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Single-residue insertion switches the quaternary structure and exciton states of cryptophyte light-harvesting proteins Stephen J. Harrop a,1 , Krystyna E. Wilk a,1 , Rayomond Dinshaw b , Elisabetta Collini c , Tihana Mirkovic b , Chang Ying Teng d , Daniel G. Oblinsky b , Beverley R. Green d , Kerstin Hoef-Emden e , Roger G. Hiller f , Gregory D. Scholes b , and Paul M. G. Curmi a,g,2 a School of Physics, The University of New South Wales, Sydney, NSW 2052, Australia; b Department of Chemistry, University of Toronto, Toronto, ON, Canada M5S 3H6; c Department of Chemical Sciences, University of Padua, 35131 Padua, Italy; d Department of Botany, University of British Columbia, Vancouver, BC, Canada V6T 1Z4; e Botanical Institute, Cologne Biocenter, University of Cologne, 50674 Cologne, Germany; f Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia; and g Centre for Applied Medical Research, St Vincents 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 (2D ES) of photosynthetic proteins has led researchers to ask whether nontrivial quantum phenomena are biologically sig- nificant. Coherent oscillations have been reported for the soluble light-harvesting phycobiliprotein (PBP) antenna isolated from cryptophyte algae. To probe the link between spectral properties and protein structure, we determined crystal structures of three PBP light-harvesting complexes isolated from different species. Each PBP is a dimer of αβ subunits in which the structure of the αβ monomer is conserved. However, we discovered two dramatically distinct quaternary conformations, one of which is specific to the genus Hemiselmis. Because of steric effects emerging from the insertion of a single amino acid, the two αβ monomers are rotated by 73° to an openconfiguration in contrast to the closedconfiguration of other cryptophyte PBPs. This structural change is significant for the light-harvesting function because it disrupts the strong excitonic coupling between two central chromophores in the closed form. The 2D ES show marked cross-peak oscillations assigned to electronic and vibrational coherences in the closed- form PC645. However, such features appear to be reduced, or perhaps absent, in the open structures. Thus cryptophytes have evolved a structural switch controlled by an amino acid insertion to modulate excitonic interactions and therefore the mechanisms used for light harvesting. X-ray crystallography | quantum coherence | protein evolution | excitonic switching L ight-harvesting complexes capture and funnel the energy from light using organic chromophore molecules that are bound to scaffolding proteins. The protein structure thereby sets the relative positions and orientations of the chromophores to control excitation transport. In other words, the protein plays a deciding role in building the electronic Hamiltonian”—the electronic coupling between chromophores and the chromo- phoric energy landscape that directs energy flow. This strong connection between structural biology and physics means that ultrafast light-harvesting functions are under genetic and evolu- tionary control. Cryptophytes, a group of marine and freshwater single-celled algae, are an intriguing example, because one of their light-harvesting antenna complexes was completely re- engineered by combining a unique bilin-binding polypeptide with a single subunit from the ancestral red algal phycobilisome (1, 2). Here we report a further example of biological manipulation of this phycobiliprotein (PBP) light-harvesting system. We have discovered an elegant but powerful genetic switch that converts the common form of this PBP into a distinct structural form in which the mechanism underpinning light harvesting is vastly differentin essence because strong excitonic interactions within the PBP are switched from on to off. The crystal structure of the cryptophyte PBP phycoerythrin PE545 from Rhodomonas CS24 showed that the protein is a di- mer of two αβ monomers (3, 4), the β subunit of which has a globin fold (5, 6) and binds three linear tetrapyrroles (bilins), whereas the α subunit is a short, extended polypeptide with a single bilin chromophore. A prominent feature of this structure is the arrangement of the two central chromophores in van der Waals contact with each other on the pseudo-twofold axis, with each chromophore covalently linked to two cysteines on one of the β subunits (referred to as β50/61). This structural feature introduces excitonic coupling between the chromophores (3, 4). We are fascinated by this observation because it implies that if coherence plays a nontrivial role in light harvesting (712), it might be switched on and off (either dynamically or genetically) by controlling the separation, and hence excitonic coupling, be- tween these two central chromophores. Significance There is intense interest in determining whether coherent quantum processes play a nontrivial role in biology. This in- terest was sparked by the discovery of long-lived oscillations in 2D electronic spectra of photosynthetic proteins, including the phycobiliproteins (PBPs) from cryptophyte algae. Using X-ray crystallography, we show that cryptophyte PBPs adopt one of two quaternary structures, open or closed. The key feature of the closed form is the juxtaposition of two central chromo- phores resulting in excitonic coupling. The switch between forms is ascribed to the insertion of a single amino acid in the open-form proteins. Thus, PBP quaternary structure controls excitonic coupling and the mechanism of light harvesting. Comparing organisms with these two distinct proteins will reveal the role of quantum coherence in photosynthesis. 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., G.D.S., and P.M.G.C. designed research, performed research, analyzed data, and wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: Atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4LMS, 4LM6, and 4LMX), and DNA se- quences have been deposited in the GenBank database (accession nos. KC905456KC905459 and KF314689KF314696). 1 S.J.H. and K.E.W. contributed equally to this work. 2 To whom correspondence should be addressed. E-mail: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1402538111/-/DCSupplemental. E2666E2675 | PNAS | Published online June 16, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1402538111

