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Research

Cite this article: Mancini JA, Sheehan M,

Kodali G, Chow BY, Bryant DA, Dutton PL,

Moser CC. 2018 De novo synthetic biliprotein

design, assembly and excitation energy

transfer. J. R. Soc. Interface 15: 20180021.

http://dx.doi.org/10.1098/rsif.2018.0021

Received: 9 January 2018

Accepted: 13 March 2018

Subject Category:Life Sciences – Chemistry interface

Subject Areas:bioenergetics, biophysics, biomimetics

Keywords:synthetic protein, maquette, biliprotein,

light-harvesting, excitation energy transfer

Author for correspondence:Christopher C. Moser

e-mail: moserc@mail.med.upenn.edu

Electronic supplementary material is available

online at https://dx.doi.org/10.6084/m9.

figshare.c.4037237.

& 2018 The Author(s) Published by the Royal Society. All rights reserved.

De novo synthetic biliprotein design,assembly and excitation energy transfer

Joshua A. Mancini1, Molly Sheehan2, Goutham Kodali1, Brian Y. Chow2,Donald A. Bryant3, P. Leslie Dutton1 and Christopher C. Moser1

1Department of Biochemistry and Biophysics, and 2Department of Bioengineering, University of Pennsylvania,Philadelphia, PA, USA3Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA

JAM, 0000-0001-5151-5643; GK, 0000-0001-6356-9016; DAB, 0000-0003-2782-5988;PLD, 0000-0002-3063-3154; CCM, 0000-0003-4814-8568

Bilins are linear tetrapyrrole chromophores with a wide range of visible and

near-visible light absorption and emission properties. These properties are

tuned upon binding to natural proteins and exploited in photosynthetic

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

folding to design novel, stable and highly adaptable bilin-binding four-

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. Biliverdin, phycocyanobilin

and phycoerythrobilin bind covalently to maquette Cys in vitro. A blue-

shifted tripyrrole formed from maquette-bound phycocyanobilin displays

a quantum yield of 26%. Although unrelated in fold and sequence to natural

phycobiliproteins, bilin lyases nevertheless interact with maquettes during

co-expression in Escherichia coli to improve the efficiency of bilin binding

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-

escence yield are improved by Arg and Asp residues adjacent to the

ligating Cys on the same helix and by His residues on adjacent helices.

1. IntroductionPhycobiliproteins (PBPs) are a superfamily of proteins that bind linear tetra-

pyrrole chromophores, i.e. bilins, and that assemble into supramolecular

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

coloured proteins carries one or more linear tetrapyrrole (i.e. bilin) chromo-

phores that are usually bound to the protein through a single cysteinyl

thioether linkage to a vinyl side chain on the bilin chromophore [1]. Over the

past 20 years, the bilin lyases responsible for the specific attachment of bilin

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chromophores to PBP apoproteins have been identified [8,13].

Functionally, it is self-evident that PBPs are remarkably

versatile proteins that are exquisitely evolved to perform

a specific task, harvesting light in the wavelength range

400–750 nm for oxygenic photosynthesis [2].

A second family of bilin-containing proteins are the phy-

tochromes and cyanobacteriochromes that are associated

with light perception in cyanobacteria, algae and plants

[14–17]. In these proteins, the bilin chromophore of a GAF

domain may be singly or doubly bound to cysteine residues

to modify the number of conjugated double bonds and thus

the light-wavelength response of the chromophore. Light-

induced isomerization of the chromophore can trigger

conformational changes in the GAF domain that may be

transmitted to an output domain, often a histidine kinase

domain that can lead to downstream signalling to a response

regulator that controls the response to light [15]. They have

been recently engineered as opto-genetic infrared reporters

for deep-tissue and low-background fluorescence imaging

[18–20].

In both types of biliproteins, protein environment pro-

foundly influences bilin optical properties. By constraining

the flexibility and orientation of the pyrrole rings, the pi-

conjugation of the highest occupied and lowest unoccupied

molecular orbitals can be extended or shortened, shifting

absorption and emission to longer or shorter wavelengths

[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

increases the effective conjugation and leads to the red-shifted

absorption spectrum of AP-B compared to AP [22]. The protein

may also provide hydrogen bonds, provide aromatic residues

for pi–pi stacking with the bilin, alter the local dielectric

environment and change pyrrole nitrogen pKa values with cor-

responding effects on the absorption properties [3]. As a result

of knowledge gained over many years of study, structure-

focused, targeted mutational studies have recently succeeded

in increasing fluorescence yield substantially [23–27].

In this report, we have tested the feasibility of drawing on

the advances made with natural biliproteins to reconstruct

and engineer biliproteins in minimal, compact protein

frames supporting selected functions that are expressible in

living cells with minimal metabolic load on cellular metab-

olism. The challenge of dramatically redesigning natural

biliproteins along these lines is daunting. Most PBPs require

specific interactions with lyase proteins that must be pre-

served to attach the bilin(s) covalently to the polypeptide

[8]. Furthermore, natural PBPs only bind bilin chromophores,

which constrains the design of multifunctional proteins. They

exhibit complex quaternary interactions that are difficult to

overcome without affecting the properties of the globin

domain that binds the chromophore(s) [9]. Changes to the

bilin-binding environment to adjust absorption and fluor-

escence properties often have unintended consequences on

the folding of the protein. De novo protein design offers an

alternative means to enhance biliprotein designability to

achieve compactness and adaptable function.

