Single-Residue Insertion Switches the Quaternary Structure and Exciton States of Cryptophyte Light-Harvesting Proteins

Single-residue insertion switches the quaternary structure and exciton states of cryptophyte light-harvesting proteins

Stephen J. Harropa,1, Krystyna E. Wilka,1, Rayomond Dinshawb, Elisabetta Collinic, Tihana Mirkovicb, Chang Ying Tengd, Daniel G. Oblinskyb, Beverley R. Greend, Kerstin Hoef-Emdene, Roger G. Hillerf, Gregory D. Scholesb, and Paul M. G. Curmia,g,2

aSchool of Physics, The University of New South Wales, Sydney, NSW 2052, Australia; bDepartment of Chemistry, University of Toronto, Toronto, ON, Canada M5S 3H6; cDepartment of Chemical Sciences, University of Padua, 35131 Padua, Italy; dDepartment of Botany, University of British Columbia, Vancouver, BC, Canada V6T 1Z4; eBotanical Institute, Cologne Biocenter, University of Cologne, 50674 Cologne, Germany; fDepartment of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia; and gCentre for Applied Medical Research, St Vincent’s Hospital, Sydney, NSW 2010, Australia

Edited by Douglas C. Rees, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, and approved May 28, 2014 (received for review February 10, 2014) Observation of coherent oscillations in the 2D electronic spectra different—in essence because strong excitonic interactions within (2D ES) of photosynthetic proteins has led researchers to ask thePBPareswitchedfromontooff. whether nontrivial quantum phenomena are biologically sig- The crystal structure of the cryptophyte PBP phycoerythrin nificant. Coherent oscillations have been reported for the soluble PE545 from Rhodomonas CS24 showed that the protein is a di- light-harvesting phycobiliprotein (PBP) antenna isolated from mer of two αβ monomers (3, 4), the β subunit of which has cryptophyte algae. To probe the link between spectral properties a globin fold (5, 6) and binds three linear tetrapyrroles (bilins), and protein structure, we determined crystal structures of three whereas the α subunit is a short, extended polypeptide with PBP light-harvesting complexes isolated from different species. a single bilin chromophore. A prominent feature of this structure Each PBP is a dimer of αβ subunits in which the structure of the αβ is the arrangement of the two central chromophores in van der monomer is conserved. However, we discovered two dramatically distinct quaternary conformations, one of which is specific to the Waals contact with each other on the pseudo-twofold axis, with genus Hemiselmis. Because of steric effects emerging from the each chromophore covalently linked to two cysteines on one of insertion of a single amino acid, the two αβ monomers are rotated the β subunits (referred to as “β50/61”). This structural feature by ∼73° to an “open” configuration in contrast to the “closed” introduces excitonic coupling between the chromophores (3, 4). configuration of other cryptophyte PBPs. This structural change We are fascinated by this observation because it implies that if is significant for the light-harvesting function because it disrupts coherence plays a nontrivial role in light harvesting (7–12), it the strong excitonic coupling between two central chromophores might be switched on and off (either dynamically or genetically) in the closed form. The 2D ES show marked cross-peak oscillations by controlling the separation, and hence excitonic coupling, be- assigned to electronic and vibrational coherences in the closed- tween these two central chromophores. form PC645. However, such features appear to be reduced, or perhaps absent, in the open structures. Thus cryptophytes have Significance evolved a structural switch controlled by an amino acid insertion to modulate excitonic interactions and therefore the mechanisms used for light harvesting. There is intense interest in determining whether coherent quantum processes play a nontrivial role in biology. This interest was sparked by the discovery of long-lived oscillations in X-ray crystallography | quantum coherence | protein evolution | 2D electronic spectra of photosynthetic proteins, including the excitonic switching phycobiliproteins (PBPs) from cryptophyte algae. Using X-ray crystallography, we show that cryptophyte PBPs adopt one of ight-harvesting complexes capture and funnel the energy two quaternary structures, open or closed. The key feature of Lfrom light using organic chromophore molecules that are the closed form is the juxtaposition of two central chromo- bound to scaffolding proteins. The protein structure thereby sets phores resulting in excitonic coupling. The switch between the relative positions and orientations of the chromophores to forms is ascribed to the insertion of a single amino acid in the control excitation transport. In other words, the protein plays open-form proteins. Thus, PBP quaternary structure controls a deciding role in building the “electronic Hamiltonian”—the excitonic coupling and the mechanism of light harvesting. electronic coupling between chromophores and the chromo- Comparing organisms with these two distinct proteins will phoric energy landscape that directs energy flow. This strong reveal the role of quantum coherence in photosynthesis. connection between structural biology and physics means that Author contributions: S.J.H., K.E.W., R.D., E.C., T.M., C.Y.T., D.G.O., B.R.G., K.H.-E., R.G.H., ultrafast light-harvesting functions are under genetic and evolu- G.D.S., and P.M.G.C. designed research, performed research, analyzed data, and wrote tionary control. Cryptophytes, a group of marine and freshwater the paper. single-celled algae, are an intriguing example, because one of The authors declare no conflict of interest. their light-harvesting antenna complexes was completely re- This article is a PNAS Direct Submission. engineered by combining a unique bilin-binding polypeptide with Data deposition: Atomic coordinates and structure factors have been deposited in the a single subunit from the ancestral red algal phycobilisome (1, 2). Protein Data Bank, www.pdb.org (PDB ID codes 4LMS, 4LM6, and 4LMX), and DNA sequences have been deposited in the GenBank database (accession nos. KC905456– Here we report a further example of biological manipulation of KC905459 and KF314689–KF314696). this phycobiliprotein (PBP) light-harvesting system. We have 1S.J.H. and K.E.W. contributed equally to this work. discovered an elegant but powerful genetic switch that converts 2To whom correspondence should be addressed. E-mail: [email protected]. the common form of this PBP into a distinct structural form in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. which the mechanism underpinning light harvesting is vastly 1073/pnas.1402538111/-/DCSupplemental.