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Single-residue insertion switches the quaternarystructure and exciton states of cryptophytelight-harvesting proteinsStephen 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, CanadaM5S 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, MacquarieUniversity, 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 forreview February 10, 2014)

Observation of coherent oscillations in the 2D electronic spectra(2D ES) of photosynthetic proteins has led researchers to askwhether nontrivial quantum phenomena are biologically sig-nificant. Coherent oscillations have been reported for the solublelight-harvesting phycobiliprotein (PBP) antenna isolated fromcryptophyte algae. To probe the link between spectral propertiesand protein structure, we determined crystal structures of threePBP light-harvesting complexes isolated from different species.Each PBP is a dimer of αβ subunits in which the structure of the αβmonomer is conserved. However, we discovered two dramaticallydistinct quaternary conformations, one of which is specific to thegenus Hemiselmis. Because of steric effects emerging from theinsertion of a single amino acid, the two αβ monomers are rotatedby ∼73° to an “open” configuration in contrast to the “closed”configuration of other cryptophyte PBPs. This structural changeis significant for the light-harvesting function because it disruptsthe strong excitonic coupling between two central chromophoresin the closed form. The 2D ES show marked cross-peak oscillationsassigned to electronic and vibrational coherences in the closed-form PC645. However, such features appear to be reduced, orperhaps absent, in the open structures. Thus cryptophytes haveevolved a structural switch controlled by an amino acid insertionto modulate excitonic interactions and therefore the mechanismsused for light harvesting.

X-ray crystallography | quantum coherence | protein evolution |excitonic switching

Light-harvesting complexes capture and funnel the energyfrom light using organic chromophore molecules that are

bound to scaffolding proteins. The protein structure thereby setsthe relative positions and orientations of the chromophores tocontrol excitation transport. In other words, the protein playsa deciding role in building the “electronic Hamiltonian”—theelectronic coupling between chromophores and the chromo-phoric energy landscape that directs energy flow. This strongconnection between structural biology and physics means thatultrafast light-harvesting functions are under genetic and evolu-tionary control. Cryptophytes, a group of marine and freshwatersingle-celled algae, are an intriguing example, because one oftheir light-harvesting antenna complexes was completely re-engineered by combining a unique bilin-binding polypeptide witha single subunit from the ancestral red algal phycobilisome (1, 2).Here we report a further example of biological manipulation ofthis phycobiliprotein (PBP) light-harvesting system. We havediscovered an elegant but powerful genetic switch that convertsthe common form of this PBP into a distinct structural form inwhich the mechanism underpinning light harvesting is vastly

different—in essence because strong excitonic interactions withinthe PBP are switched from on to off.The crystal structure of the cryptophyte PBP phycoerythrin

PE545 from Rhodomonas CS24 showed that the protein is a di-mer of two αβ monomers (3, 4), the β subunit of which hasa globin fold (5, 6) and binds three linear tetrapyrroles (bilins),whereas the α subunit is a short, extended polypeptide witha single bilin chromophore. A prominent feature of this structureis the arrangement of the two central chromophores in van derWaals contact with each other on the pseudo-twofold axis, witheach chromophore covalently linked to two cysteines on one ofthe β subunits (referred to as “β50/61”). This structural featureintroduces excitonic coupling between the chromophores (3, 4).We are fascinated by this observation because it implies that ifcoherence plays a nontrivial role in light harvesting (7–12), itmight be switched on and off (either dynamically or genetically)by controlling the separation, and hence excitonic coupling, be-tween these two central chromophores.

Significance

There is intense interest in determining whether coherentquantum processes play a nontrivial role in biology. This in-terest was sparked by the discovery of long-lived oscillations in2D electronic spectra of photosynthetic proteins, including thephycobiliproteins (PBPs) from cryptophyte algae. Using X-raycrystallography, we show that cryptophyte PBPs adopt one oftwo quaternary structures, open or closed. The key feature ofthe closed form is the juxtaposition of two central chromo-phores resulting in excitonic coupling. The switch betweenforms is ascribed to the insertion of a single amino acid in theopen-form proteins. Thus, PBP quaternary structure controlsexcitonic coupling and the mechanism of light harvesting.Comparing organisms with these two distinct proteins willreveal the role of quantum coherence in photosynthesis.