De novo protein design uses first principles of protein

folding to intentionally build minimal, structurally transpar-

ent protein frameworks that have been proved robust to

extensive amino acid sequence variation. a-Helical bundles

with a binary patterning of hydrophobic and hydrophilic

residues are favoured by de novo protein engineers because

of their designability, i.e. many different sequences fold

into the same structure [28,29]. Structural stability is built

in, resisting temperatures up to boiling [30]. These frames

are readily adapted to accommodate many natural and

non-natural cofactors to achieve diverse functionality through

informed, iterative design [31–37]. They readily lend them-

selves to progressive engineering directed towards further

synthetic applications [30,36,37]. This approach allows the

exploitation of design and sequence space undiscovered by

natural selection.

The work reported here seeks to define some initial prin-

ciples to determine whether bilins can be effectively and

efficiently bound to completely artificial proteins with no

sequence similarity to natural proteins, and if so, how the

optical properties of the resulting proteins can be manipu-

lated and optimized. Here we report initial designs to

artificial biliproteins to optimize three factors that control

the key optical properties of absorption, emission and fluor-

escence yield: (i) the type of bilin; (ii) Cys localization for

bilin binding; and (iii) the bilin binding environment. This

work initiates the development of basic design rules for

these choices and tests the performance of initial de novo bili-

protein light-harvesting maquettes in excitation energy

transfer (EET) from different bilins to cyclic tetrapyrrole

acceptors bound within the same maquette.

2. Results and discussion2.1. De novo biliprotein design choicesThere are three principal choices in designing de novo bili-

proteins to control the key optical properties of absorption,

emission and fluorescence yield. The most important choice

is bilin type. The different conjugation lengths of the various

bilins set the expected absorption and emission range, with

the longest absorption and emission wavelengths of red to

near IR for biliverdin (BV), shorter wavelengths for PCB

and phycoerythrobilin (PEB), and the shortest wavelengths

for tripyrroles (figure 1). Although not discussed here,

other bilin choices are available [39,40] for in vivo ligation

to the de novo bundle scaffold.

The second choice is the location of the ligating Cys resi-

due within the 4-helix bundle framework and the identity of

adjacent amino acids that control the yield of in vivo bilin

binding during expression. These selections modulate the

accessibility of the Cys residue for interacting with free bilin

or bilin lyase and the ability of the region of the bundle to

unfold and permit access to the bundle hydrophobic core

during the ligation between the Cys sulfhydryl group and

bilin vinyl moiety.

The third choice is to select residues that control the

environment of the bound bilin to modulate the bilin

conformation—specifically, the extension of the tetrapyrrole

chain, the angle of rotation between the pyrrole rings [22]

and the bilin rigidity. These factors critically influence the

effective degree of pi-electron conjugation in the molecular

orbitals of the bilin and thereby its optical properties [3].

This is a special design challenge presented by bilin cofactors

that is not a concern for previously studied rigid maquette

cofactors, including porphyrins, chlorins, iron–sulfur clusters

and flavins [31].

wavelength (nm)

0.5

1.0

protein

bound

0300 400 500 600 700 800

TPB

abso

rban

ce

PEB PCBBV

0

0.1

0.2 free

wavelength (nm)300 400 500 600 700 800

visible

(b)

(a)

(c)

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 inphosphate-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 subunitfused 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.

heptad position:

negative helix: positive helix: neutral loop: positive loop: short loop:

helix 1

helix 2

helix 3

helix 4

loop 2

loop 3

loop 1

polar

non polar 12

3 4

view downhelix axis

a

bc

d

e

f

g

net charge: –4 +1

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 thebundle 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-polarheptad 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 connectedby either long or short loops (bottom).

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2.2. Binary patterning in helical bundle designMaquettes exploit first principles of protein folding by using

repeating amino acid heptads (figure 2) [42]. In an a-helical

fold, seven amino acids form complementary hydrogen

bonds to complete two helical turns. Binary patterning of

these heptads places non-polar residues along one face of

the helix with polar residues on the other. Ends of helices

are connected with simple glycine (Gly)-rich loops. In aqu-

eous solution, this single polypeptide chain spontaneously

sequesters the non-polar residues to form the core of a

CGEI

2CGRI

3

1

CGRD

CX X X

abso

rban

ce

55 6

6

4

4

9

9

8

8

7

7

0.2

0.1

0.3

wavelength (nm)400 500 600 700 400 500 600 700

wavelength (nm)

loop CXXX helix CLRD

aminoterminus

acidterminus

(b)(a)

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 aCLRD 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, thuslarger absorbances correspond to larger ligation efficiency. Binding yields are given in table 1.

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4-helix bundle. Maquette frames are highly tolerant to

changes in sequences that maintain the general binary pat-

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

Cys, change the net charge of individual helices, alter lengths

of loops and make changes in the hydrophobic core, all with-

out compromising the highly tolerant folding of the protein.

