys positions in the middle loop or second helix. rsif.royalsocietypublishing.org

Cys location His position on helix relative binding yield in vivo

ID Cys insertion helix loop position 1 2 3 4 PCB Vis/UV

1 CGEI 2 3 6 6 6 6 36% 0.38 2 CGRI 2 3 6 6 6 6 72% 0.54 3 CGRD 2 3 6 6 6 6 54% 0.47 4 CLRD 2 1 6 6 6 6 100% (0.9% absolute) 0.62 Interface Soc. R. J. 5 CLRD 2 2 6 6 6 6 72% 0.57 6 CLRD 2 3 6 — 6 6 82% 0.70 7 CLRD 2 23 6 6 6 6 21% 0.43

8 CLRD 2 24 6 6 6 6 16% 0.21 15 9 CLRD 2 25 6 6 6 6 15% 0.31 20180021 :

Table 2. Relative binding yields and optical qualities of maquette designs shown in figure 4. CXRD and the reverse DRXC sequences are compared. Absolute yield of bilin binding was determined by protein unfolding in acidic urea (see the electronic supplementary material, figure S3) and analytical HPLC.

Cys location His position on helix binding abs. Cys yield quantum Vis/ ID insertion helix loop position 1 2 3 4 in vivo yield (%) UV

4 CLRD 2 1 6 6 6 6 0.9 2.9 0.76 10 CLRD 2 3 6 6 6 6 4.2 0.7 1.65 11 CLRD 4 9 6 6 6 6 1.1 1.0 2.03 12 CLRK 4 9 6 6 6 6 0.3 2.5 0.61 13 DRLC 4 9 6 6 6 — 3.1 1.3 1.27 14 DRAC 4 9 6 6 6 — 3.2 0.9 1.02 15 DRLC 4 9 — 6 6, 26 — 3.4 1.6 1.68

Arg and Asp to the ligating Cys is more important than the motif and has a higher fluorescence quantum yield, consist- sequence order. ent with constraint by a better packed, less mobile Structural insight on this equality can be found by super- hydrophobic core. imposing the bilin-binding Cys-containing helices of the If maquette-bound bilin orients similarly to the PCB of crystal structure of the alpha subunit of the natural biliprotein the alpha subunit of allophycocyanin, the D ring pyrrole allophycocyanin from the cyanobacterium Phormidium sp. should approach position 26 in adjacent helix 3. Indeed, A09DM (4RMP) [47] with a molecular dynamics structure replacing Leu with His in this position noticeably sharpens of the maquette helical bundles (figure 4c) [48]. Despite the the Vis absorbance band and increases the Vis/UV ratio reverse order of the sequences, the arginine of both the allo- (table 2 and figure 4c). This is consistent with a pi-stacking phycocyanin and the maquette are in positions close enough or hydrogen bonding interaction between the helix 3 His to interact with the B ring propionate, while the aspartate of and ring D of PCB. This third-helix His improves both both motifs are close enough to interact with the ring B and C binding and fluorescence quantum yield. pyrrole nitrogens. Furthermore, the nearly parallel orien- Figure 5b shows that changing the external charge tations of adjacent helices near the bound bilin in both patterning from near-neutral to negative modestly improves allophycocyanin and the maquette accommodate intercala- the fluorescence quantum yield from 2 to 3%. Quantum tion of roughly planar PCB between adjacent helices. yields are comparable to that observed for the PCB-binding domain of a natural cyanobacteriochrome [51]. Shortening the loops has little effect, but breaking the central loop (17), 2.8. Modulating bili-maquette fluorescence so that the 4-helix bundle assembles from two separate Figure 5a compares the absorption and fluorescence emission subunits, doubles the yield of bilin binding per Cys, presum- (excitation at 580 nm) spectra of three maquette sequences ably through greater lyase accessibility. However, described in figure 4d. A higher Vis/UV absorbance ratio is fluorescence quantum yield was relatively poor, probably correlated with a more rigid and extended bilin conformation reflecting higher mobility of the PCB when a single maquette [3,49,50]. The DRLC motif binds more PCB than the DRAC chain is divided into two. Downloaded from http://rsif.royalsocietypublishing.org/ on June 13, 2018

(a) helical conformations seen in free bilins have relatively weak 7 absorbance emission long wavelength absorptions; more extended conformations

0.08 4 rsif.royalsocietypublishing.org imposed by bilin-binding sites provide longer pi-conjugation 0.06 3 for more intense absorptions, particularly when torsion 15 angles allow pi-orbitals to overlap optimally [3,49,50,53]. 13 Suppression of bilin flexibility by binding-site confinement 0.04 14 2 and specific interactions with nearby residues inhibits excited absorbance 0.02 1 state quenching and supports higher fluorescence emission 580 emission (arb. units) quantum yields [19]. Because maquettes have minimal sequence similarity to (b) 0 0 400 500 600 700 4 natural biliproteins, they are not expected to form specific 0.08 Interface Soc. R. J. 17 complexes with natural lyases. Nevertheless, lyases do confer different maquette bilin geometries than seen in 0.06 3 lyase-free autocatalysis. Autocatalytic PCB binding to maquettes produces spectral forms with relatively large 0.04 2 18 absorption bands around 650 nm, similar to the absorption