E2666–E2675 | PNAS | Published online June 16, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1402538111 Downloaded by guest on September 30, 2021 To gain a better understanding of the interrelation between The results of 2D electronic spectroscopy (2D ES) from all three PNAS PLUS protein structure, chromophore arrangement, quantum coher- cryptophyte PBPs are reported here. We conclude that the ence, and biological evolution, we have determined crystal struc- mechanism of light harvesting and whether effects arising from tures of three cryptophyte PBPs: phycocyanin PC645 from electronic coherence are significant depend strongly on the Chroomonas sp. (strain CCMP 270), PC612 from Hemiselmis structure. It is notable that each of these complexes harvests light virescens (strain CCAC 1635 B), and PE555 from Hemiselmis differently but apparently successfully. andersenii (strain CCMP 644) (13). The PC645 dimer has the From the protein sequences and structures, it appears that the same architecture as PE545, which we call the “closed” form, in Hemiselmis proteins with the open form differ from the crypto- which the two central β50/61 chromophores are in physical phyte closed-form proteins by the insertion of a single amino contact. In contrast, the structures from the two Hemiselmis acid in a conserved region just before the chromophore attach- species, PC612 and PE555, show a dramatically different dimer ment site in the α subunit. This insertion results in a rotation of structure in which the αβ monomers have been rotated by ∼73° part of the chromophore, and this rotation, in turn, precludes the compared with the closed form. In this open form, the central formation of the closed-form dimer, ultimately resulting in the β50/61 chromophores are separated by a water-filled channel. new, open-form dimer structure. We compare the open and BIOPHYSICS AND COMPUTATIONAL BIOLOGY

Fig. 1. Structures of the open and closed forms of cryptophyte PBPs. (A and B) Stereo cartoon diagrams of (A) the closed-form PC645 α1β.α2β dimer and (B) the open-form PC612 (αβ)2 dimer. The α chains are colored blue and red; the corresponding β chains are magenta and cyan. In PC645, α1 is blue, and α2 red. Chromophores are shown as CPK models. The view is along the quasi-twofold axis with the two doubly linked chromophores proximal to the viewer. (C) Superposition of the stereo cartoon diagrams of the αβ monomers from all available cryptophyte PBPs. The β subunits are shown in light orange; α subunits

are coded: PE545 α1, green; PE545 α2, lime green; PC645 α1, magenta; PC645 α2, purple; PE555 α1, blue; PE555 α2, cyan; PC612 α subunits (two chains in crystal structure), red and salmon. Chromophores are shown in atom colors. (D and E) Two orthogonal views of the closed-form PC645 dimer (monomers are shown in red and green) (D) and the open-form PC612 dimer (monomers shown in magenta and cyan) (E), where the 90° rotation between views is about the vertical axis in the plane of the page. (F) Electrostatic surface of PC612 dimer rotated 180° about the vertical axis in the plane of the page as compared with B.

Harrop et al. PNAS | Published online June 16, 2014 | E2667 Downloaded by guest on September 30, 2021 closed PBP structures using quantum chemical calculations and over ∼210 Cα atoms). The sequences of the β subunits are highly spectroscopic measurements. These findings open the door for conserved (79–92% identity). The sequences of the examined determining the role of quantum coherence in a real-life bi- α subunit are much more divergent, with 23–82% identity, but ological system and gaining a better understanding of how these their conformations still are very similar (rmsd of 0.8–2.6 Å). distinct light-harvesting proteins might have evolved from the elaborate ancestral phycobilisome structure. Comparison of Open- and Closed-Form Quaternary Structures. Al- though the protein structure and chromophore arrangement Results within the αβ monomer are conserved, the open and closed Crystal Structure of Phycocyanin PC645 from Chroomonas sp. The quaternary structures are radically different. The transformation crystal structure of phycocyanin PC645 from Chroomonas sp. that relates the open- and closed-form quaternary structures is CCMP270 was determined at 1.35-Å resolution (Fig. 1A, Fig. a rotation of one αβ monomer by ∼73° around an axis perpen- A α β α β S1 , and Table S1). The molecule consists of an 1 . 2 dimer in dicular to the dimer pseudo-twofold axis (Fig. 1 D and E and which each α subunit is covalently linked to a mesobiliverdin Table S2). The centers of mass of the two αβ monomers are chromophore (MBV α18), and each β subunit has a doubly slightly more separated in the open form: The center of mass-to- linked dibiliverdin chromophore, DBV β50/61, and two singly center of mass separation for the closed form was 23.4 Å for linked phycocyanobilins, PCB β82 and PCB β158 (Table 1). The PE545 and 22.2 Å for PC645, compared with 24.4 Å for the two DBV β50/61 chromophores are in van der Waals contact at open form of PC612 and 24.8 Å for the open form of PE555. the pseudo-twofold axis of the dimer where the pyrrole A rings Transitions between the two quaternary forms of the same are offset stacked (Figs. S1A and S2A). protein are unlikely to occur, and there is no evidence that The structure of the PC645 dimer is very similar to the pre- either closed- or open-form proteins are in equilibrium with viously published closed structure of phycoerythrin PE545 from a measurable monomer pool. In the closed-form dimer, the Rhodomonas CS24 (73% sequence identity; rmsd 0.85Å on 453 monomer–monomer interaction buries a substantial surface Cα atoms) (3, 4). (PE545: 1,060 Å2 per monomer; PC645: 1,230 Å2 per monomer), indicating that the dimer is very stable. In the open-form Crystal Structure of Phyocyanin PC612 from H. virescens. The crystal dimer, the monomer–monomer interaction buries a smaller but structure of phycocyanin PC612 from H. virescens CCAC 1635 still significant surface area (PE555: 618 Å2 per monomer; was determined at 1.7-Å resolution (Fig. 1B, Fig. S1B, and Table PC612: 511 Å2 per monomer). Although this result suggests S1). This light-harvesting complex also exists as an αβ.αβ dimer, that the open-form dimer may be less stable than the closed but, in contrast to PE545 and PC645, it has nearly perfect two- – fold symmetry, and the two α subunit sequences are identical. form, we see no evidence of monomer dimer equilibrium on The αβ monomers in PC612 are very similar to those observed in size-exclusion chromatography. the closed-form structures (71% overall sequence identity with The main effect of the change from the closed to the open form is the separation of the central, doubly linked β50/61 PC645; rmsd 1.11 Å on 213 Cα atoms; Fig. 1C). The positions of the chromophores in the αβ monomer are equivalent to those in chromophores. In the closed-form structures, these two chro- the closed-form structures. The only chromophore difference mophores are in van der Waals contact with the pyrrole A rings, between PC612 and PC645 is the α chromophore, which in which are offset stacked (closest approach of 3.8 Å between A A A PC612 is PCB instead of MBV (Table 1). atoms in pyrrole A rings; Fig. 1 and Figs. S1 and S2 ). Because the quaternary arrangement of the two αβ monomers However, in the open form, these two chromophores are well in PC612 is so distinct from the closed form observed in PC645 separated (closest approach between atoms in pyrrole A ring B B C and PE545 (in Fig. 1, compare A with B and D with E), we refer of 10.0 Å in PC612 and 11.0Å in PE555; Fig. 1 , Fig. S2 and , to it as the “open” form. The two αβ monomers in the PC612 and Table 2). structure form a dome or cup-like structure which contains a central cavity (Fig. 1 B and F). Dimer Interface in the Closed Form. The dimer interface interactions in the closed form are mediated by the α subunit, the α Crystal Structure of Phycoerythrin PE555 from H. andersenii. The chromophore, or the β50/61 chromophore with no direct protein– crystal structure of phycoerythrin PE555 from H. andersenii protein interactions between the two β subunits. There are CCMP 644 was determined at 1.8-Å resolution (Table S1). The three key interaction sites. First, the pyrrole A rings of the two β A structure shows a near symmetric α1β.α2β dimer that is nearly 50/61 chromophores pack against each other (Fig. S2 ). In identical to the open-state structure of PC612 (84% overall se- addition, the loop connecting helices hG and hH (the GH loop) quence identity; rmsd 0.92 Å on 464 Cα atoms). However, in of the β subunit and the C-terminal loop of α1 pack against the terms of chromophores, PE555 contains three phycoerythrobilins face of the β50/61 chromophore from the α2β monomer (Fig. (PEBs) that replace the PCBs of PC612 (Table 1). S2A). The second interaction site is centered on pyrrole rings A and B of the α chromophore. These sit in a hydrophobic pocket αβ Monomer Structure and Chromophore Arrangement Are Conserved. formed by the C-terminal tail of the opposite α subunit, the C Each αβ monomer is composed of a β subunit with a globin terminus of the opposite α subunit helix, and the loop connecting fold and an extended α subunit which lies along the β subunit helix hB to hE of the opposite β subunit (Figs. 2A and 4A). Third, (Fig. 1C). Apart from loops (particularly around the α chromo- the α subunit helix makes polar interactions with the break be- phore), there is little deviation in the αβ monomer when com- tween helices hA and hB and with helix hE in the opposite β – paring PE545, PC645, PC612, and PE555 (rmsd of 0.78 1.6 Å subunit plus the opposite α subunit (Fig. 2 B and C).