2.3. Bilin lyase co-expressionWe exploited the expression system developed previously

[40,43,44], which imports bilin synthesis and attachment

machinery for ligating diverse phycobilins to PBP apopro-

teins expressed into E. coli. The plasmids code for the

following: haem oxygenase-1 to catalyse BV synthesis from

haem; a reductase to reduce BV to either PCB or PEB; and

a bilin lyase. We added plasmid PJ414 encoding a maquette

to this system. Despite the completely unnatural sequence

of the designed bundle, successful coupling between the

bilin lyase and the maquette is clear. The higher copy

number of the maquette PJ414 expression vector tends to out-

strip the bilin cofactor biosynthetic machinery on the low

copy number pACYCDuet vector. Nevertheless, expression

of the bilin S-type lyase CpcS from Thermosynechococcus elon-gatus [44] greatly increases the amount of bilin bound.

Without PCB lyase, purified maquettes carried minimal

amounts of bound PCB along with small amounts of

bound BV and haem (see the electronic supplementary

material, figure S1, for spectra).

In this study, we show the bilins BV, PCB and PEB all

bind covalently to Cys residues in maquettes in vivo (figures 1

and 3; and electronic supplementary material, figure S20) and

in vitro (figure 6). We selected the PCB plasmid for the most

extensive analysis.

2.4. Modulating bilin binding through Cys exposureTo test the hypothesis that surface exposure of the ligating

Cys residue may modulate bilin binding, Cys residues were

inserted at different positions in the helical heptad. Even

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

Cys residues are inserted in the middle of a helix, both hydro-

phobic core sites (heptad positions a or d ) and surface

exposed sites (heptad positions b or c) lead to similar bilin-

binding yields (see the electronic supplementary material,

figure S2 and table S1). On the other hand, Cys residues

inserted in a conformationally flexible loop position dis-

played greater absorbance and enhanced binding yield.

Spontaneous PCB binding without lyase is negligible. These

observations support the hypothesis that local unfolding of

the protein sequence is more important than Cys exposure

in interfacing with bilin lyases in vivo. Local unfolding has

been proposed in a structural model that docks the lyase

CpcT with the natural biliprotein CpcB; in this model the

Cys-containing sequence is inserted into the bilin-carrying

cleft of the lyase, which brings the Cys-sulfhydryl and the

bilin vinyl into apposition for catalytic ligation [45].

2.5. Modulating bilin binding through nearby Argand Asp

Cys-containing sequences in natural biliproteins are highly

diverse and different sequence classes are associated with

specific bilin lyase families [8,13]. Restricting a biliprotein

DD C RR

C L R D

LL

400 600500 700

DRLChelix

CLRKhelix

CLRDloop

CLRDhelix

0.2

0.1

0

Abs

/Abs

280

nm

Abs

/Abs

280

nm

wavelength (nm)400 600500 700 800

0.1

0DC R

C L R D

X

H

A

DRLChelix

DRLCadd His

DRAChelix

CLRDloop

13

11

12

10

10

15

13

14

(b)(a)

(c) (d )

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 yielddramatically (magenta). (c) Overlay of X-ray structure of natural allophycocyanin (green backbone ribbon, purple bilin) with molecular dynamics structure of themaquette (grey) by superimposing allophycocyanin CLRD onto maquette DRLC. Cys (yellow), Arg (blue), Asp (red). (d ) Corresponding spectra.

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BLAST search to those associated with the S-type lyase used

here indicates that Arg and Asp residues are usually found

downstream from the Cys in a Cys-Xxx-Arg-Asp (CXRD)

pattern, where Xxx is typically a non-polar residue [46].

Arginines can form charge-pairs with bilin propionates,

while Asp can interact with pyrrole nitrogens [44].

Figure 3a shows that introducing an Arg two positions after

a loop Cys boosts the bilin-binding yield by a factor of 2

and adds structure to the absorption spectrum. Longer wave-

length absorption features in natural biliproteins are

associated with more extensive pi-orbital conjugations of

the more extended bilin conformations and those with smal-

ler dihedral angles between the pyrrole rings, especially

between the rings C and D. However, adding an Asp in a

loop CXRD motif did not improve binding yield.

2.6. Modulating bilin-binding yield with CXRD positionWhile cysteines in CXRD motifs are comparably effective in

binding PCB at both surface-exposed or core buried helix

heptad positions (figure 3b), there is a conspicuous asymme-

try in bilin-binding efficiency of the amino end of the helix

compared to the carboxyl end of the helix. Bilin-binding

yield increases by nearly fivefold when CXRD is placed

near the amino rather than the carboxyl terminal of helices.

Helical unwinding for insertion of Cys into the lyase for lig-

ation is expected to be easier near either end of the helix.

However, when the Cys was placed within two turns or

10.5 A from the carboxyl terminus of a helix, bilin-binding

yield decreased and the absorption broadens (brown spectra

in figure 3b). Cys anchoring close to the carboxyl terminus

(less than the 13 A of an extended bilin) may force a twisted

geometry.

2.7. Modulating bilin binding with CXRD orderWhen the Cys is positioned in the middle of a helix, introduc-

tion of a negatively charged Asp two residues downstream

greatly improves bilin binding in maquettes relative to posi-

tively charged lysine (Lys; figure 4a). This is evidence that

in contrast to Asp in loops, the carboxylate of Asp in a

helix is positioned for favourable interactions with the nitro-

gens of pyrrole ring B/C. When the Asp carboxylate is

replaced with a Glu carboxylate, analogous to the CXRE

motif found in some BV-binding bacteriophytochromes,

PCB binds less favourably, while comparable amounts of

BV are bound (electronic supplementary material, figure S21).