absorbance 16

4 seen in autocatalytic binding of PCB to the apo-biliprotein 15 0.02 1 emission (arb. units) PecA [54]. Four examples are shown as solid lines in 20180021 : 580 figure 7. Maquette designs with a Cys in the central loop 0 0 400 500 600 700 show a similar spectrum (dashed black line figure 7) upon wavelength (nm) in vivo expression with the lyase. However, for maquette Figure 5. (a) The DRLC motif (13,15) binds more PCB and has higher flu- Cys in helix positions, lyase-assisted bilin binding generates orescence quantum yield than a DRAC motif (14). (b) Changing external shorter wavelength, relatively broad and less-structured charge patterning and loop length (4, 16, 18) had minimal effects on absorption bands (orange, purple and blue dashed lines bilin attachment and quantum yield. Breaking the 4-helix bundle into a figure 7). Apparently for helical ligation sites in maquettes, dimer of two helices (17) increases bilin ligation efficiency but lowers fluor- lyase interaction favours less extended and potentially more escence quantum yield. Spectra were normalized to 50 mM protein. varied bilin geometries. These absorption maxima are well within the 535–700 nm range observed for natural PCB bili- proteins [26] and match the typical 610–710 nm range of 2.9. Lyase strengthens CLRD motif effectiveness the majority of natural PCB biliproteins (see electronic The chaperoning influence of bilin lyase during in vivo supplementary material, figure S12) [1,12]. expression is absent with in vitro ligation. In such cases, Lyases also appear to influence E–Z isomerization at the loop Cys position provides the best yield for attachment for C15–C16 or possibly the C4–C5 double bond of PCB in all bilins (figure 6, sequence (2)). Without a lyase, cysteine maquettes. Under denaturing conditions that largely remove exposure facilitates binding. Although the binding yield the influence of the protein environment on the bilin absorption was lower, burying the Cys in the core (20) generally (see electronic supplementary material, figures S13 and S14), enhanced fluorescence quantum yield, indicating that the PCB largely assumes a Z isomer (absorbance maximum core restricts quenching associated with bilin mobility. 661 nm [3]) when ligated to maquettes in the presence of To test the hypothesis that CLRD motifs improve in vitro lyase in vivo. By contrast, in vitro ligation of the Z isomer bilin binding, we inserted the motif downstream from the of PCB purified by high-performance liquid chromatography buried helical core position at the beginning and end (HPLC) yields predominantly E isomers (593 nm [55]). (sequences 5 and 9) of the second helix. Neither CLRD pos- Although photoisomerization between Z and E configurations ition improved bilin binding or fluorescence quantum of PCB is a critical part of the signalling process in phyto- yields compared to Cys alone. Indeed, BV binding decreased. chromes [15,56], there is so far no evidence of analogous This contrast with the results for in vivo binding supports photoisomerization in bili-maquettes. the view that a CLRD motif gains significance through modulating interactions with bilin lyases. 2.11. Tripyrrole and phycocyanobilin tetrapyrrole to excitation energy transfer 2.10. Lyase–maquette interactions The Q band absorbance maxima and the UV/Vis absorbance The tendency of the hydrophobic core of elementary 4-helix ratios for high-yield bili-maquettes lie within the range of natu- bundle synthetic protein designs to associate with bilins even ral CpcB and PecB previously produced in E. coli [57], an without Cys present probably facilitates the autocatalytic indication that PCB is bound rigidly. However, fluorescence thioether linkage formation to bilin vinyl groups when Cys is quantum yields in maquettes of 1 to 3% (compared to 12 and introduced. This reaction is analogous to the previously demon- 32% of a range of PCB-binding PBPs [57,58]) show room for strated ligation of haem vinyls to Cys during the formation of improving chromophore rigidity. Nevertheless, these fluor- cytochrome c maquettes [52]. However, the autocatalytic bilin escence levels are adequate for adapting bili-maquette reaction in vitro is generally slow compared to in vivo lyase- designs towards voltage sensors and energy-transfer agents. catalysed ligation. By co-expressing lyases with bilin synthases, We demonstrate EET from a maquette-bound bilin to the total amount of bilin available for ligation improves. another maquette-bound chromophore, analogous to EET in In natural systems, lyases boost bilin ligation yield maquettes fused to a PCB-containing phycocyanin a subunit and modulate the stereochemistry of bilin ligation and corre- [38]. We created a Zn-tetrapyrrole-macrocycle binding site by sponding bilin absorption and emission properties [8,13]. The inserting a potential His ligand at position 6 on helix 3, Downloaded from http://rsif.royalsocietypublishing.org/ on June 13, 2018

8 DRLC

ADEC rsif.royalsocietypublishing.org 9 20 5 2 DRLC CGRI

(a) (b) 2 absorbance excitation emission BV 3 10 Interface Soc. R. J. ) 20 –2

2

5 15 absorbance (×10 1 20180021 : fluorescence (arb.units) 5 9 0 0 400 500 600 700 600 700 PEB ) –2

1 5 absorbance (×10 fluorescence (arb.units)

0 0 400 500 600 700 600 700 PCB 5.0 )

–2 2

2.5 1 absorbance (×10 fluorescence (arb.units)

0 0 300 400 500600 700 800 500600 700 800 wavelength (nm) wavelength (nm) Figure 6. Absorption (a) and emission (b) properties of representative bili-maquettes ligated in vitro with BV, PEB or PCB with Cys in the helix core (blue) or loop (green) and HPLC-purified. Absorption spectra are normalized for protein concentration. Emission spectra are normalized for absorbance at excitation wavelength: 600, 560 and 580 nm for BV, PEB and PCB, respectively. Excitation spectra are normalized for 600, 575 and 575 nm for BV, PEB and PCB, respectively. Binding yields are given in table 4.

approximately 32 A˚ from the bilin-ligating Cys. Two remain- energy-transfer distance of approximately 50 A˚ [38]. If chro- ing interior His residues were replaced with alanine to mophores were isotropically oriented, EET efficiency would preclude competing ligation sites (sequence (19); table 3). be above 90%. The transition dipoles of bilins, commonly A range of light-active Zn-tetrapyrrole macrocycles ligate to assumed to be along the direction of the B to D pyrroles His in this site [59]. [60], would be constrained to be mostly parallel to the long The EET distance between maquette-bound bilin and Zn helical axis if buried in the bundle core in an extended confor- tetrapyrrole is much shorter than the calculated Fo¨rster mation. A test of this orientation uses a chromophore with a Downloaded from http://rsif.royalsocietypublishing.org/ on June 13, 2018

Table 3. Binding yields and optical qualities of maquette designs shown in figure 5b. 9 rsif.royalsocietypublishing.org Cys location His position on helix Cys binding yield abs. quantum Vis/ ID insertion helix pos. 1 2 3 4 in vivo (%) yield (%) UV

16 CLRD 2 1 6 6 6 6 1.3 3.0 0.96 17 CLRD 2 1 6 6 5.6 0.9 4.49 18 CLRD 2 1 6 6 6 6 2.1 2.3 1.45 19 CLRD 2 1 — 6 6 — 4.4 2.0 .R o.Interface Soc. R. J.