Dimer Interface in the Open Form. As in the closed form, all dimer Table 1. Chromophores interface contacts in the two open-form structures are mediated Organism Strain PBP αβ50,61 β82 β158 by either the α subunits or the α subunit chromophore with no direct interactions between the two β subunits. A major contact is Rhodomonas sp. CS24 CS24 PE545 DBV PEB PEB PEB made by the α chromophore where pyrrole ring A sits in a hy- Hemiselmis andersenii CCMP 644 PE555 PEB DBV PEB PEB drophobic pocket in the opposite αβ monomer (Fig. 2D). The Hemiselmis virescens M1635 PC612 PCB DBV PCB PCB pocket is formed by the N-terminal portions of helix hE in the β Chroomonas sp. CCMP 270 PC645 MBV DBV PCB PCB subunit and the helix in the α subunit, plus the hydrophobic

E2668 | www.pnas.org/cgi/doi/10.1073/pnas.1402538111 Harrop et al. Downloaded by guest on September 30, 2021 Table 2. Electronic couplings (unscreened) and center-to-center distances for selected phycobiliprotein pairs in PNAS PLUS PE545, PC645, PC612, and PE555 PE545 PC645 PC612 PE555

Bilin pair (interdimer) PEBβ50/61PEBβ50/61 DBVβ50/61DBVβ50/61 DBVβ50/61DBVβ50/61 DBVβ50/61DBVβ50/61 Center-to-center separation, Å 15.11 13.17 19.48 19.91 Coupling, cm−1 166 647 29 4

Bilin pair (interdimer) DBVα20PEBβ50/61 MBVα18DBVβ50/61 PCBα20DBVβ50/61 PEBα20DBVβ50/61 Center-to-center separation, Å 22.74 23.49 30.43 29.30 − Coupling, cm 1 −64 −74 −5 −8

Bilin pair (intradimer) DBVα20PEBβ158 MBVα18 PCBβ158 PCBα20PCBβ158 PEBα20PEBβ158 Center-to-center separation, Å 20.55 18.34 18.23 19.35 − Coupling, cm 1 51 151 146 68