Unexpectedly, the reverse motif, DRXC, appears equally

effective in driving bilin ligation in maquettes (figure 4a).

While the amount of bilin bound to the maquette varies

depending upon details of growth conditions and the relative

rate of cellular production of both the bilin chromophore and

the maquette protein frame, under controlled conditions of

parallel growth and purification, there is comparable PCB

binding yield for the DRLC and CLRD motifs (table 2).

These results indicate that within a helix, proximity of the

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

ID Cys insertion

Cys location His position on helix relative binding yield in vivo

Vis/UVhelix loop position 1 2 3 4 PCB

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

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

9 CLRD 2 25 6 6 6 6 15% 0.31

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

IDCysinsertion

Cys location His position on helix bindingyieldin vivo

abs.quantumyield (%)

Vis/UVhelix loop position 1 2 3 4

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

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Arg and Asp to the ligating Cys is more important than the

sequence order.

Structural insight on this equality can be found by super-

imposing the bilin-binding Cys-containing helices of the

crystal structure of the alpha subunit of the natural biliprotein

allophycocyanin from the cyanobacterium Phormidium sp.

A09DM (4RMP) [47] with a molecular dynamics structure

of the maquette helical bundles (figure 4c) [48]. Despite the

reverse order of the sequences, the arginine of both the allo-

phycocyanin and the maquette are in positions close enough

to interact with the B ring propionate, while the aspartate of

both motifs are close enough to interact with the ring B and C

pyrrole nitrogens. Furthermore, the nearly parallel orien-

tations of adjacent helices near the bound bilin in both

allophycocyanin and the maquette accommodate intercala-

tion of roughly planar PCB pyrroles between adjacent helices.

2.8. Modulating bili-maquette fluorescenceFigure 5a compares the absorption and fluorescence emission

(excitation at 580 nm) spectra of three maquette sequences

described in figure 4d. A higher Vis/UV absorbance ratio is

correlated with a more rigid and extended bilin conformation

[3,49,50]. The DRLC motif binds more PCB than the DRAC

motif and has a higher fluorescence quantum yield, consist-

ent with constraint by a better packed, less mobile

hydrophobic core.

If maquette-bound bilin orients similarly to the PCB of

the alpha subunit of allophycocyanin, the D ring pyrrole

should approach position 26 in adjacent helix 3. Indeed,

replacing Leu with His in this position noticeably sharpens

the Vis absorbance band and increases the Vis/UV ratio

(table 2 and figure 4c). This is consistent with a pi-stacking

or hydrogen bonding interaction between the helix 3 His

and ring D of PCB. This third-helix His improves both

binding and fluorescence quantum yield.

Figure 5b shows that changing the external charge

patterning from near-neutral to negative modestly improves

the fluorescence quantum yield from 2 to 3%. Quantum

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),

so that the 4-helix bundle assembles from two separate

subunits, doubles the yield of bilin binding per Cys, presum-

ably through greater lyase accessibility. However,

fluorescence quantum yield was relatively poor, probably

reflecting higher mobility of the PCB when a single maquette

chain is divided into two.

emissionabsorbance

18

1413

15

164

17

0.08

0.06

0.04

0.02

0

abso

rban

ce

0.08

0.06

0.04

0.02

0

abso

rban

ce

400 500

580

580

600 700

400 500 600 700wavelength (nm)

emis

sion

(ar

b. u

nits

)em

issi

on (

arb.

uni

ts)

0

1

2

3

4

0

1

2

3

4

(a)

(b)

Figure 5. (a) The DRLC motif (13,15) binds more PCB and has higher flu-orescence quantum yield than a DRAC motif (14). (b) Changing externalcharge patterning and loop length (4, 16, 18) had minimal effects onbilin attachment and quantum yield. Breaking the 4-helix bundle into adimer of two helices (17) increases bilin ligation efficiency but lowers fluor-escence quantum yield. Spectra were normalized to 50 mM protein.

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2.9. Lyase strengthens CLRD motif effectivenessThe chaperoning influence of bilin lyase during in vivoexpression is absent with in vitro ligation. In such cases,

loop Cys position provides the best yield for attachment for

all bilins (figure 6, sequence (2)). Without a lyase, cysteine

exposure facilitates binding. Although the binding yield

was lower, burying the Cys in the core (20) generally

enhanced fluorescence quantum yield, indicating that the

core restricts quenching associated with bilin mobility.

To test the hypothesis that CLRD motifs improve in vitrobilin binding, we inserted the motif downstream from the

buried helical core position at the beginning and end

(sequences 5 and 9) of the second helix. Neither CLRD pos-

ition improved bilin binding or fluorescence quantum

yields compared to Cys alone. Indeed, BV binding decreased.

This contrast with the results for in vivo binding supports

the view that a CLRD motif gains significance through

modulating interactions with bilin lyases.

2.10. Lyase – maquette interactionsThe tendency of the hydrophobic core of elementary 4-helix

bundle synthetic protein designs to associate with bilins even

without Cys present probably facilitates the autocatalytic

thioether linkage formation to bilin vinyl groups when Cys is

introduced. This reaction is analogous to the previously demon-

strated ligation of haem vinyls to Cys during the formation of

cytochrome c maquettes [52]. However, the autocatalytic bilin

reaction in vitro is generally slow compared to in vivo lyase-

catalysed ligation. By co-expressing lyases with bilin synthases,

the total amount of bilin available for ligation improves.