Table 4. Binding yields and optical qualities of maquette designs shown in figure 6.

Cys location (%) binding yield in vitro quantum yield (%) 15

ID Cys insertion helix loop position BV PEB PCB 20180021 :

2 CGRI 2 3 100 (0.3) 100 (1.8) 100 (1.3) 5 CLRD 2 2 9.7 (0.2) 65 (1.7) 82 (1.1) 9 CLRD 2 25 7.1 (0.7) 77 (1.5) 97 (0.9) 20 CEDA 2 6 70 (1.1) 69 (1.8) 88 (2.0)

0.30 supplementary material, figure S11). Figure 9 shows absorp- tion spectra for individual energy-transfer pigments. As ZnChl was titrated into the TPB maquette, singular value 0.20 decomposition (SVD) of the absorption spectra confirmed 5 the presence of two spectral forms corresponding to the 2 bound and unbound ZnChl. Figure 10a,b shows binding 0.10 and saturation of the site at a 1 : 1 stoichiometry. Fitting to a single binding-site model confirms nM binding of ZnChl relative absorbance relative 9 with and without bound TPB. 20 To characterize EET from the bilin to the Zn tetrapyrrole, 0 400 500 600 700 800 each titration step was excited at 550 nm. Figure 10c,d shows wavelength (nm) that SVD analysis of the emission spectra reveals three com- ponents fitting a simple binding-site model. The first Figure 7. Maquette-bound PCB has longer wavelength absorption when spectrum corresponds to TPB emission in the maquette with- ligated in vitro (solid lines) compared to lyase-assisted in vivo (dashed). out ZnChl with a maximum near 590 nm (red, figure 10c). As Colours correspond to maquette sequences in figure 6. ZnChl is added, this emission spectrum is replaced with a second emission spectrum with two peaks of approximately transition dipole unfavourably oriented nearly perpendicular equal intensity at 590 and 675 nm corresponding to emission to the helical-bundle axis, Zn- a (ZnChl). from TPB and histidine-ligated ZnChl, respectively (green, For EET donors, we compared two bilin chromophores: figure 10c). As excess ZnChl is added, a third spectral com- PCB and a blue-shifted tripyrrole bilin (TPB) derived from ponent grows that quenches the remaining TPB emission PCB. By raising the pH of a denaturing 6 M guanadinium sol- and shows a blue-shifted emission maximum at 665 nm, cor- ution of PCB-bound maquette-19–8.5, the absorption responding to ZnChl not ligated to the maquette histidine redshifts to 739 nm (figure 8), presumably because of chro- (blue, figure 10c). At a 1 : 1 stoichiometry of His-ligated mophore deprotonation [3]. Over 30 min, the absorption ZnChl per maquette, approximately 30% of the TPB fluor- blue-shifts to 586 nm. Mass spectrometry revealed a mass escence is quenched by energy transfer, indicating that the loss of 109 Da, consistent with scission of one pyrrole ring transition dipoles are relatively constrained and unfavourably to form a TPB [3,61] (see the electronic supplementary oriented. At high concentration, ZnChl as a relatively hydro- material, figure S10). The detailed chemical structure of this phobic pigment will partition into the hydrophobic core. readily generated tripyrrole derivative has not yet been deter- Once the His site is filled with ZnChl, the additional loosely mined, but the high quantum yield of fluorescence (26%) bound ZnChl will not be held as rigidly or as far from the facilitates energy-transfer measurements. bound bilin, permitting more efficient fluorescence quench- For the energy-transfer measurements, the bilin-bound ing. Figure 11 shows that ZnChl is more effective at maquette was HPLC-purified to enrich the fraction of quenching the fluorescence of PCB. At a 1 : 1 stoichiometry maquette containing bound TPB to about 50%. ZnChl bind- of ZnChl per maquette, PCB is 75% quenched, thus the ing (Kd 4 + 3 nM) to either the PCB or TPB maquette does PCB transition dipole is oriented less orthogonal to the not distort the bilin absorption spectrum (see the electronic dipole of ZnChl. Downloaded from http://rsif.royalsocietypublishing.org/ on June 13, 2018

0.4 10

TPB PCB 0.2 rsif.royalsocietypublishing.org

pH 8 0.2 0.1 absorbance absorbance

586 739 pH 8.5 .R o.Interface Soc. R. J.

640 323 345 424 586 606 0 669 0 400 500 600 700 800 500 600 700 800 wavelength (nm) wavelength (nm) Figure 9. Spectra of individual energy transfer pigments in maquette 19: Figure 8. Formation of TPB by alkaline scission of PCB bound to maquette 15