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.,G.D.S., and P.M.G.C. designed research, performed research, analyzed data, and wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: Atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID codes 4LMS, 4LM6, and 4LMX), and DNA se-quences have been deposited in the GenBank database (accession nos. KC905456–KC905459 and KF314689–KF314696).1S.J.H. and K.E.W. contributed equally to this work.2To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1402538111/-/DCSupplemental.

E2666–E2675 | PNAS | Published online June 16, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1402538111

To gain a better understanding of the interrelation betweenprotein structure, chromophore arrangement, quantum coher-ence, and biological evolution, we have determined crystal struc-tures of three cryptophyte PBPs: phycocyanin PC645 fromChroomonas sp. (strain CCMP 270), PC612 from Hemiselmisvirescens (strain CCAC 1635 B), and PE555 from Hemiselmisandersenii (strain CCMP 644) (13). The PC645 dimer has thesame architecture as PE545, which we call the “closed” form, inwhich the two central β50/61 chromophores are in physicalcontact. In contrast, the structures from the two Hemiselmisspecies, PC612 and PE555, show a dramatically different dimerstructure in which the αβ monomers have been rotated by ∼73°compared with the closed form. In this open form, the centralβ50/61 chromophores are separated by a water-filled channel.

The results of 2D electronic spectroscopy (2D ES) from all threecryptophyte PBPs are reported here. We conclude that themechanism of light harvesting and whether effects arising fromelectronic coherence are significant depend strongly on thestructure. It is notable that each of these complexes harvests lightdifferently but apparently successfully.From the protein sequences and structures, it appears that the

Hemiselmis proteins with the open form differ from the crypto-phyte closed-form proteins by the insertion of a single aminoacid in a conserved region just before the chromophore attach-ment site in the α subunit. This insertion results in a rotation ofpart of the chromophore, and this rotation, in turn, precludes theformation of the closed-form dimer, ultimately resulting in thenew, open-form dimer structure. We compare the open and

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 subunitsare 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 crystalstructure), red and salmon. Chromophores are shown in atom colors. (D and E) Two orthogonal views of the closed-form PC645 dimer (monomers are shownin 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 verticalaxis 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.

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closed PBP structures using quantum chemical calculations andspectroscopic measurements. These findings open the door fordetermining the role of quantum coherence in a real-life bi-ological system and gaining a better understanding of how thesedistinct light-harvesting proteins might have evolved fromthe elaborate ancestral phycobilisome structure.

ResultsCrystal Structure of Phycocyanin PC645 from Chroomonas sp. Thecrystal structure of phycocyanin PC645 from Chroomonas sp.CCMP270 was determined at 1.35-Å resolution (Fig. 1A, Fig.S1A, and Table S1). The molecule consists of an α1β.α2β dimer inwhich each α subunit is covalently linked to a mesobiliverdinchromophore (MBV α18), and each β subunit has a doublylinked dibiliverdin chromophore, DBV β50/61, and two singlylinked phycocyanobilins, PCB β82 and PCB β158 (Table 1). Thetwo DBV β50/61 chromophores are in van der Waals contact atthe pseudo-twofold axis of the dimer where the pyrrole A ringsare offset stacked (Figs. S1A and S2A).The structure of the PC645 dimer is very similar to the pre-

viously published closed structure of phycoerythrin PE545 fromRhodomonas CS24 (73% sequence identity; rmsd 0.85Å on 453Cα atoms) (3, 4).

Crystal Structure of Phyocyanin PC612 from H. virescens. The crystalstructure of phycocyanin PC612 from H. virescens CCAC 1635was determined at 1.7-Å resolution (Fig. 1B, Fig. S1B, and TableS1). This light-harvesting complex also exists as an αβ.αβ dimer,but, in contrast to PE545 and PC645, it has nearly perfect two-fold symmetry, and the two α subunit sequences are identical.The αβ monomers in PC612 are very similar to those observed inthe closed-form structures (71% overall sequence identity withPC645; rmsd 1.11 Å on 213 Cα atoms; Fig. 1C). The positions ofthe chromophores in the αβ monomer are equivalent to those inthe closed-form structures. The only chromophore differencebetween PC612 and PC645 is the α chromophore, which inPC612 is PCB instead of MBV (Table 1).Because the quaternary arrangement of the two αβ monomers

in PC612 is so distinct from the closed form observed in PC645and PE545 (in Fig. 1, compare A with B and D with E), we referto it as the “open” form. The two αβ monomers in the PC612structure form a dome or cup-like structure which contains acentral cavity (Fig. 1 B and F).