residue that precedes the α subunit helix by two residues (Met47 with PE555; Table 2). This increased separation dramatically in PC612 α, Phe45 in PE555 α1, and Met45 in PE555 α2). weakens electronic couplings in the open-form PBPs compared The other major dimer interface interaction is centered on the with those determined for the closed structures (Table 2). In C terminus of the α subunit α helix, which makes polar contacts particular, the very strong Coulombic interaction calculated with the GH loop in the opposite β subunit (Fig. 2 E and F). within the central β50/61 bilin pair of the closed-form structures − − Additionally, in the PE555 structure, the C-terminal residue of of PC645 (647 cm 1) and PE545 (166 cm 1) is absent in the −1 −1 α2, Leu62, makes van der Waals contacts with the C-terminal tail open-form PBPs, PC612 (29 cm ) and PE555 (4 cm ) (Table of α1 and the GH loop of the opposite β subunit (Fig. 2F and 2). Previous work has shown that the strong couplings calculated Fig. S2C). This interaction is adjacent to the polar interface for the closed-form PC645 and PE545 are consistent with spec- mentioned above. troscopic data (15, 16). Steady-state spectra for the PBPs are documented in Fig. S3. These data include circular dichroism Sequence and Structural Changes Around the α Chromophore Dictate spectra, which are good experimental indicators of exciton Quaternary Structure. Given the structural conservation of the αβ interactions. However, the primary derivative-like feature in the dimer, it is not immediately obvious why the two Hemiselmis PBP spectra comes from subtle interactions among the periph- PBPs assemble in the open-form dimer rather than the closed eral chromophores, not the central dimer, and therefore is form. An examination of the sequences of the α subunits shows present in spectra of both the closed and open forms of the that in the Hemiselmis PBPs an aspartic acid (Asp18) has been protein. A decrease is observed for all interdimer αβ–αβ cou- inserted in the highly conserved FDxRGC motif that links the plings, whereas the intradimer αβ couplings are relatively un- first β strand to the α chromophore attachment site (Fig. 3C). In affected by the different quaternary structure (Table 2 and the closed-form structures, this motif forms a network of hy- Table S4). drogen bonds that determine the orientation of pyrrole ring A in the α chromophore with respect to the β sheet (Fig. 3A). The Spectroscopy of the Closed-State PC645 Versus the Open-State PC612. insertion of an aspartic acid in the Hemiselmis α subunits alters The general features of the absorption spectra of PC645, PC612, the hydrogen bonding network (Fig. 3B). The net effect of this and PE555 (Fig. 5 A–C) are consistent with the chromophore change is that the plane of pyrrole ring A with respect to pyrrole compositions (Table 1). Previous modeling of the spectroscopy ring B in the α chromophore is rotated in a counterclockwise of PC645 suggests a model for the absorption band positions (the fashion by ∼29° in the closed form, whereas it is rotated in a center positions of absorptions indicated in Fig. 5D) (16). The

clockwise fashion by ∼40° in the open form (Table S3). Thus, three different chromophore types (DBV, MBV, and PCB) pro- BIOPHYSICS AND

the sequence insertion results in an ∼69° rotation in pyrrole vide the primary spectral broadening and establish an energy COMPUTATIONAL BIOLOGY ring A of the open-form αβ monomer as compared with the funnel from the core to periphery of the complex (17). PC612 is closed form. similar to PC645 in that its absorption spectrum is broad, with How does the rotation of pyrrole ring A in the α subunit two distinct peaks, and absorbs in the same region, but it does chromophore determine the quaternary structure? In both open not contain MBV chromophores (Table 1). Based on the relative and closed forms, pyrrole ring A of the α chromophore makes absorption energies of the individual bilins and the assignment a major contribution to the dimer interface (see Figs. 2A and 4A of chromophore absorption energies in PC645, we expect that for the closed form and Fig. 2D for the open form). Superposi- the higher energy peak is dominated by DBVs and that the lower tion of the open-form αβ monomer on a closed-form quaternary energy peak is dominated by PCBs (Fig. 5E) (18). structure shows that the rotation of pyrrole ring A results in Two types of spectral shift are evident in the PC645 spectrum. a steric clash with conserved Pro64 in the opposite β subunit First, the DBV chromophores are positioned closely in the (Fig. 4B). Thus, the open-form αβ monomer is prevented from closed-form crystal structure and are particularly strongly adopting the closed-form dimer structure. coupled (Table 2). This electronic coupling splits the DBV absorption bands into the two exciton states labeled DBV(+) Quantum Chemical Calculations Reveal Excitonic Switching. Using and DBV(−). Second, the degeneracy of the PCB absorption the high-resolution crystal structures of the PBPs, we calculated bands is broken. Atomistic modeling of PE545 indicates that the gas-phase couplings (Table S4) and transition dipole mo- spectral shifts are caused mainly by perturbations of the ments (Table S5). Note that these electronic couplings do not chromophore conformation with smaller effects caused by include dielectric screening effects that tend to reduce their electrostatic interactions with the local protein environment magnitude by a factor of about two (14). For the central, doubly (15). Neither of these two spectral broadening features linked β50/61 chromophores, the center-to-center separation appears to be evident in the PC612 spectrum. For example, increases when comparing closed and open-form PBPs by 6.3Å the absorption spectrum recorded with the sample at 77 K in the phycocyanin proteins (compare PC645 with PC612; Table (Fig. 5B) clearly reveals only two bands and a vibronic tail on 2) and by 4.8Å in the phycoerythrin proteins (compare PE545 the blue side of the spectrum. Exciton splitting is absent in the

Harrop et al. PNAS | Published online June 16, 2014 | E2669 Downloaded by guest on September 30, 2021 Fig. 2. Dimer contacts in the closed- and open-form structures. (A–C) Dimer contacts in the closed-form PC645 structure. (D–F) Dimer contacts in the open- form structures of PC612 (D and E) and PE555 (F). (A) Pyrrole rings A and B of the PC645 α chromophore (green carbon atoms) lie in a hydrophobic pocket formed by the C terminus of the neighboring α subunit (red cartoon) and the loop between helices hB and hE in the neighboring β subunit (cyan cartoon). (B) Helix from PC645 α1 (blue) makes polar side-chain contacts with residues from the opposite αβ monomer [β subunit helices hA, hB, and hE (cyan cartoon) and the α2 linker between β strand s2 and helix (red cartoon)]. Residues from the α1 subunit are labeled in black; residues from the neighboring αβ monomer are labeled in red and blue, respectively. (C) Helix from PC645 α2 (red) makes similar polar contacts with the opposite αβ monomer (hA and hB, magenta, and α1, blue). Residues from α2 are labeled in black; residues from the opposite αβ monomer are labeled in blue and magenta, respectively. (D) The open-form PC612 showing the hydrophobic contact between pyrrole ring A of the α chromophore (green) and the pocket in the opposite αβ monomer formed by β subunit helix hE (cyan) and the N terminus of the α subunit helix (red). (E) PC612 showing dimer interface between the C terminus of the α subunit helix (blue) and the GH loop from the opposite β subunit (cyan). Residues from the α subunit are labeled in black; residues from the β subunit are labeled in cyan. (F) The same interaction in PE555 between the helix from α2 (red) and the opposite GH loop (magenta). Residues from the α2 subunit are labeled in black; residues from the β subunit are labeled in magenta. Hydrogen bonds/salt links are shown as dotted lines.