tripyrrole (pink), PCB (blue) and ZnChl (green). 20180021 : 19. Bound PCB in 6 M guanidinium (pH 8) has a broad absorption around 650 nm (cyan). Adjusting the pH to 8.5 rapidly shifts to 739 nm (blue); this converts on a minutes timescale to the 586 nm absorbing species especially bundle loops, although helical positions are also (purple) associated with a tripyrrole. competent. Arg placed two residues away presumably inter- acts with the propionate side chains of rings B/C to constrain 2.12. Compact biliprotein design motivations bilin orientation partially and improve fluorescence yield. The relatively simple in vitro rules for creating bili- When natural biliproteins are exploited for use maquettes are altered in the more complex environment of as red to near-infrared fluorescent proteins, their utility and the cell where interactions with natural bilin lyases boost performance can be limited due to intrinsic biophysical the speed, and alter the yield and stereochemistry of bilin lig- reasons. As photosensory signalling proteins, they have not ation. The S-type lyases operate by partially unfolding both evolved to be naturally fluorescent, but rather, their struc- natural and artificial proteins to expose the ligating Cys to tures were selected to convert their photonic energy into the A-ring vinyl of the bilin carried by the lyase. Such unfold- large-scale structural rearrangements. While they have ing and insertion occurs for Cys in most regions of the bundle been engineered by directed evolution for enhanced fluor- with the exception of the carboxy terminal ends of the helices. escence by minimizing this Nature-prescribed motion and In the other helical locations along the helices, Arg and Asp restricted water access in the bilin-binding GAF domain two and three residues away are well positioned to interact [18], there is a limit to size reduction [62] due to the structural with the propionate carboxylates and pyrrole nitrogens, requirements for bilin binding and stabilization. By using respectively, of the bilin to constrain motion and favour the compact artificial protein platform presented here more extended chromophore geometries with desirable opti- (approx. half the molecular weight of green fluorescent cal changes in absorption and fluorescence emission protein), energy-transfer distances can be shortened and the quantum efficiency. Arg and Asp are equally effective in a cellular metabolic burden can be limited when applied as natural CXRD and an unnatural DRXC motif. cell-expressible transcriptional reporters and fusion tags. Engineering bilin binding in maquettes is a modular, local This work establishes key determinants of biliprotein design issue that leaves the rest of the protein framework free holo-protein formation in E. coli using genetically encoded for other functions, such as introduction of a second chromo- components. Companion eukaryotic work has established phore binding site for EET, or for fusion to natural proteins bili-maquette expression in mammalian neuronal cells. for cellular localization. Improvement in optical performance That work, aimed at developing maquette biliproteins for can be expected by adding to these rules, as required for any opto-genetic studies, shows that maquette technology can specific design application. be compacted to a 9 kDa maquette while preserving useful BV fluorescence. 4. Methods 3. Summary 4.1. Gene constructions This work extends the fundamental rules for designing Codon optimized synthetic genes were obtained from DNA2.0 in protein maquettes to include a new and versatile class of a PJ414 expression vector. Mutations to parent vector were made light-active cofactors, bilins, thus enabling the engineering with primers synthesized by IDT or Invitrogen (see electronic of a broad spectral selection of light-activated artificial pro- supplementary material, tables S2–S4). Sequences were verified teins (e.g. [40]). Unlike the His ligation used to bind using UPENN DNA sequencing core. metalated cyclic tetrapyrrole cofactors to selected positions in maquette helical bundles, Cys ligation is used to anchor 4.2. Maquette expression bilins, analogous to the Cys binding to haem vinyls by Bilin biosynthetic plasmids were CpcS lyase [44] and PcyA and maquettes with CXXCH motifs to produce c-type cyto- HO1 [43]. His-tagged maquettes and bilin biosynthetic and chromes. Ligation of various bilins in vitro is favoured by attachment enzymes were expressed in E. coli BL21 (DE3). Strains placing Cys in relatively exposed and flexible positions, containing bilin biosynthetic plasmids were made competent to Downloaded from http://rsif.royalsocietypublishing.org/ on June 13, 2018

(a) absorbance (c) emission 11 8

10 rsif.royalsocietypublishing.org 1.5 0.2 bound 0.5 4

D Abs 0.1 8 ) 423–408 nm

concentration (mM) 0.4 emission 680 nm –1 05 1.0 0 5 concentration (mM)

cm 6 0.3 –1 4 emission

absorbance unbound 0.2

0.5 e ( m M 0.1 2 bilin 0 0 0 Interface Soc. R. J. 300400 500 600 700 800 600650 700 750 800 wavelength (nm) wavelength (nm) (b) (d) 0.6 0.6 1:1 1:1 SVD 0.4 stoichiometry 0.4 stoichiometry component 2 15

0.2 0.2 SVD 20180021 : SVD component 2 component 3 0 0.0 654321 654321 m m –0.2 concentration ( M) concentration ( M)

SVD amplitude –0.2 SVD amplitude SVD component 1 –0.4 –0.4 SVD component 1 –0.6 –0.6

Figure 10. Binding titration of ZnChl into TPB maquette demonstrates EET by fluorescence quenching of the tripyrrole derivative of PCB. (a) Absorbance of bili- maquette (purple) and successive additions of ZnChl. PBS buffer, 1 M NaCl (pH 7.4). (b) SVD analysis fits to a single His-ligating site with a nanomolar range Kd. Spectra of His-ligated (red) and non-His ligated ZnChl are superimposed on graph A. (c) Emission spectra of each addition upon excitation at 550 nm. (d) SVD analysis of emission spectra reveals three components fitting to a single His-binding model. The spectra corresponding to bilin-only maquette (red), His-ligated ZnChl (green) and non-His-ligated ZnChl (blue) are superimposed on graph C.

take up the maquette plasmid using the Hanahan method [63]. (a) 0.20 0.04 Cells were grown in Terrific Broth [64] to OD at 600 nm between 0.8 and 1 at 378C at 205 r.p.m. and then induced with 1 mM iso- 0.02 propyl-thiogalactopyranoside for 21 h at 208C at 260 r.p.m. in

0.15 D Abs bevelled flasks with culture volumes of 100 ml.

435–399 nm concentration (mM) 0 42 0.10 4.3. High-performance liquid chromatography methods

absorbance Solvents used for pigment and protein purification were a mix- 0.05 ture of acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA) in water. A Waters HPLC was used with a Vydac C18 analytical and preparatory reverse-phase column. 0 300 400 500 600 700 800 wavelength (nm) (b) 4.4. Protein purification 4 Proteins expressed with His tags were purified by resuspending Em 626 nm pelleted cells in 300 mM NaCl, 20 mM imidazole, 50 mM 4 Em 676 nm 676 nm 2 NaH2PO4 (pH 8) buffer, homogenized and microtip sonicated.

emission 626 nm Lysate was centrifuged at 25 000g for 25 min, with supernatant 0 2 4 applied to a Qiagen Ni-NTA superflow resin and purified via concentration (mM) the gravity column method. One litre of cell culture yields approximately 20 mg of maquette. PCB bili-maquette 19 without emission 2 His tag was purified via solvent extraction, by washing cell pel- lets with 0.1% TFA in water and then sonicating with 50% ACN, 0.1% TFA in water. Initial extractions lacked blue colour and were discarded; after approximately 60 ml, subsequent blue 0 600 650 700 750 800 extractions were pooled and rotovapped until dry. wavelength (nm) Unless otherwise noted, both tagged and untagged proteins were then HPLC-purified using a 30 ml gradient from 30 to 42% Figure 11. EET is demonstrated by fluorescence quenching of PCB. (a) Absor- ACN; spectra collected during the gradient quantified the effi- bance of ZnChl titrated into 2.7 mM maquette, 10 mM NaPO4 (pH 7.4). ciency of PCB attached to mutants in figure 4a (see electronic Insert: PCB and ZnChl peak emission intensities at 626 (red) and 676 nm supplementary material, figure S4). Purified bili-maquette was (blue). (b) Corresponding emission spectra during titration. collected, lyophilized and stored at 2208C in the dark until Downloaded from http://rsif.royalsocietypublishing.org/ on June 13, 2018