Crystal Structure of Phycoerythrin PE555 from H. andersenii. Thecrystal structure of phycoerythrin PE555 from H. anderseniiCCMP 644 was determined at 1.8-Å resolution (Table S1). Thestructure shows a near symmetric α1β.α2β dimer that is nearlyidentical to the open-state structure of PC612 (84% overall se-quence identity; rmsd 0.92 Å on 464 Cα atoms). However, interms of chromophores, PE555 contains three phycoerythrobilins(PEBs) that replace the PCBs of PC612 (Table 1).

αβMonomer Structure and Chromophore Arrangement Are Conserved.Each αβ monomer is composed of a β subunit with a globinfold and an extended α subunit which lies along the β subunit(Fig. 1C). Apart from loops (particularly around the α chromo-phore), there is little deviation in the αβ monomer when com-paring PE545, PC645, PC612, and PE555 (rmsd of 0.78–1.6 Å

over ∼210 Cα atoms). The sequences of the β subunits are highlyconserved (79–92% identity). The sequences of the examinedα subunit are much more divergent, with 23–82% identity, buttheir conformations still are very similar (rmsd of 0.8–2.6 Å).

Comparison of Open- and Closed-Form Quaternary Structures. Al-though the protein structure and chromophore arrangementwithin the αβ monomer are conserved, the open and closedquaternary structures are radically different. The transformationthat relates the open- and closed-form quaternary structures isa rotation of one αβ monomer by ∼73° around an axis perpen-dicular to the dimer pseudo-twofold axis (Fig. 1 D and E andTable S2). The centers of mass of the two αβ monomers areslightly more separated in the open form: The center of mass-to-center of mass separation for the closed form was 23.4 Å forPE545 and 22.2 Å for PC645, compared with 24.4 Å for theopen form of PC612 and 24.8 Å for the open form of PE555.Transitions between the two quaternary forms of the same

protein are unlikely to occur, and there is no evidence thateither closed- or open-form proteins are in equilibrium witha measurable monomer pool. In the closed-form dimer, themonomer–monomer interaction buries a substantial surface(PE545: 1,060 Å2 per monomer; PC645: 1,230 Å2 per mono-mer), indicating that the dimer is very stable. In the open-formdimer, the monomer–monomer interaction buries a smaller butstill significant surface area (PE555: 618 Å2 per monomer;PC612: 511 Å2 per monomer). Although this result suggeststhat the open-form dimer may be less stable than the closedform, we see no evidence of monomer–dimer equilibrium onsize-exclusion chromatography.The main effect of the change from the closed to the open

form is the separation of the central, doubly linked β50/61chromophores. In the closed-form structures, these two chro-mophores are in van der Waals contact with the pyrrole A rings,which are offset stacked (closest approach of 3.8 Å betweenatoms in pyrrole A rings; Fig. 1A and Figs. S1A and S2A).However, in the open form, these two chromophores are wellseparated (closest approach between atoms in pyrrole A ringof 10.0 Å in PC612 and 11.0Å in PE555; Fig. 1B, Fig. S2 B and C,and Table 2).

Dimer Interface in the Closed Form. The dimer interface inter-actions in the closed form are mediated by the α subunit, the αchromophore, or the β50/61 chromophore with no direct protein–protein interactions between the two β subunits. There arethree key interaction sites. First, the pyrrole A rings of the twoβ50/61 chromophores pack against each other (Fig. S2A). Inaddition, the loop connecting helices hG and hH (the GH loop)of the β subunit and the C-terminal loop of α1 pack against theface of the β50/61 chromophore from the α2β monomer (Fig.S2A). The second interaction site is centered on pyrrole rings Aand B of the α chromophore. These sit in a hydrophobic pocketformed by the C-terminal tail of the opposite α subunit, the Cterminus of the opposite α subunit helix, and the loop connectinghelix hB to hE of the opposite β subunit (Figs. 2A and 4A). Third,the α subunit helix makes polar interactions with the break be-tween helices hA and hB and with helix hE in the opposite βsubunit plus the opposite α subunit (Fig. 2 B and C).

Dimer Interface in the Open Form. As in the closed form, all dimerinterface contacts in the two open-form structures are mediatedby either the α subunits or the α subunit chromophore with nodirect interactions between the two β subunits. A major contact ismade by the α chromophore where pyrrole ring A sits in a hy-drophobic pocket in the opposite αβ monomer (Fig. 2D). Thepocket is formed by the N-terminal portions of helix hE in the βsubunit and the helix in the α subunit, plus the hydrophobic

Table 1. Chromophores

Organism Strain PBP α β50,61 β82 β158

Rhodomonas sp. CS24 CS24 PE545 DBV PEB PEB PEBHemiselmis andersenii CCMP 644 PE555 PEB DBV PEB PEBHemiselmis virescens M1635 PC612 PCB DBV PCB PCBChroomonas sp. CCMP 270 PC645 MBV DBV PCB PCB

E2668 | www.pnas.org/cgi/doi/10.1073/pnas.1402538111 Harrop et al.

residue that precedes the α subunit helix by two residues (Met47in PC612 α, Phe45 in PE555 α1, and Met45 in PE555 α2).The other major dimer interface interaction is centered on the

C terminus of the α subunit α helix, which makes polar contactswith the GH loop in the opposite β subunit (Fig. 2 E and F).Additionally, in the PE555 structure, the C-terminal residue ofα2, Leu62, makes van der Waals contacts with the C-terminal tailof α1 and the GH loop of the opposite β subunit (Fig. 2F andFig. S2C). This interaction is adjacent to the polar interfacementioned above.