open-form structure of PC612 (Table 2) because the doubly PC645 has proven to be a remarkable system because of the linked β50/61 DBV chromophores are widely separated. clear coherent oscillations seen in cross-peaks in the 2D ES as

E2670 | www.pnas.org/cgi/doi/10.1073/pnas.1402538111 Harrop et al. Downloaded by guest on September 30, 2021 PNAS PLUS

Fig. 3. Insertion of an aspartic acid in the open-form α subunit results in the rotation of pyrrole ring A in the α chromophore as compared with the closed form. (A) Stereo view of the α chromophore pocket of the closed-form PC645 showing the hydrogen bonding network. (B) Identical view showing the open- form PC612 pocket. (C) Structure-based alignment of all mature α subunit sequences reported in this paper. The red arrow indicates the chromophore attachment site; the blue arrow indicates Glu16 that coordinates the central pyrrole nitrogens in the open-form structures. Red type indicates identity; blue type indicates similarity. Breaks in the alignment mark the ends of secondary structure elements. Note that the helix length is variable at its C terminus.

a function of pump-probe waiting time (10, 19, 20). Comple- mates of the absorption energies. Based on the relative site en- mentary nonlinear experiments also have identified these co- ergies of the PEBs and DBVs in PE545, the higher-energy herences (21, 22). A representative total, real 2D ES spectrum is shoulder is attributed to the PEBs, and the lower-energy, main shown in Fig. 5D. The spectrum shows numerous features of absorption band is attributed to DBVs. This ordering agrees with interest, most distinctly an off-diagonal cross-peak (located at previous assignment (Fig. 5F) (23, 24). The narrow, congested excitation wavelength 570 nm and signal wavelength 600 nm in absorption spectrum suggests that the absorption bands of in- Fig. 5D) that oscillates strongly as a function of the waiting time dividual chromophores overlap significantly and that exciton (Fig. 5G) (10). An extensive analysis of the oscillations has been splitting and energy shifts caused by different bilin conformations BIOPHYSICS AND reported (19). Using a procedure involving the separation of the are minimal. As in PC612, this observation is consistent with the COMPUTATIONAL BIOLOGY total spectrum into its rephasing and nonrephasing components idea that the open-form complex is more symmetric than the (20), we concluded that the oscillations involve both vibrational closed-form complex. The 2D ES total, real spectrum (Fig. 5F) and electronic coherences (19). corroborates this expectation, appearing qualitatively similar to Unlike PC645, little is known about the photophysics of the that recorded for PC612. Oscillations in the amplitude were open-form PBPs PC612 and PE555. For PC612, we specifically weak compared with the other complexes (Fig. 5I;seethetrace photoexcited the DBV states to compare the 2D ES measure- for excitation wavelength 540 nm and signal wavelength 560 nm in ments directly with those of PC645. The 2D ES total, real Fig. 5F) and appear most consistent with vibrational coherences. spectrum (Fig. 5E) shows a rectangular feature, centered at the DBV bleach, suggesting substantial coupling between vibronic Discussion transitions throughout this spectral region; however, a striking Crystallographic analyses reveal two very different quaternary oscillating cross-peak like that noted for PC645 is not evident in structures that are adopted by distinct cryptophyte PBPs: the these data. In Fig. 5H, a trace at an off-diagonal position (exci- open and the closed forms. To date, both open-form PBPs come tation wavelength 550 nm and signal wavelength 600 nm in Fig. E from Hemiselmis spp., whereas the two closed-form PBPs come 5 ) shows damped oscillations that have frequencies consistent αβ with the vibrational beats (19). from two distinct cryptophyte subclades (25). The monomer structure is conserved in all cryptophyte PBP structures that have Spectroscopy of the Open-State PE555. In contrast to PC645 and been determined. This structural conservation includes the ar- PC612, the PE555 absorption spectrum is narrow and nearly rangement of chromophores in both α and β subunits. The in- featureless, showing minimal change between the room tem- terface between the two αβ monomers forming the dimer depends perature and 77-K spectra (Fig. 5C). The presence of two distinct mainly on contacts between α subunits and particularly the α types of chromophores in PE555 (singly bonded PEBs and subunit chromophore, which makes key contacts across the di- doubly bonded DBVs) suggests that there could be at least two mer interface. Thus, the α subunit mediates dimer formation and distinct absorption energies. By fitting the room temperature and hence determines the quaternary structure of the cryptophyte 77-K absorption spectra using two Gaussians, we obtained esti- light-harvesting protein.