use. Molecular weight was assayed by both SDS PAGE (Invitro- guanadinium-HCl (pH 8) and raised to pH 8.5 with NaOH. 12 gen Novex NuPAGE electrophoresis system, 4–12% Bis-Tris gels, After 30 min, the solution was HPLC-purified using a 500 ml MES running buffer) and MALDI [30] (see the electronic sup- gradient from 20 to 70% ACN. rsif.royalsocietypublishing.org plementary material, figure S5). 4.7. Protein, phycocyanobilin concentration and 4.5. Pigment purification PCB was extracted using a modified procedure of [65] via over- quantum yield determination night methanol refluxing of 50 mg of a gene fusion between UV/Vis absorbance was measured using a Varian Cary-50 spec- CpcA and maquette [38]. The rotovapped reflux mixture was trophotometer. Fluorescence spectra were obtained using a extracted with 1 ml of ethanol, and purified by HPLC using a Horiba Fluorolog 2 fluorimeter at 208C. The PCB concentration 500 ml 20–70% ACN gradient on a C-18 column (see the elec- was calculated by denaturing in 8 M urea (pH 2), using an e

21 21 Interface Soc. R. J. tronic supplementary material, figure S16). MALDI-MS verified 660 of 35.4 mM cm [70]. HPLC spectra show that PCB absor- the mass of PCB (see the electronic supplementary material, bance at 280 nm is minor compared to Trp absorbance. Relative figure S17). bilin yield was assayed by normalizing 660 nm absorbance PEB was extracted from a His-tagged CpcA modified to delete in acidic urea to the 280 nm Trp absorbance in PBS after the covalently ligating Cys (electronic supplementary material, purification. table S5). After His tag purification, 25 mg of protein was precipi- The quantum yield of PEB and PCB bound to maquettes tated by adding methanol at 50% followed by a half volume of used the method in [43], after PD-10 size-exclusion chromato- 15 graphy using cresyl violet perchlorate (quantum yield 0.54 in 20180021 : CHCl3 and then drying by rotary evaporation at 408C. MALDI mass spectrometry used 2,5-dihydroxybenzoic acid matrix (see methanol) [71] as a standard. For BV, four dilutions of each electronic supplementary material, figure S18). HPLC verified BV-maquette and Cy5 standard in PBS were made. Absorbance purity (see electronic supplementary material, figure S19). at 600 nm was plotted against integrated emission from 635 nm Pigment concentration assay used extinction coefficients (1) for to 830 nm. Quantum yield was determined using the slope of free PEB of 25.2 mM21 cm21 at 591 nm [66] and for free PCB the dilution lines relative to that of Cy5 (quantum yield of Cy5 1 ¼ 37.9 mM21 cm21 at 690 nm in 5% HCl/methanol. of 0.27) [72]. ZnChl was prepared by addition of Zn to (Frontier Scientific) as described [67]. DMSO-solubilized ZnChl Data accessibility. The datasets supporting this article have been stock concentrations were determined by dilution in methanol uploaded as part of the electronic supplementary material. 21 21 using an extinction coefficient of 64 000 M cm at 656 nm [68]. Authors’ contributions. J.A.M. engineered and performed bili-maquette molecular biology, purification and physical and spectroscopic characterization, and contributed to manuscript drafting. M.S. 4.6. In vitro bilin attachment assayed BV binding. G.K. assisted in experimental design and engin- Maquettes were reduced with 5 mM DTT, followed by PD-10 eering of bili-maquettes and synthesized ZnChl pigment. B.Y.C. size-exclusion chromatography before addition of bilin. BV reac- supervised in vitro BV binding measurements. D.A.B. provided tion was performed in PBS buffer at pH 7.4 for 4 h with 100 mM CpcS, haem oxygenase and PcyA overexpression bacterial strains. protein and 500 mM BV. Free BV was separated from BV- P.L.D. supervised maquette design. C.C.M. assisted in bili-maquette design and analysis, and directed manuscript drafting. All the maquette by a size-exclusion column equilibrated with PBS authors gave their final approval for publication. (pH 7.4). PEB in vitro treatment was performed overnight in Competing interests. We declare we have no competing interests. 60 mM protein 50 mM Tris (pH 8), while PCB attachment was Funding. performed in 30 mM protein 50 mM NaPO4 (pH 7) as described This research was carried out as part of the Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research in [69]. In vitro PEB/maquette attachment reactions were purified Center funded by the US Department of Energy, Office of Science, using a 700 ml gradient from 30 to 60% ACN for sequences 5, 9 Office of Basic Energy Sciences, under Award DESC0001035 support- and 20 and a 500 ml gradient from 20 to 70% ACN for ing J.A.M., M.S., G.K., D.A.B., P.L.D. and C.C.M. in maquette sequence 2. PCB in vitro maquette attachment reactions were pur- construction and characterization as well as chlorin synthesis. ified using a 500 ml gradient from 20 to 70% ACN. To form the National Science Foundation (CBET 126497) supported M.S. and tripyrrole, sequence 19 with bound PCB was solubilized in 6 M B.Y.C. in biliverdin maquette design development.