Sequence and Structural Changes Around the α Chromophore DictateQuaternary Structure. Given the structural conservation of the αβdimer, it is not immediately obvious why the two HemiselmisPBPs assemble in the open-form dimer rather than the closedform. An examination of the sequences of the α subunits showsthat in the Hemiselmis PBPs an aspartic acid (Asp18) has beeninserted in the highly conserved FDxRGC motif that links thefirst β strand to the α chromophore attachment site (Fig. 3C). Inthe closed-form structures, this motif forms a network of hy-drogen bonds that determine the orientation of pyrrole ring A inthe α chromophore with respect to the β sheet (Fig. 3A). Theinsertion of an aspartic acid in the Hemiselmis α subunits altersthe hydrogen bonding network (Fig. 3B). The net effect of thischange is that the plane of pyrrole ring A with respect to pyrrolering B in the α chromophore is rotated in a counterclockwisefashion by ∼29° in the closed form, whereas it is rotated in aclockwise fashion by ∼40° in the open form (Table S3). Thus,the sequence insertion results in an ∼69° rotation in pyrrolering A of the open-form αβ monomer as compared with theclosed form.How does the rotation of pyrrole ring A in the α subunit

chromophore determine the quaternary structure? In both openand closed forms, pyrrole ring A of the α chromophore makesa major contribution to the dimer interface (see Figs. 2A and 4Afor the closed form and Fig. 2D for the open form). Superposi-tion of the open-form αβ monomer on a closed-form quaternarystructure shows that the rotation of pyrrole ring A results ina steric clash with conserved Pro64 in the opposite β subunit(Fig. 4B). Thus, the open-form αβ monomer is prevented fromadopting the closed-form dimer structure.

Quantum Chemical Calculations Reveal Excitonic Switching. Usingthe high-resolution crystal structures of the PBPs, we calculatedthe gas-phase couplings (Table S4) and transition dipole mo-ments (Table S5). Note that these electronic couplings do notinclude dielectric screening effects that tend to reduce theirmagnitude by a factor of about two (14). For the central, doublylinked β50/61 chromophores, the center-to-center separationincreases when comparing closed and open-form PBPs by 6.3Åin the phycocyanin proteins (compare PC645 with PC612; Table2) and by 4.8Å in the phycoerythrin proteins (compare PE545

with PE555; Table 2). This increased separation dramaticallyweakens electronic couplings in the open-form PBPs comparedwith those determined for the closed structures (Table 2). Inparticular, the very strong Coulombic interaction calculatedwithin the central β50/61 bilin pair of the closed-form structuresof PC645 (647 cm−1) and PE545 (166 cm−1) is absent in theopen-form PBPs, PC612 (29 cm−1) and PE555 (4 cm−1) (Table2). Previous work has shown that the strong couplings calculatedfor the closed-form PC645 and PE545 are consistent with spec-troscopic data (15, 16). Steady-state spectra for the PBPs aredocumented in Fig. S3. These data include circular dichroismspectra, which are good experimental indicators of excitoninteractions. However, the primary derivative-like feature in thePBP spectra comes from subtle interactions among the periph-eral chromophores, not the central dimer, and therefore ispresent in spectra of both the closed and open forms of theprotein. A decrease is observed for all interdimer αβ–αβ cou-plings, whereas the intradimer αβ couplings are relatively un-affected by the different quaternary structure (Table 2 andTable S4).

Spectroscopy of the Closed-State PC645 Versus the Open-State PC612.The general features of the absorption spectra of PC645, PC612,and PE555 (Fig. 5 A–C) are consistent with the chromophorecompositions (Table 1). Previous modeling of the spectroscopyof PC645 suggests a model for the absorption band positions (thecenter positions of absorptions indicated in Fig. 5D) (16). Thethree different chromophore types (DBV, MBV, and PCB) pro-vide the primary spectral broadening and establish an energyfunnel from the core to periphery of the complex (17). PC612 issimilar to PC645 in that its absorption spectrum is broad, withtwo distinct peaks, and absorbs in the same region, but it doesnot contain MBV chromophores (Table 1). Based on the relativeabsorption energies of the individual bilins and the assignmentof chromophore absorption energies in PC645, we expect thatthe higher energy peak is dominated by DBVs and that the lowerenergy peak is dominated by PCBs (Fig. 5E) (18).Two types of spectral shift are evident in the PC645 spectrum.