Harrop et al. PNAS | Published online June 16, 2014 | E2671 Downloaded by guest on September 30, 2021 structure, where these two chromophores are separated by a water- filled central channel, reducing the excitonic coupling (Table 2). The common chromophore across each of the PBPs in- vestigated in this work is DBV, in each case the doubly linked β50/61 chromophore. Consequently, strong electronic coupling among the central bilins is the key distinguishing feature of the closed- versus open-form PBPs with respect to light harvesting. Spectral shifts resulting from a combination of local environment and conformational effects extend the PC645 absorption ∼25 nm further to the red than that of PC612. PC645 further incorpo- rates MBV chromophores to absorb at ∼600 nm. In comparison, the absorption cross-section of PE555 is unusually narrow, about half the spectral width of PC645. The great diversity of solutions to light harvesting in the cryptophyte algae, in particular the combination of different chromophores and significantly different structural combinations, are quite extraordinary. The ancestral cryptophyte alga acquired its chloroplast by engulfing and taming a red algal endosymbiont, which would have had at least a primitive phycobilisome (7, 24). Extant cya- nobacterial and red algal phycobilisomes are complex structures made up of stacked rods of several types of trimeric PBPs (αβ)3 rings attached to the stromal surface of the thylakoid membrane (26), however, the phycobilisome α subunits are globin proteins that are completely unrelated to the cryptophyte α subunits. At some point during the integration of the red algal plastid, the phycobilisome structure disappeared, and the original globin- fold α subunit was replaced by an unrelated polypeptide of un- known evolutionary origin and was retargeted to the thylakoid lumen (2). This replacement resulted in the first radical rearrangement in quaternary structure: the formation of a crypto- Fig. 4. Rotation of pyrrole ring A in the open-form α chromophore pre- phyte progenitor (αβ)2 dimer (4). Here we report that among cludes formation of the closed form via a steric clash. (A) Stereo view of the the cryptophytes there are two radically different forms of the packing interaction between pyrrole ring A and the opposite αβ monomer in α1β.α2β dimer, the open-form dimer, which appears to be con- the closed-form PC645. (B) Model of PE555 in which the two αβ monomers Hemiselmis have been rotated so that they overlay the closed-form structure of PC645. fined to the lineage, and the closed form. The emer- Pyrrole ring A of the α subunit chromophore (green stick model) is rotated gence of two forms appears to have been caused by a single by ∼70° compared with that of PC645, and this rotation results in a steric insertional/deletional mutation in the new cryptophyte α subunit. clash with conserved Pro64 in the opposite β subunit. A central biological question is whether the presence of long- lived electronic coherence in the light-harvesting proteins results in a selective advantage for the algae—for example, is coherence Sequence and structural analysis shows that the insertion of an important for efficient light harvesting? If the emergence of long- aspartic acid residue just before the covalent chromophore at- lived electronic coherence gives cryptophytes containing the tachment site (Cys20) in the Hemiselmis α subunits results in closed-form PBP a selective advantage over the ancestral cyano- a rotation of the first pyrrole ring A of the α chromophore by bacteria and red algae, it would seem that the Hemiselmis cryp- ∼69° compared with the closed form. This rotation precludes the tophytes, with their open-form PBPs, have lost this advantage. Our assembly of the Hemiselmis αβ monomers into the closed-form results suggest that successful light harvesting can be achieved in dimer, because such assembly would result in a severe steric diverse ways, with or without coherent molecular excitons delo- clash. It appears that the observed open form is the next avail- calized over pairs of chromophores. Nevertheless, it is apparent able dimeric state in terms of minimizing free energy. The buried that the excitonic interactions in the PBPs are switched profoundly surface area between monomers in the open-form dimer is ap- (over an order of magnitude) by the structural change from open proximately half that observed for closed-form dimers, making the to closed and that this exciton switch is genetically controlled. open form a less stable structure. However, we note that we have observed only dimers with no evidence of free αβ monomers. Materials and Methods The closed-form cryptophyte PBP dimers are clearly asym- Growth of Cryptophytes. Chroomonas sp. Strain CCMP 270 (Provasoli-Guillard metric in structure and α subunit sequence, with the long α National Center for Marine Algae and Microbiota, Bigelow Laboratory for – subunit providing a C-terminal extension that mimics the loop Ocean Sciences) was cultured in modified Fe medium (27) at 22 24 °C under β continuous aeration with a 12-h light/12-h dark cycle at a light intensity of between helices G and H in the subunit and interacts with the – · −2· −1 × 8 – β 80 100 mE m s . When cultures reached a density of 5 10 cells/L (4 6 doubly linked 50/61 chromophore (4). In contrast, the open- wk), cells were harvested by centrifugation. The pellet was resuspended in form Hemiselmis PBP dimers are nearly symmetric in structure 25 mM phosphate buffer (pH 7) and was stored at −80 °C. and sequence. Only minor structural differences are observed in H. andersenii. CCMP 644 (Provasoli-Guillard National Center for Marine Algae PE555, and no significant differences are observed in PC612. and Microbiota, Bigelow Laboratory for Ocean Sciences) was grown in GSe The key difference between the two quaternary forms in terms of medium (28) at 22–24 °C under a 12-h light/12-h dark cycle at a light intensity −2 −1 chromophore arrangement is the van der Waals contact between of 80–100 μE·m ·s . Cells were harvested after 4–6 wk by centrifugation − the two doubly linked β50/61 chromophores on the pseudo-twofold and were stored at 80 °C. H. virescens. CCAC 1635B (Culture Collection of Algae at the University of axis of the closed-form PBPs. This arrangement is unique to closed- Cologne) cultures were grown in aerated ASP-H medium (29, 30) at 16 °C form cryptophyte PBPs, and it results in the strong excitonic cou- under a 14-h light/10-h dark cycle with light intensities of ∼50 μmol pho- − − pling of these two chromophores (Table 2). This pairing of chro- tons·m 2·s 1. Cultures were harvested by flow-through centrifugation and mophores is completely disrupted in the open-form quaternary were stored at−80 °C.

E2672 | www.pnas.org/cgi/doi/10.1073/pnas.1402538111 Harrop et al. Downloaded by guest on September 30, 2021 PNAS PLUS

Fig. 5. Electronic spectroscopy of the closed- and open-form PBPs. (A–C) Electronic absorption spectra of closed-form PC645 (A) and open-form PC612 (B)and PE555 (C) PBPs. Spectra were recorded at 295 K (red trace) and 77 K (blue trace). (D–F) Representative 2D electronic spectra at 295 K for the closed-form PC645 = = =

(D, at waiting time T 55 fs), the open-form PC612 (E, at waiting time T 100 fs) and the open-form PE555 (F, at waiting time T 100 fs). The spectra are the BIOPHYSICS AND real part of the total signal, plotted with 33 evenly spaced contours. The estimated exciton energies of the chromophores are plotted on the 295-K absorption spectra which are superimposed onto the excitation and emission axes. (G–I) Magnitude of the 2D ES amplitude at selected cross-peaks as a function of COMPUTATIONAL BIOLOGY waiting time taken as a trace from the absolute value 2D ES spectra (the first 15 fs are omitted to avoid possible nonresonant solvent response). Cross-peak coordinates (excitation, detection) in nanometers are approximately PC645: (570, 600), PC612: (550, 600), and PE555 (540, 560). Error bars indicate one SD as determined from three trials for PC645 and PC612 and six trials for PE555.