References

1. Sidler WA. 1994 Phycobilisome and phycobiliprotein 5. Glazer AN. 1989 Light guides. Directional energy Switzerland: Springer International Publishing. structures. In The molecular biology of cyanobacteria (ed. transfer in a photosynthetic antenna. J. Biol. Chem. (doi:10.1007/978-3-319-51365-2_9) DA Bryant), pp. 139–216. Dordrecht, The Netherlands: 264,1–4. 9. Zhang J, Ma J, Liu D, Qin S, Sun S, Zhao J, Sui S-F. Springer. (doi:10.1007/978-94-011-0227-8_7) 6. Beale SI, Cornejo J. 1991 Biosynthesis of 2017 Structure of phycobilisome from the red alga 2. Bryant DA, Canniffe DP. 2018 How nature designs phycobilins—3(Z)-phycoerythrobilin and 3(Z)- Griffithsia pacifica. Nature 551, 57–63. (doi:10. light-harvesting antenna systems: design principles phycocyanobilin are intermediates in the formation 1038/nature24278) and functional realization in chlorophototrophic of 3(E)-phycocyanobilin from biliverdin-IX-alpha. 10. Gan F, Zhang S, Rockwell NC, Martin SS, Lagarias JC, prokaryotes. J. Phys. B 51, 033001. (doi:10.1088/ J. Biol. Chem. 266, 22 333–22 340. Bryant DA. 2014 Extensive remodeling of a 1361-6455/aa9c3c) 7. Beale SI. 1993 Biosynthesis of phycobilins. Chem. cyanobacterial photosynthetic apparatus in far-red 3. Falk H. 1989 The chemistry of linear oligopyrroles Rev. 93, 785–802. (doi:10.1021/cr00018a008) light. Science 345, 1312–1317. (doi:10.1126/ and bile pigments. Vienna, Austria: Springer Science 8. Ledermann B, Aras M, Frankenberg-Dinkel N. 2017 science.1256963) & Business Media. Biosynthesis of cyanobacterial light-harvesting 11. Li Y, Lin Y, Garvey CJ, Birch D, Corkery RW, Loughlin 4. Glazer AN. 1985 Light harvesting by phycobilisomes. pigments and their assembly into phycobiliproteins. PC, Scheer H, Willows RD, Chen M. 2016 Annu. Rev. Biophys. Biophys. Chem. 14, 47–77. In Modern topics in the phototrophic prokaryotes Characterization of red-shifted phycobilisomes (doi:10.1146/annurev.bb.14.060185.000403) (ed. PC Hallenbeck), pp. 305–340. Cham, isolated from the f-containing Downloaded from http://rsif.royalsocietypublishing.org/ on June 13, 2018

cyanobacterium Halomicronema hongdechloris. 24. Auldridge ME, Satyshur KA, Anstrom DM, Forest KT. proteins. J. R. Soc. Interface 14, 20160896. 13 Biochim. Biophys. Acta 1857, 107–114. (doi:10. 2012 Structure-guided engineering enhances a (doi:10.1098/rsif.2016.0896) rsif.royalsocietypublishing.org 1016/j.bbabio.2015.10.009) phytochrome-based infrared fluorescent protein. 39. Schluchter WM, Bryant DA. 2002 Analysis and 12. Ho M-Y, Gan F, Shen G, Bryant DA. 2017 Far-red J. Biol. Chem. 287, 7000–7009. (doi:10.1074/jbc. reconstitution of phycobiliproteins: methods for the light photoacclimation (FaRLiP) in Synechococcus sp M111.295121) characterization of bilin attachment reactions. In PCC 7335. II. Characterization of phycobiliproteins 25. Shcherbakova DM, Verkhusha VV. 2013 Near- , chlorophyll, and bilins (eds AG Smith, M produced during acclimation to far-red light. infrared fluorescent proteins for multicolor in vivo Witty), pp. 311–334. Totowa, NJ: Humana Press. Photosynth. Res. 131, 187–202. (doi:10.1007/ imaging. Nat. Meth. 10, 751–754. (doi:10.1038/ (doi:10.1385/1-59259-243-0:311) s11120-016-0303-5) nmeth.2521) 40. Alvey RM, Biswas A, Schluchter WM, Bryant DA. 13. Schluchter WM, Shen G, Alvey RM, Biswas A, 26. Velazquez Escobar F et al. 2014 Structural 2011 Attachment of noncognate chromophores to Saune´e NA, Williams SR, Mille CA, Bryant DA. 2010 parameters controlling the fluorescence properties CpcA of Synechocystis sp. PCC 6803 and Phycobiliprotein biosynthesis in cyanobacteria: of . Biochemistry 53, 20–29. (doi:10. Synechococcus sp. PCC 7002 by heterologous Interface Soc. R. J. structure and function of enzymes involved in post- 1021/bi401287u) expression in Escherichia coli. Biochemistry 50, translational modification. In Recent advances in 27. Baloban M, Shcherbakova DM, Pletnev S, Pletnev 4890–4902. (doi:dx.doi.org/10.1021/bi200307s) phototropic prokaryotes (ed. P Hallenbeck), VZ, Lagarias JC, Verkhusha VV. 2017 Designing 41. Huang SS, Gibney BR, Stayrook SE, Dutton PL, Lewis pp. 211–228. New York, NY: Springer. (doi:10. brighter near-infrared fluorescent proteins: insights M. 2003 X-ray structure of a maquette scaffold.

1007/978-1-4419-1528-3_12) from structural and biochemical studies. Chem. Sci. J. Mol. Biol. 326, 1219–1225. (doi:10.1016/S0022- 15