In natural systems, lyases boost bilin ligation yield

and modulate the stereochemistry of bilin ligation and corre-

sponding bilin absorption and emission properties [8,13]. The

helical conformations seen in free bilins have relatively weak

long wavelength absorptions; more extended conformations

imposed by bilin-binding sites provide longer pi-conjugation

for more intense absorptions, particularly when torsion

angles allow pi-orbitals to overlap optimally [3,49,50,53].

Suppression of bilin flexibility by binding-site confinement

and specific interactions with nearby residues inhibits excited

state quenching and supports higher fluorescence emission

quantum yields [19].

Because maquettes have minimal sequence similarity to

natural biliproteins, they are not expected to form specific

complexes with natural lyases. Nevertheless, lyases do

confer different maquette bilin geometries than seen in

lyase-free autocatalysis. Autocatalytic PCB binding to

maquettes produces spectral forms with relatively large

absorption bands around 650 nm, similar to the absorption

seen in autocatalytic binding of PCB to the apo-biliprotein

PecA [54]. Four examples are shown as solid lines in

figure 7. Maquette designs with a Cys in the central loop

show a similar spectrum (dashed black line figure 7) upon

in vivo expression with the lyase. However, for maquette

Cys in helix positions, lyase-assisted bilin binding generates

shorter wavelength, relatively broad and less-structured

absorption bands (orange, purple and blue dashed lines

figure 7). Apparently for helical ligation sites in maquettes,

lyase interaction favours less extended and potentially more

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

the majority of natural PCB biliproteins (see electronic

supplementary material, figure S12) [1,12].

Lyases also appear to influence E–Z isomerization at the

C15–C16 or possibly the C4–C5 double bond of PCB in

maquettes. Under denaturing conditions that largely remove

the influence of the protein environment on the bilin absorption

(see electronic supplementary material, figures S13 and S14),

PCB largely assumes a Z isomer (absorbance maximum

661 nm [3]) when ligated to maquettes in the presence of

lyase in vivo. By contrast, in vitro ligation of the Z isomer

of PCB purified by high-performance liquid chromatography

(HPLC) yields predominantly E isomers (593 nm [55]).

Although photoisomerization between Z and E configurations

of PCB is a critical part of the signalling process in phyto-

chromes [15,56], there is so far no evidence of analogous

photoisomerization in bili-maquettes.

2.11. Tripyrrole and phycocyanobilin tetrapyrrole tochlorin excitation energy transfer

The Q band absorbance maxima and the UV/Vis absorbance

ratios for high-yield bili-maquettes lie within the range of natu-

ral CpcB and PecB previously produced in E. coli [57], an

indication that PCB is bound rigidly. However, fluorescence

quantum yields in maquettes of 1 to 3% (compared to 12 and

32% of a range of PCB-binding PBPs [57,58]) show room for

improving chromophore rigidity. Nevertheless, these fluor-

escence levels are adequate for adapting bili-maquette

designs towards voltage sensors and energy-transfer agents.

We demonstrate EET from a maquette-bound bilin to

another maquette-bound chromophore, analogous to EET in

maquettes fused to a PCB-containing phycocyanin a subunit

[38]. We created a Zn-tetrapyrrole-macrocycle binding site by

inserting a potential His ligand at position 6 on helix 3,

abso

rban

ce (

×10

–2)

abso

rban

ce (

×10

–2)

abso

rban

ce (

×10

–2)

(b)(a)

20

2

5

9

excitation emission

BV

wavelength (nm) wavelength (nm)

PEB

absorbance

PCB

1

2

0

1

0

1

2

0

3

fluo

resc

ence

(ar

b.un

its)

600 700

600 700

600 700500 800600 700500 800300 400

5

10

0

5

0

2.5

5.0

0

600 700500400

600 700500400

20

2

59

CGRI

DRLC

ADECDRLC

fluo

resc

ence

(ar

b.un

its)

fluo

resc

ence

(ar

b.un

its)

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 yieldsare given in table 4.

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approximately 32 A from the bilin-ligating Cys. Two remain-

ing interior His residues were replaced with alanine to

preclude competing ligation sites (sequence (19); table 3).

A range of light-active Zn-tetrapyrrole macrocycles ligate to

His in this site [59].

The EET distance between maquette-bound bilin and Zn

tetrapyrrole is much shorter than the calculated Forster

energy-transfer distance of approximately 50 A [38]. If chro-

mophores were isotropically oriented, EET efficiency would

be above 90%. The transition dipoles of bilins, commonly

assumed to be along the direction of the B to D pyrroles

[60], would be constrained to be mostly parallel to the long

helical axis if buried in the bundle core in an extended confor-

mation. A test of this orientation uses a chromophore with a

Table 3. Binding yields and optical qualities of maquette designs shown in figure 5b.

IDCysinsertion

Cys location His position on helix

binding yieldin vivo (%)

abs. quantumyield (%)

Vis/UVhelix pos. 1 2 3 4

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

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

ID Cys insertion

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

helix loop position BV PEB PCB

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)

400 500 600 700 8000

0.10

0.20

0.30

wavelength (nm)

rela

tive

abso

rban

ce

20

2

5

9

Figure 7. Maquette-bound PCB has longer wavelength absorption whenligated in vitro (solid lines) compared to lyase-assisted in vivo (dashed).Colours correspond to maquette sequences in figure 6.