Protein Purification. Algal cell pellets were thawed, resuspended in two to Data Collection. Crystals were transferred to the cryoprotectant solution of three volumes of 25 mM phosphate buffer (pH 7), and homogenized with reservoir plus 15% glycerol and then were flash-cooled in liquid nitrogen a Teflon glass homogenizer at 30 rpm. Cells were disrupted in a French press and mounted in a Cryostream cooler (Oxford Cryosystems) for data col- at 1,000 psi and centrifuged at 23,000 × g for 1 h at 4 °C. The supernatant was lection: PC645 on beamline 9.2, Stanford Synchrotron Radiation Light- purified via ammonium sulfate cuts (0–50%, 50–60%, 60–70%, and 70–80%) source; PE555 on beamline 23ID-D, Advanced Photon Source; and PC612 by adding solid ammonium sulfate, stirring for 1 h at 4 °C, and centrifuging om beamline MX1, Australian Synchrotron (Table S1). Diffraction images at 23,000 × g for 30 min at 4 °C. The 70–80% pellets were resuspended in for PC645 and PE555 were collected on a MarCCD (Rayonix) detector, and 25 mM phosphate buffer (pH 7), filtered, dialyzed against the same buffer, diffraction images for PC612 were collected on an ADSC Q210 (Area De- and loaded onto a Q Sepharose HiLoad 26/10 anion exchange column (GE tector Systems Corporation) detector. Data collection was carried out using Healthcare). The fractions containing the majority of the light-harvesting Blu-Ice (31). protein were selected using the absorbance at 280 nm and concentrated on a 10-kDa Centriprep (Millipore). The protein was purified by size-exclusion Data Reduction and Structure Determination. All data were processed using chromatography using a Superdex 200 HiLoad 26/60 column (GE Health- XDS (32) and SCALA [CCP4 (33)]. Phasing, auto building, and refinement care). Proteins eluted as a single peak and were concentrated using a 10-kDa were carried out using PHENIX (34). A single β subunit from the structure of cutoff (Centriprep; Millipore) before snap freezing and storage at −80 °C. PE545 (3) was used as a molecular replacement probe using PHASER (35) as implemented in PHENIX. Manual adjustments were carried out using COOT Crystallization. The proteins were crystallized using vapor diffusion under the (36). Structural figures were created using PYMOL (37). following conditions: PC645, 20% PEG 4 k, 20% isopropanol, and 0.1 M

sodium citrate (pH 5.6); PE555, 20% PEG 10 k in 0.1 M Hepes (pH 7.5); and Structure of PC645. The PC645 structure contains one α1β.α2β dimer in the PC612, 25% PEG 3,350 in 0.1 M Hepes (pH 7.5). asymmetric unit. The complete α1 molecule is visible in the electron density

Harrop et al. PNAS | Published online June 16, 2014 | E2673 Downloaded by guest on September 30, 2021 69 70 map, whereas the last two residues of α2 (Lys –Lys ) are disordered. The 2D Electronic Spectroscopy. A 5-kHz Ti:sapphire amplified laser system pro- C-terminal residue of each β subunit (Ala177) is disordered. The first 14 res- duces 150-fs pulses centered at 800 nm with ∼0.6 mJ. About 0.15 mJ seeds idues of the β subunit (helix hX) are absent in the electron density, their a noncollinear optical parametric amplifier (NOPA), producing pulses with position being occupied by an ordered PEG molecule that extends across a bandwidth of 60 nm (spectral intensity FWHM) centered in the Vis range, a crystallographic twofold axis. The only modified residue is Asn72 in the β nearly free from angular dispersion. A combination of a folded 4-f grating subunit where the side-chain nitrogen is methylated (5). compressor and a single-prism prism compressor is used to compress the pulse from the NOPA. Pulse compression is determined by measuring the αβ Structure of PC612. The PC612 structure contains a single ( )2 homodimer in nonresonant third-order response from methanol in the sample position. the asymmetric unit. Electron density is seen for the complete α subunits The pulse duration, central wavelength, and bandwidth used for each pro- β β and one of the subunits. In the second subunit, electron density starts at tein sample are summarized in Table S6. The beam is attenuated by the Asp3 and continues to the C terminus. combination of a broadband half-waveplate and a 0.7-mm-thick wire-grid polarizer before entering the four-wave-mixing setup (20). α β α β Structure of PE555. The PE555 structure has three copies of the ( 1 ).( 2 ) A spherical mirror with a 50-cm focal length focuses the beam on a 2D cross- heterodimer in the asymmetric unit. The first heterodimer shows clear hatched phase mask (UV fused silica substrate). The four first-order diffraction α electron density for the full 2 subunit and all but the last residue (Val67) of beams, each about 12% of the input power, are arranged in the BOXCARS α . The β chain associated with α is complete, whereas the remaining β chain 1 2 configuration and are directed by a small steering mirror toward the mirror starts at Asp3. The heterodimer structure is nearly symmetric. with a 50-cm focal length. The steering mirror allows us to use the large The other two copies of the heterodimer appear to be superpositions of an spherical mirror at an angle of exactly 0°. The large spherical mirror collimates (α β).(α β) dimer with an (α β).(α β) dimer (i.e., rotation of 180° about the 1 2 2 1 and makes parallel the four beams, which then pass above, below, and to the pseudo-twofold axis). In each α subunit, electron density is seen for both sides of the steering mirror. Three of the beams traverse antiparallel pairs of possible C-terminal regions [as seen in the asymmetric (α1β).(α2β) structure]. 1°, uncoated UV fused silica glass wedges for pulse delays. One wedge from There are five residues within the first 60 that distinguish α1 from α2. These residues show density (or lack of density) for both possible α chains, in each pair is mounted on a computer-controlled delay stage allowing a step accuracy of ∼850 zeptoseconds. The pulse that serves as the final excitation particular α1Phe45/α2Met45. field interaction is chopped at a frequency of 25 Hz; the chopper also triggers Sequence Determination for H. virescens PC612, Chroomonas sp. CCMP270, and the detector, which acquires signal for 20 ms (100 laser shots). The fourth 4 H. andersenii CCMP644. Algal cells were grown to exponential stage at 20°, beam, the local oscillator (LO), is attenuated by 10 and interacts with the − − ∼ 20 μmol photons·m 2·s 1 on a 12-h light/12-h dark cycle. RNA was isolated sample 250 fs before the final excitation field, thus limiting its use to a ref- using RNAqueous 4 PCR (Ambion) or Total RNA Isolation Reagent (Advanced erence field. The four pulses then encounter a second spherical mirror used at Biotechnologies). cDNA was generated using SuperScript III Reverse Tran- 0° but with a focal length of 20 cm. The pulses focus and cross in the sample scriptase (Invitrogen Life Technologies) at 50 °C with random hexamer/ plane (beam waist diameter ∼50 μm) after encountering another small nonamer or oligo (dT) as primers and was used for degenerate PCR. The β steering mirror. The signal and LO are collimated by a curved mirror with a 20- subunit degenerate primers were designed based on the alignment of DNA cm focal length and are directed to an imaging spectrometer (f = 16.3 cm) sequences from cryptophytes Guillardia theta and Rhodomonas salina and coupled to a CCD detector with 1,024 pixels in the dispersed dimension. The all the red algal β subunit sequences in GenBank. The α subunit degenerate spectrometer is calibrated using an Hg/Ar lamp. The phase stability of the primer pairs were based on Edman N-terminal sequencing (CCMP270) or on apparatus is λ/350 short term (5 min) and λ/200 long term (2 h). All 2D ES the best partial amino acid sequences derived from electron-density maps. experiments are performed with pulse energies of ≤5 nJ. The coherence time