14. Rockwell NC, Su Y-S, Lagarias JC. 2006 Phytochrome 8, 4546–4557. (doi:10.1039/C7SC00855D) 2836(02)01441-9) 20180021 : structure and signaling mechanisms. Ann. Rev. Plant 28. Grigoryan G, DeGrado WF. 2011 Probing 42. Regan L, DeGrado WF. 1988 Characterization of a Physiol. Plant Molec. Biol. 57, 837–858. (doi:10. designability via a generalized model of helical helical protein designed from first principles. 1146/annurev.arplant.56.032604.144208) bundle geometry. J. Mol. Biol. 405, 1079–1100. Science 241, 976–978. (doi:10.1126/science. 15. Rockwell NC, Lagarias JC. 2010 A brief history of (doi:10.1016/j.jmb.2010.08.058) 3043666) phytochromes. ChemPhysChem 11, 1172–1180. 29. Beasley JR, Hecht MH. 1997 Protein design: the 43. Biswas A, Vasquez YM, Dragomani TM, Kronfel ML, (doi:10.1002/cphc.200900894) choice of de novo sequences. J. Biol. Chem. 272, Williams SR, Alvey RM, Bryant DA, Schluchter WM. 16. Murphy JT, Lagarias JC. 1997 The phytofluors: a 2031–2034. (doi:10.1074/jbc.272.4.2031) 2010 Biosynthesis of cyanobacterial new class of fluorescent protein probes. Curr. Biol. 30. Farid TA et al. 2013 Elementary tetrahelical protein phycobiliproteins in Escherichia coli: 7, 870–876. (doi:10.1016/S0960-9822(06)00375-7) design for diverse oxidoreductase functions. Nat. chromophorylation efficiency and specificity of all 17. Rockwell NC, Martin SS, Lagarias JC. 2012 Red/ Chem. Biol. 9, 826–833. (doi:10.1038/nchembio. bilin lyases from Synechococcus sp. strain PCC 7002. green cyanobacteriochromes: sensors of color and 1362) Appl. Environ. Microbiol. 76, 2729–2739. (doi:10. power. Biochemistry 51, 9667–9677. (doi:10.1021/ 31. Moser CC, Sheehan MM, Ennist NM, Kodali G, Bialas 1128/AEM.03100-09) bi3013565) C, Englander MT, Discher BM, Dutton PL. 2016 De 44. Kronfel CM et al. 2013 Structural and biochemical 18. Shu X, Royant A, Lin MZ, Aguilera TA, Lev-Ram V, novo construction of redox active proteins. Meth. characterization of the bilin lyase CpcS from Steinbach PA, Tsien RY. 2009 Mammalian expression Enzymol. 580, 365–388. (doi:10.1016/bs.mie.2016. Thermosynechococcus elongatus. Biochemistry 52, of infrared fluorescent proteins engineered from a 05.048) 8663–8676. (doi:10.1021/bi401192z) bacterial phytochrome. Science 324, 804–807. 32. Goparaju G, Fry BA, Chobot SE, Wiedman G, Moser 45. Zhou W, Ding W-L, Zeng X-L, Dong LL, Zhao B, (doi:10.1126/science.1168683) CC, Dutton PL, Discher BM. 2016 First principles Zhou M, Scheer H, Zhao KH, Yang X. 2014 Structure 19. Bhattacharya S, Auldridge ME, Lehtivuori H, design of a core bioenergetic transmembrane and mechanism of the phycobiliprotein lyase CpcT. Ihalainen JA, Forest KT. 2014 Origins of fluorescence electron-transfer protein. Biochim. Biophys. Acta J. Biol. Chem. 289, 26 677–26 689. (doi:10.1074/ in evolved bacteriophytochromes. J. Biol. Chem. 1857, 503–512. (doi:10.1016/j.bbabio.2015.12. jbc.M114.586743) 289, 32 144–32 152. (doi:10.1074/jbc.M114. 002) 46. Zhao KH et al. 2007 :cystein-84 biliprotein 589739) 33. Bialas C et al. 2016 Engineering an artificial lyase, a near-universal lyase for cysteine-84-binding 20. Piatkevich KD, Subach FV, Verkhusha VV. 2013 flavoprotein magnetosensor. J. Am. Chem. Soc. 138, sites in cyanobacterial phycobiliproteins. Proc. Natl Engineering of bacterial phytochromes for near- 16 584–16 587. (doi:10.1021/jacs.6b09682) Acad. Sci. USA 104, 14 300–14 305. (doi:10.1073/ infrared imaging, sensing, and light-control in 34. Lichtenstein BR, Bialas C, Cerda JF, Fry BA, Dutton pnas.0706209104) mammals. Chem. Soc. Rev. 42, 3441–3452. (doi:10. PL, Moser CC. 2015 Designing light-activated 47. Sonani RR, Gupta GD, Madamwar D, Kumar V. 2015 1039/C3CS35458J) charge-separating proteins with a naphthoquinone Crystal structure of allophycocyanin from marine 21. Senge MO, MacGowan SA, O’Brien JM. 2015 amino acid. Angew. Chem. Int. Ed. 127, cyanobacterium Phormidium sp. A09DM. PLoS ONE Conformational control of cofactors in nature—the 13 830–13 833. (doi:10.1002/anie.201507094) 10, e0124580. (doi:10.1371/journal.pone.0124580) influence of protein-induced macrocycle distortion 35. Lichtenstein BR et al. 2012 Engineering 48. Fry BA, Solomon LA, Dutton PL, Moser CC. on the biological function of . Chem. oxidoreductases: maquette proteins designed from 2016 Design and engineering of a man-made Commun. 51, 17 031–17 063. (doi:10.1039/ scratch. Biochem. Soc. Trans. 40, 561–566. (doi:10. diffusive electron-transport protein. Biochim. C5CC06254C) 1042/BST20120067) Biophys. Acta 1857, 513–521. (doi:10.1016/j. 22. Peng PP, Dong LL, Sun YF, Zeng X-L, Ding W-L, 36. Koder RL, Anderson JLR, Solomon LA, Reddy KS, bbabio.2015.09.008) Scheer H, Yang X, Zhao KH. 2014 The structure of Moser CC, Dutton PL. 2009 Design and engineering 49. Glazer AN, Fang S. 1973 Chromophore content of

allophycocyanin B from Synechocystis PCC 6803 of an O2 transport protein. Nature 458, 305–309. blue-green algal phycobiliproteins. J. Biol. Chem. reveals the structural basis for the extreme redshift (doi:10.1038/nature07841) 248, 659–662. of the terminal emitter in phycobilisomes. Acta 37. Robertson DE et al. 1994 Design and synthesis of 50. Glazer AN. 1976 Phycocyanins: structure and Crystallogr. D Biol. Crystallogr. 70, 2558–2569. multi-heme proteins. Nature 368, 425–431. function. In Photochemical and photobiological (doi:10.1107/S1399004714015776) (doi:10.1038/368425a0) reviews (ed. KC Smith), pp. 71–115. Boston, MA: 23. Filonov GS, Piatkevich KD, Ting L-M, Zhang J, Kim 38. Mancini JA, Kodali G, Jiang J, Reddy KR, Lindsey JS, Springer. (doi:10.1007/978-1-4684-2574-1_2) K, Verkhusha VV. 2011 Bright and stable near- Bryant DA, Dutton PL, Moser CC. 2017 Multi-step 51. Kupka M, Scheer H. 2008 Unfolding of infrared fluorescent protein for in vivo imaging. Nat. excitation energy transfer engineered in genetic C-phycocyanin followed by loss of non-covalent Biotechnol. 29, 757–761. (doi:10.1038/nbt.1918) fusions of natural and synthetic light-harvesting chromophore–protein interactions. Biochim. Downloaded from http://rsif.royalsocietypublishing.org/ on June 13, 2018