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transition dipole unfavourably oriented nearly perpendicular

to the helical-bundle axis, Zn-chlorophyllide a (ZnChl).

For EET donors, we compared two bilin chromophores:

PCB and a blue-shifted tripyrrole bilin (TPB) derived from

PCB. By raising the pH of a denaturing 6 M guanadinium sol-

ution of PCB-bound maquette-19–8.5, the absorption

redshifts to 739 nm (figure 8), presumably because of chro-

mophore deprotonation [3]. Over 30 min, the absorption

blue-shifts to 586 nm. Mass spectrometry revealed a mass

loss of 109 Da, consistent with scission of one pyrrole ring

to form a TPB [3,61] (see the electronic supplementary

material, figure S10). The detailed chemical structure of this

readily generated tripyrrole derivative has not yet been deter-

mined, but the high quantum yield of fluorescence (26%)

facilitates energy-transfer measurements.

For the energy-transfer measurements, the bilin-bound

maquette was HPLC-purified to enrich the fraction of

maquette containing bound TPB to about 50%. ZnChl bind-

ing (Kd 4+3 nM) to either the PCB or TPB maquette does

not distort the bilin absorption spectrum (see the electronic

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

decomposition (SVD) of the absorption spectra confirmed

the presence of two spectral forms corresponding to the

bound and unbound ZnChl. Figure 10a,b shows binding

and saturation of the site at a 1 : 1 stoichiometry. Fitting to

a single binding-site model confirms nM binding of ZnChl

with and without bound TPB.

To characterize EET from the bilin to the Zn tetrapyrrole,

each titration step was excited at 550 nm. Figure 10c,d shows

that SVD analysis of the emission spectra reveals three com-

ponents fitting a simple binding-site model. The first

spectrum corresponds to TPB emission in the maquette with-

out ZnChl with a maximum near 590 nm (red, figure 10c). As

ZnChl is added, this emission spectrum is replaced with a

second emission spectrum with two peaks of approximately

equal intensity at 590 and 675 nm corresponding to emission

from TPB and histidine-ligated ZnChl, respectively (green,

figure 10c). As excess ZnChl is added, a third spectral com-

ponent grows that quenches the remaining TPB emission

and shows a blue-shifted emission maximum at 665 nm, cor-

responding to ZnChl not ligated to the maquette histidine

(blue, figure 10c). At a 1 : 1 stoichiometry of His-ligated

ZnChl per maquette, approximately 30% of the TPB fluor-

escence is quenched by energy transfer, indicating that the

transition dipoles are relatively constrained and unfavourably

oriented. At high concentration, ZnChl as a relatively hydro-

phobic pigment will partition into the hydrophobic core.

Once the His site is filled with ZnChl, the additional loosely

bound ZnChl will not be held as rigidly or as far from the

bound bilin, permitting more efficient fluorescence quench-

ing. Figure 11 shows that ZnChl is more effective at

quenching the fluorescence of PCB. At a 1 : 1 stoichiometry

of ZnChl per maquette, PCB is 75% quenched, thus the

PCB transition dipole is oriented less orthogonal to the

dipole of ZnChl.

500 600 700 8000

0.2

0.4ab

sorb

ance

wavelength (nm)

586

640

739

PCB

pH 8

pH 8.5

TPB

Figure 8. Formation of TPB by alkaline scission of PCB bound to maquette19. Bound PCB in 6 M guanidinium ( pH 8) has a broad absorption around650 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( purple) associated with a tripyrrole.

400 500 600 700 800

0

0.1

0.2

abso

rban

ce

wavelength (nm)

323 345 424669

586 606

Figure 9. Spectra of individual energy transfer pigments in maquette 19:tripyrrole ( pink), PCB (blue) and ZnChl (green).

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2.12. Compact biliprotein design motivationsWhen natural phytochrome biliproteins are exploited for use

as red to near-infrared fluorescent proteins, their utility and

performance can be limited due to intrinsic biophysical

reasons. As photosensory signalling proteins, they have not

evolved to be naturally fluorescent, but rather, their struc-

tures were selected to convert their photonic energy into

large-scale structural rearrangements. While they have

been engineered by directed evolution for enhanced fluor-

escence by minimizing this Nature-prescribed motion and

restricted water access in the bilin-binding GAF domain

[18], there is a limit to size reduction [62] due to the structural

requirements for bilin binding and stabilization. By using

the compact artificial protein platform presented here

(approx. half the molecular weight of green fluorescent

protein), energy-transfer distances can be shortened and the

cellular metabolic burden can be limited when applied as

cell-expressible transcriptional reporters and fusion tags.

This work establishes key determinants of biliprotein

holo-protein formation in E. coli using genetically encoded

components. Companion eukaryotic work has established

bili-maquette expression in mammalian neuronal cells.

That work, aimed at developing maquette biliproteins for

opto-genetic studies, shows that maquette technology can

be compacted to a 9 kDa maquette while preserving useful

BV fluorescence.