PCR products were cloned into T-vectors, and isolated colonies were selected (τ1) was scanned in intervals of 0.2 fs (0.15 fs) from −45 to 45 fs. The waiting

randomly for sequencing. The resulting sequences were used to design time (τ2)rangedfrom0–400 fs in 5-fs steps for PC645 and PC612 measure- outward-directed PCR primer pairs for cDNA-based inverse PCR according to ments; the PE555 measurement was monitored at 10-fs intervals. The meas- Huang and Chen (38). For the complete α subunit sequences, 5′ RACE was urements were conducted at 298 K, and the sample was flowed using done with the FirstChoice RLM -RACE kit (Ambion) or ExactSTART kit (Epi- a peristaltic pump at a flow rate that guaranteed a fresh spot at each pulse. ′ β centre). For the 3 end of the subunit, genomic DNA-based inverse PCR was Protein samples were stored at −75 °C until required and then were performed. The assembled sequences were confirmed by PCR from the start thawed and diluted in a suitable buffer to the appropriate OD for spec- codon to beyond the stop codon using specific nondegenerate primers. troscopic measurements (ODλ max <0.4).

Quantum Chemical Calculations. The initial conformations of the phycobilins ACKNOWLEDGMENTS. We thank Michael Melkonian for giving K.H.-E. ac- were extracted from the Protein Data Bank file with the covalently bound cess to the Melkonian group’s research facilities. This research was under- cysteine residue. The cysteine residues were capped with an acetyl and taken on the MX1 beamline at the Australian Synchrotron, Victoria, N-methyl amino group. Each of the tetrapyrrole nitrogens was protonated Australia. The access to major research facilities program is supported by (resulting in a +1 charge), and the two solvent-exposed carbocyclic acid the Commonwealth of Australia under the International Science Linkages chains were deprotonated (resulting in a −2 charge) with an overall mo- program. Portions of this research were carried out at the Stanford Synchro- lecular charge of −1 on all phycobilins. The molecular charge also is consis- tron Radiation Lightsource, a Directorate of the Stanford Linear Accelerator tent with polarizable continuum pK calculations implemented using the Center National Accelerator Laboratory and an Office of Science User Facility a operated for the US Department of Energy (DOE) Office of Science by Stan- universal solvation model designated for solvation model density (39). Hy- ford University. The Stanford Synchrotron Radiation Lightsource Structural drogen atoms were added and optimized using b3lyp/cc-pvtz, followed Molecular Biology Program is supported by the DOE Office of Biological and by a bond-length optimization with dihedral angles restrained using the Environmental Research and by the National Institutes of Health, National Gaussian 09 software package (Gaussian, Inc.). Institute of General Medical Sciences (including P41GM103393). Use of the The phycobilin transition density from the S0 to the S1 state was obtained Advanced Photon Source, an Office of Science User Facility operated for the from a configuration interaction singles (CIS)/cc-pvtz calculation, again using US DOE Office of Science by Argonne National Laboratory, was supported by the Gaussian 09 software package. Transition density cubes were inspected the US DOE under Contract DE-AC02-06CH11357. This work was supported visually to ensure the proper excited state was probed. The gas-phase cou- by grants from the Australian Research Council. G.D.S. and B.R.G. received support from Defense Advanced Research Projects Agency (Quantum Effects plings were computed from the transition densities using the methodology in Biological Environments) and the Natural Sciences and Engineering outlined by Krueger et al. (40). No scaling of the couplings was used to Research Council of Canada. E.C. received financial support from the correct for the overall overestimation of the transition dipole moments by European Research Council (ERC) under the European Community’sSev- the CIS because they were overestimated by only ∼9% compared with the enth Framework Programme (FP7/2007-2013) with the ERC Starting Grant experimental value of 2.34 eÅ (41). QUENTRHEL (Grant Agreement 278560).

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