First, the DBV chromophores are positioned closely in theclosed-form crystal structure and are particularly stronglycoupled (Table 2). This electronic coupling splits the DBVabsorption bands into the two exciton states labeled DBV(+)and DBV(−). Second, the degeneracy of the PCB absorptionbands is broken. Atomistic modeling of PE545 indicates thatspectral shifts are caused mainly by perturbations of thechromophore conformation with smaller effects caused byelectrostatic interactions with the local protein environment(15). Neither of these two spectral broadening featuresappears to be evident in the PC612 spectrum. For example,the absorption spectrum recorded with the sample at 77 K(Fig. 5B) clearly reveals only two bands and a vibronic tail onthe blue side of the spectrum. Exciton splitting is absent in the

Table 2. Electronic couplings (unscreened) and center-to-center distances for selected phycobiliprotein pairs inPE545, 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 19.91Coupling, 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 29.30Coupling, 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 19.35Coupling, cm−1 51 151 146 68

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open-form structure of PC612 (Table 2) because the doublylinked β50/61 DBV chromophores are widely separated.

PC645 has proven to be a remarkable system because of theclear coherent oscillations seen in cross-peaks in the 2D ES as

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 pocketformed 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) andthe 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 arelabeled 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 PC612showing the hydrophobic contact between pyrrole ring A of the α chromophore (green) and the pocket in the opposite αβ monomer formed by β subunithelix 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 theGH 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 sameinteraction in PE555 between the helix from α2 (red) and the opposite GH loop (magenta). Residues from the α2 subunit are labeled in black; residues fromthe β subunit are labeled in magenta. Hydrogen bonds/salt links are shown as dotted lines.

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a function of pump-probe waiting time (10, 19, 20). Comple-mentary nonlinear experiments also have identified these co-herences (21, 22). A representative total, real 2D ES spectrum isshown in Fig. 5D. The spectrum shows numerous features ofinterest, most distinctly an off-diagonal cross-peak (located atexcitation wavelength 570 nm and signal wavelength 600 nm inFig. 5D) that oscillates strongly as a function of the waiting time(Fig. 5G) (10). An extensive analysis of the oscillations has beenreported (19). Using a procedure involving the separation of thetotal spectrum into its rephasing and nonrephasing components(20), we concluded that the oscillations involve both vibrationaland electronic coherences (19).Unlike PC645, little is known about the photophysics of the

open-form PBPs PC612 and PE555. For PC612, we specificallyphotoexcited the DBV states to compare the 2D ES measure-ments directly with those of PC645. The 2D ES total, realspectrum (Fig. 5E) shows a rectangular feature, centered at theDBV bleach, suggesting substantial coupling between vibronictransitions throughout this spectral region; however, a strikingoscillating cross-peak like that noted for PC645 is not evident inthese data. In Fig. 5H, a trace at an off-diagonal position (exci-tation wavelength 550 nm and signal wavelength 600 nm in Fig.5E) shows damped oscillations that have frequencies consistentwith the vibrational beats (19).

Spectroscopy of the Open-State PE555. In contrast to PC645 andPC612, the PE555 absorption spectrum is narrow and nearlyfeatureless, showing minimal change between the room tem-perature and 77-K spectra (Fig. 5C). The presence of two distincttypes of chromophores in PE555 (singly bonded PEBs anddoubly bonded DBVs) suggests that there could be at least twodistinct absorption energies. By fitting the room temperature and77-K absorption spectra using two Gaussians, we obtained esti-

mates of the absorption energies. Based on the relative site en-ergies of the PEBs and DBVs in PE545, the higher-energyshoulder is attributed to the PEBs, and the lower-energy, mainabsorption band is attributed to DBVs. This ordering agrees withprevious assignment (Fig. 5F) (23, 24). The narrow, congestedabsorption spectrum suggests that the absorption bands of in-dividual chromophores overlap significantly and that excitonsplitting and energy shifts caused by different bilin conformationsare minimal. As in PC612, this observation is consistent with theidea that the open-form complex is more symmetric than theclosed-form complex. The 2D ES total, real spectrum (Fig. 5F)corroborates this expectation, appearing qualitatively similar tothat recorded for PC612. Oscillations in the amplitude wereweak compared with the other complexes (Fig. 5I; see the tracefor excitation wavelength 540 nm and signal wavelength 560 nm inFig. 5F) and appear most consistent with vibrational coherences.