3. SummaryThis work extends the fundamental rules for designing

protein maquettes to include a new and versatile class of

light-active cofactors, bilins, thus enabling the engineering

of a broad spectral selection of light-activated artificial pro-

teins (e.g. [40]). Unlike the His ligation used to bind

metalated cyclic tetrapyrrole cofactors to selected positions

in maquette helical bundles, Cys ligation is used to anchor

bilins, analogous to the Cys binding to haem vinyls by

maquettes with CXXCH motifs to produce c-type cyto-

chromes. Ligation of various bilins in vitro is favoured by

placing Cys in relatively exposed and flexible positions,

especially bundle loops, although helical positions are also

competent. Arg placed two residues away presumably inter-

acts with the propionate side chains of rings B/C to constrain

bilin orientation partially and improve fluorescence yield.

The relatively simple in vitro rules for creating bili-

maquettes are altered in the more complex environment of

the cell where interactions with natural bilin lyases boost

the speed, and alter the yield and stereochemistry of bilin lig-

ation. The S-type lyases operate by partially unfolding both

natural and artificial proteins to expose the ligating Cys to

the A-ring vinyl of the bilin carried by the lyase. Such unfold-

ing and insertion occurs for Cys in most regions of the bundle

with the exception of the carboxy terminal ends of the helices.

In the other helical locations along the helices, Arg and Asp

two and three residues away are well positioned to interact

with the propionate carboxylates and pyrrole nitrogens,

respectively, of the bilin to constrain motion and favour

more extended chromophore geometries with desirable opti-

cal changes in absorption and fluorescence emission

quantum efficiency. Arg and Asp are equally effective in a

natural CXRD and an unnatural DRXC motif.

Engineering bilin binding in maquettes is a modular, local

design issue that leaves the rest of the protein framework free

for other functions, such as introduction of a second chromo-

phore binding site for EET, or for fusion to natural proteins

for cellular localization. Improvement in optical performance

can be expected by adding to these rules, as required for any

specific design application.

4. Methods4.1. Gene constructionsCodon optimized synthetic genes were obtained from DNA2.0 in

a PJ414 expression vector. Mutations to parent vector were made

with primers synthesized by IDT or Invitrogen (see electronic

supplementary material, tables S2–S4). Sequences were verified

using UPENN DNA sequencing core.

4.2. Maquette expressionBilin biosynthetic plasmids were CpcS lyase [44] and PcyA and

HO1 [43]. His-tagged maquettes and bilin biosynthetic and

attachment enzymes were expressed in E. coli BL21 (DE3). Strains

containing bilin biosynthetic plasmids were made competent to

e (m

M–1

cm

–1)

0.5

0.4

0.3

0.2

0.1

1.5

400300 500 600 700 800

1.0

0.5

0

0.2

0.1

0

wavelength (nm)

abso

rban

ceabsorbance emission

bound

unbound

bilin

654321

concentration (mM)

0.6

0.4

0.2

0

–0.2

–0.4

–0.6

SVD

am

plitu

de

SVD component 1

SVD component 2

1:1stoichiometry

D A

bs

423–

408

nm

50

em

issi

on 6

80 n

m

concentration (mM)

concentration (mM)0 5

4

8

emis

sion

10

8

6

4

2

0600 700 750 800650

wavelength (nm)

(a)

(b)

(c)

(d )0.6

0.4

0.2

0.0

–0.2

–0.4

–0.6SV

D a

mpl

itude

654321concentration (mM)

1:1stoichiometry

SVD component 1

SVD component 3

SVD component 2

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 ) SVDanalysis 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.

Em 626 nmEm 676 nm

abso

rban

ceem

issi

on

600 650 700 750 800wavelength (nm)

0.05

0.10

0.15

0.20

0

0

0.02

0.04

concentration (mM)

D A

bs

435–

399

nm

42

0

2

4

300 400 500 600 700 800wavelength (nm)

626 nm

676 nm

emis

sion

2concentration (mM)

40

2

4(b)

(a)

Figure 11. EET is demonstrated by fluorescence quenching of PCB. (a) Absor-bance of ZnChl titrated into 2.7 mM maquette, 10 mM NaPO4 ( pH 7.4).Insert: PCB and ZnChl peak emission intensities at 626 (red) and 676 nm(blue). (b) Corresponding emission spectra during titration.

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take up the maquette plasmid using the Hanahan method [63].

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-

propyl-thiogalactopyranoside for 21 h at 208C at 260 r.p.m. in

bevelled flasks with culture volumes of 100 ml.

4.3. High-performance liquid chromatography methodsSolvents used for pigment and protein purification were a mix-

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.

4.4. Protein purificationProteins expressed with His tags were purified by resuspending

pelleted cells in 300 mM NaCl, 20 mM imidazole, 50 mM

NaH2PO4 (pH 8) buffer, homogenized and microtip sonicated.

Lysate was centrifuged at 25 000g for 25 min, with supernatant

applied to a Qiagen Ni-NTA superflow resin and purified via

the gravity column method. One litre of cell culture yields

approximately 20 mg of maquette. PCB bili-maquette 19 without

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

extractions were pooled and rotovapped until dry.

Unless otherwise noted, both tagged and untagged proteins

were then HPLC-purified using a 30 ml gradient from 30 to 42%

ACN; spectra collected during the gradient quantified the effi-

ciency of PCB attached to mutants in figure 4a (see electronic

supplementary material, figure S4). Purified bili-maquette was

collected, lyophilized and stored at 2208C in the dark until

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use. Molecular weight was assayed by both SDS PAGE (Invitro-

gen Novex NuPAGE electrophoresis system, 4–12% Bis-Tris gels,

MES running buffer) and MALDI [30] (see the electronic sup-

plementary material, figure S5).