Biophys. Acta 1777, 94–103. (doi:10.1016/j.bbabio. 58. Saune´e NA, Williams SR, Bryant DA, Schluchter WM. 65. Fu E, Friedman L, Siegelman HW. 1979 Mass- 14 2007.10.009) 2008 Biogenesis of phycobiliproteins II. CpcS-I AND spectral identification and purification of rsif.royalsocietypublishing.org 52. Anderson JLR et al. 2014 Constructing a man-made CpcU comprise the heterodimeric bilin lyase that phycoerythrobilin and phycocyanobilin. Biochem. J. c-type cytochrome maquette in vivo: electron attaches phycocyanobilin to cys-82 of b- 179, 1–6. (doi:10.1042/bj1790001) transfer, oxygen transport and conversion to a phycocyanin and cys-81 of allophycocyanin 66. Chapman DJ, Cole WJ, Siegelman HW. 1967 photoactive light harvesting maquette. Chem. Sci. 5, subunits in Synechococcus sp. pcc 7002. J. Biol. Structure of phycoerythrobilin. J. Am. Chem. Soc. 507–514. (doi:10.1039/C3SC52019F) Chem. 283, 7513–7522. (doi:10.1074/jbc. 89, 5976–5977. (doi:10.1021/ja00999a058) 53. Braslavsky SE, Holzwarth AR, Schaffner K. 1983 M708165200) 67. Hartwich G, Fiedor L, Simonin I, Cmiel E, Scha¨fer W, Solution conformations, photophysics, and 59. Kodali G et al. 2017 Design and engineering Noy D, Scherz A, Scheer H. 1998 Metal-substituted photochemistry of bile pigments; and of water-soluble light-harvesting protein . 1. Preparation and influence biliverdin, dimethyl esters and related linear maquettes. Chem. Sci. 8, 316–324. (doi:10.1039/ of metal and coordination on spectra. J. Am. tetrapyrroles. Angew. Chem. Int. Ed. 22, 656–674. C6SC02417C) Chem. Soc. 120, 3675–3683. (doi:10.1021/ Interface Soc. R. J. (doi:10.1002/anie.198306561) 60. Duerring M, Huber R, Bode W, Ruembeli R. 1990 ja970874u) 54. Bo¨hm S, Endres S, Scheer H, Zhao KH. 2007 Refined three-dimensional structure of 68. Gerola AP, Tsubone TM, Santana A, de Oliveira HPM, Biliprotein chromophore attachment: chaperone-like phycoerythrocyanin from the cyanobacterium Hioka N, Caetano W. 2011 Properties of chlorophyll function of the PecE subunit of alpha- Mastigocladus laminosus at 2.7A˚. J. Mol. Biol. and derivatives in homogeneous and

phycoerythrocyanin lyase. J. Biol. Chem. 282, 211, 633–644. (doi:10.1016/0022-2836(90) microheterogeneous systems. J. Phys. Chem. B 115, 15

25 357–25 366. (doi:10.1074/jbc.M702669200) 90270-v) 7364–7373. (doi:10.1021/jp201278b) 20180021 : 55. Zhang J, Wu XJ, Wang ZB, Chen Y, Wang X, Zhou 61. Beru¨ter J, Colombo JP, Schlunegger UP. 1975 69. Arciero DM, Bryant DA, Glazer AN. 1988 In vitro M, Scheer H, Zhao KH. 2010 Fused-gene approach Isolation and identification of the urinary pigment attachment of bilins to apophycocyanin. I. Specific to photoswitchable and fluorescent biliproteins. uroerythrin. Eur. J. Biochem. 56, 239–244. (doi:10. covalent adduct formation at cysteinyl residues Angew. Chem. Int. Ed. 49, 5456–5458. (doi:10. 1111/j.1432-1033.1975.tb02226.x) involved in phycocyanobilin binding in C- 1002/anie.201001094) 62. Rumyantsev KA, Shcherbakova DM, Zakharova NI, phycocyanin. J. Biol. Chem. 263, 18 343–18 349. 56. Rudiger W, Thummler F, Cmiel E, Schneider S. 1983 Emelyanov AV, Turoverov KK, Verkhusha VV. 2015 70. Glazer AN. 1988 Phycobiliproteins. Meth. Enzymol. Chromophore structure of the physiologically active Minimal domain of bacterial phytochrome required 167, 291–303. (doi:10.1016/0076-6879(88) form (Pfr) of phytochrome. Proc. Natl Acad. Sci. USA for chromophore binding and fluorescence. Sci. Rep. 67034-0) 80, 6244–6248. (doi:10.1073/pnas.80.20.6244) 5, 52. (doi:10.1038/srep18348) 71. Magde D, Brannon JH, Cremers TL, Olmsted J. 1979 57. Zhao KH, Su P, Li J, Tu JM, Zhou M, Bubenzer C, 63. Hanahan D. 1983 Studies on transformation Absolute luminescence yield of cresyl violet. A Scheer H. 2006 Chromophore attachment to of Escherichia coli with plasmids. J. Mol. Biol. standard for the red. J. Phys. Chem. 83, 696–699. phycobiliprotein beta-subunits—phycocyanobilin : 166, 557–580. (doi:10.1016/S0022- (doi:10.1021/j100469a012) cysteine-beta 84 phycobiliprotein lyase activity of 2836(83)80284-8) 72. Mujumdar RB, Ernst LA, Mujumdar SR, Lewis CJ, CpeS-like protein from Anabaena sp PCC7120. 64. Elbing K, Brent R. 2001 Media preparation and Waggoner AS. 2002 Cyanine dye labeling reagents: J. Biol. Chem. 281, 8573–8581. (doi:10.1074/jbc. bacteriological tools. Hoboken, NJ: John Wiley & sulfoindocyanine succinimidyl esters. Bioconjugate M513796200) Sons, Inc. (doi:10.1002/0471142727.mb0101s59) Chem. 4, 105–111. (doi:10.1021/bc00020a001)