DiscussionCrystallographic analyses reveal two very different quaternarystructures that are adopted by distinct cryptophyte PBPs: theopen and the closed forms. To date, both open-form PBPs comefrom Hemiselmis spp., whereas the two closed-form PBPs comefrom two distinct cryptophyte subclades (25). The αβ monomerstructure is conserved in all cryptophyte PBP structures that havebeen determined. This structural conservation includes the ar-rangement of chromophores in both α and β subunits. The in-terface between the two αβ monomers forming the dimer dependsmainly on contacts between α subunits and particularly the αsubunit chromophore, which makes key contacts across the di-mer interface. Thus, the α subunit mediates dimer formation andhence determines the quaternary structure of the cryptophytelight-harvesting protein.

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 closedform. (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 at-tachment site; the blue arrow indicates Glu16 that coordinates the central pyrrole nitrogens in the open-form structures. Red type indicates identity; blue typeindicates similarity. Breaks in the alignment mark the ends of secondary structure elements. Note that the helix length is variable at its C terminus.

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Sequence and structural analysis shows that the insertion of anaspartic acid residue just before the covalent chromophore at-tachment site (Cys20) in the Hemiselmis α subunits results ina rotation of the first pyrrole ring A of the α chromophore by∼69° compared with the closed form. This rotation precludes theassembly of the Hemiselmis αβ monomers into the closed-formdimer, because such assembly would result in a severe stericclash. It appears that the observed open form is the next avail-able dimeric state in terms of minimizing free energy. The buriedsurface area between monomers in the open-form dimer is ap-proximately half that observed for closed-form dimers, making theopen form a less stable structure. However, we note that we haveobserved only dimers with no evidence of free αβ monomers.The closed-form cryptophyte PBP dimers are clearly asym-

metric in structure and α subunit sequence, with the long αsubunit providing a C-terminal extension that mimics the loopbetween helices G and H in the β subunit and interacts with thedoubly linked β50/61 chromophore (4). In contrast, the open-form Hemiselmis PBP dimers are nearly symmetric in structureand sequence. Only minor structural differences are observed inPE555, and no significant differences are observed in PC612.The key difference between the two quaternary forms in terms of

chromophore arrangement is the van der Waals contact betweenthe two doubly linked β50/61 chromophores on the pseudo-twofoldaxis of the closed-form PBPs. This arrangement is unique to closed-form cryptophyte PBPs, and it results in the strong excitonic cou-pling of these two chromophores (Table 2). This pairing of chro-mophores is completely disrupted in the open-form quaternary

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 linkedβ50/61 chromophore. Consequently, strong electronic couplingamong the central bilins is the key distinguishing feature of theclosed- versus open-form PBPs with respect to light harvesting.Spectral shifts resulting from a combination of local environmentand conformational effects extend the PC645 absorption ∼25 nmfurther 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, abouthalf the spectral width of PC645. The great diversity of solutionsto light harvesting in the cryptophyte algae, in particular thecombination of different chromophores and significantly differ-ent structural combinations, are quite extraordinary.The ancestral cryptophyte alga acquired its chloroplast by

engulfing and taming a red algal endosymbiont, which wouldhave had at least a primitive phycobilisome (7, 24). Extant cya-nobacterial and red algal phycobilisomes are complex structuresmade up of stacked rods of several types of trimeric PBPs (αβ)3rings attached to the stromal surface of the thylakoid membrane(26), however, the phycobilisome α subunits are globin proteinsthat are completely unrelated to the cryptophyte α subunits. Atsome point during the integration of the red algal plastid, thephycobilisome 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 thylakoidlumen (2). This replacement resulted in the first radical rear-rangement in quaternary structure: the formation of a crypto-phyte progenitor (αβ)2 dimer (4). Here we report that amongthe cryptophytes there are two radically different forms of theα1β.α2β dimer, the open-form dimer, which appears to be con-fined to the Hemiselmis lineage, and the closed form. The emer-gence of two forms appears to have been caused by a singleinsertional/deletional mutation in the new cryptophyte α subunit.A central biological question is whether the presence of long-

lived electronic coherence in the light-harvesting proteins resultsin a selective advantage for the algae—for example, is coherenceimportant for efficient light harvesting? If the emergence of long-lived electronic coherence gives cryptophytes containing theclosed-form PBP a selective advantage over the ancestral cyano-bacteria and red algae, it would seem that the Hemiselmis cryp-tophytes, with their open-form PBPs, have lost this advantage. Ourresults suggest that successful light harvesting can be achieved indiverse ways, with or without coherent molecular excitons delo-calized over pairs of chromophores. Nevertheless, it is apparentthat the excitonic interactions in the PBPs are switched profoundly(over an order of magnitude) by the structural change from opento closed and that this exciton switch is genetically controlled.