4.5. Pigment purificationPCB was extracted using a modified procedure of [65] via over-

night methanol refluxing of 50 mg of a gene fusion between

CpcA and maquette [38]. The rotovapped reflux mixture was

extracted with 1 ml of ethanol, and purified by HPLC using a

500 ml 20–70% ACN gradient on a C-18 column (see the elec-

tronic supplementary material, figure S16). MALDI-MS verified

the mass of PCB (see the electronic supplementary material,

figure S17).

PEB was extracted from a His-tagged CpcA modified to delete

the covalently ligating Cys (electronic supplementary material,

table S5). After His tag purification, 25 mg of protein was precipi-

tated by adding methanol at 50% followed by a half volume of

CHCl3 and then drying by rotary evaporation at 408C. MALDI

mass spectrometry used 2,5-dihydroxybenzoic acid matrix (see

electronic supplementary material, figure S18). HPLC verified

purity (see electronic supplementary material, figure S19).

Pigment concentration assay used extinction coefficients (1) for

free PEB of 25.2 mM21 cm21 at 591 nm [66] and for free PCB

1 ¼ 37.9 mM21 cm21 at 690 nm in 5% HCl/methanol.

ZnChl was prepared by addition of Zn to pheophorbide a(Frontier Scientific) as described [67]. DMSO-solubilized ZnChl

stock concentrations were determined by dilution in methanol

using an extinction coefficient of 64 000 M21 cm21 at 656 nm [68].

4.6. In vitro bilin attachmentMaquettes were reduced with 5 mM DTT, followed by PD-10

size-exclusion chromatography before addition of bilin. BV reac-

tion was performed in PBS buffer at pH 7.4 for 4 h with 100 mM

protein and 500 mM BV. Free BV was separated from BV-

maquette by a size-exclusion column equilibrated with PBS

(pH 7.4). PEB in vitro treatment was performed overnight in

60 mM protein 50 mM Tris (pH 8), while PCB attachment was

performed in 30 mM protein 50 mM NaPO4 (pH 7) as described

in [69]. In vitro PEB/maquette attachment reactions were purified

using a 700 ml gradient from 30 to 60% ACN for sequences 5, 9

and 20 and a 500 ml gradient from 20 to 70% ACN for

sequence 2. PCB in vitro maquette attachment reactions were pur-

ified using a 500 ml gradient from 20 to 70% ACN. To form the

tripyrrole, sequence 19 with bound PCB was solubilized in 6 M

guanadinium-HCl (pH 8) and raised to pH 8.5 with NaOH.

After 30 min, the solution was HPLC-purified using a 500 ml

gradient from 20 to 70% ACN.

4.7. Protein, phycocyanobilin concentration andquantum yield determination

UV/Vis absorbance was measured using a Varian Cary-50 spec-

trophotometer. Fluorescence spectra were obtained using a

Horiba Fluorolog 2 fluorimeter at 208C. The PCB concentration

was calculated by denaturing in 8 M urea (pH 2), using an e

660 of 35.4 mM21 cm21 [70]. HPLC spectra show that PCB absor-

bance at 280 nm is minor compared to Trp absorbance. Relative

bilin yield was assayed by normalizing 660 nm absorbance

in acidic urea to the 280 nm Trp absorbance in PBS after

purification.

The quantum yield of PEB and PCB bound to maquettes

used the method in [43], after PD-10 size-exclusion chromato-

graphy using cresyl violet perchlorate (quantum yield 0.54 in

methanol) [71] as a standard. For BV, four dilutions of each

BV-maquette and Cy5 standard in PBS were made. Absorbance

at 600 nm was plotted against integrated emission from 635 nm

to 830 nm. Quantum yield was determined using the slope of

the dilution lines relative to that of Cy5 (quantum yield of Cy5

of 0.27) [72].

Data accessibility. The datasets supporting this article have beenuploaded as part of the electronic supplementary material.

Authors’ contributions. J.A.M. engineered and performed bili-maquettemolecular biology, purification and physical and spectroscopiccharacterization, and contributed to manuscript drafting. M.S.assayed BV binding. G.K. assisted in experimental design and engin-eering of bili-maquettes and synthesized ZnChl pigment. B.Y.C.supervised in vitro BV binding measurements. D.A.B. providedCpcS, haem oxygenase and PcyA overexpression bacterial strains.P.L.D. supervised maquette design. C.C.M. assisted in bili-maquettedesign and analysis, and directed manuscript drafting. All theauthors gave their final approval for publication.

Competing interests. We declare we have no competing interests.

Funding. This research was carried out as part of the PhotosyntheticAntenna Research Center (PARC), an Energy Frontier ResearchCenter funded by the US Department of Energy, Office of Science,Office of Basic Energy Sciences, under Award DESC0001035 support-ing J.A.M., M.S., G.K., D.A.B., P.L.D. and C.C.M. in maquetteconstruction and characterization as well as chlorin synthesis.National Science Foundation (CBET 126497) supported M.S. andB.Y.C. in biliverdin maquette design development.

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