Materials and MethodsGrowth of Cryptophytes. Chroomonas sp. Strain CCMP 270 (Provasoli-GuillardNational Center for Marine Algae and Microbiota, Bigelow Laboratory forOcean Sciences) was cultured in modified Fe medium (27) at 22–24 °C undercontinuous aeration with a 12-h light/12-h dark cycle at a light intensity of80–100 mE·m−2·s−1. When cultures reached a density of 5 × 108 cells/L (4–6wk), cells were harvested by centrifugation. The pellet was resuspended in25 mM phosphate buffer (pH 7) and was stored at −80 °C.H. andersenii. CCMP 644 (Provasoli-Guillard National Center for Marine Algaeand Microbiota, Bigelow Laboratory for Ocean Sciences) was grown in GSemedium (28) at 22–24 °C under a 12-h light/12-h dark cycle at a light intensityof 80–100 μE·m−2·s−1. Cells were harvested after 4–6 wk by centrifugationand were stored at −80 °C.H. virescens. CCAC 1635B (Culture Collection of Algae at the University ofCologne) cultures were grown in aerated ASP-H medium (29, 30) at 16 °Cunder a 14-h light/10-h dark cycle with light intensities of ∼50 μmol pho-tons·m−2·s−1. Cultures were harvested by flow-through centrifugation andwere stored at−80 °C.

Fig. 4. Rotation of pyrrole ring A in the open-form α chromophore pre-cludes formation of the closed form via a steric clash. (A) Stereo view of thepacking interaction between pyrrole ring A and the opposite αβmonomer inthe closed-form PC645. (B) Model of PE555 in which the two αβ monomershave been rotated so that they overlay the closed-form structure of PC645.Pyrrole ring A of the α subunit chromophore (green stick model) is rotatedby ∼70° compared with that of PC645, and this rotation results in a stericclash with conserved Pro64 in the opposite β subunit.

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Protein Purification. Algal cell pellets were thawed, resuspended in two tothree volumes of 25 mM phosphate buffer (pH 7), and homogenized witha Teflon glass homogenizer at 30 rpm. Cells were disrupted in a French pressat 1,000 psi and centrifuged at 23,000 × g for 1 h at 4 °C. The supernatant waspurified via ammonium sulfate cuts (0–50%, 50–60%, 60–70%, and 70–80%)by adding solid ammonium sulfate, stirring for 1 h at 4 °C, and centrifugingat 23,000 × g for 30 min at 4 °C. The 70–80% pellets were resuspended in25 mM phosphate buffer (pH 7), filtered, dialyzed against the same buffer,and loaded onto a Q Sepharose HiLoad 26/10 anion exchange column (GEHealthcare). The fractions containing the majority of the light-harvestingprotein were selected using the absorbance at 280 nm and concentrated ona 10-kDa Centriprep (Millipore). The protein was purified by size-exclusionchromatography using a Superdex 200 HiLoad 26/60 column (GE Health-care). Proteins eluted as a single peak and were concentrated using a 10-kDacutoff (Centriprep; Millipore) before snap freezing and storage at −80 °C.

Crystallization. The proteins were crystallized using vapor diffusion under thefollowing conditions: PC645, 20% PEG 4 k, 20% isopropanol, and 0.1 Msodium citrate (pH 5.6); PE555, 20% PEG 10 k in 0.1 M Hepes (pH 7.5); andPC612, 25% PEG 3,350 in 0.1 M Hepes (pH 7.5).

Data Collection. Crystals were transferred to the cryoprotectant solution ofreservoir plus 15% glycerol and then were flash-cooled in liquid nitrogenand mounted in a Cryostream cooler (Oxford Cryosystems) for data col-lection: PC645 on beamline 9.2, Stanford Synchrotron Radiation Light-source; PE555 on beamline 23ID-D, Advanced Photon Source; and PC612om beamline MX1, Australian Synchrotron (Table S1). Diffraction imagesfor PC645 and PE555 were collected on a MarCCD (Rayonix) detector, anddiffraction images for PC612 were collected on an ADSC Q210 (Area De-tector Systems Corporation) detector. Data collection was carried out usingBlu-Ice (31).

Data Reduction and Structure Determination. All data were processed usingXDS (32) and SCALA [CCP4 (33)]. Phasing, auto building, and refinementwere carried out using PHENIX (34). A single β subunit from the structure ofPE545 (3) was used as a molecular replacement probe using PHASER (35) asimplemented in PHENIX. Manual adjustments were carried out using COOT(36). Structural figures were created using PYMOL (37).

Structure of PC645. The PC645 structure contains one α1β.α2β dimer in theasymmetric unit. The complete α1 molecule is visible in the electron density

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) andPE555 (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 thereal part of the total signal, plotted with 33 evenly spaced contours. The estimated exciton energies of the chromophores are plotted on the 295-K absorptionspectra which are superimposed onto the excitation and emission axes. (G–I) Magnitude of the 2D ES amplitude at selected cross-peaks as a function ofwaiting 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-peakcoordinates (excitation, detection) in nanometers are approximately PC645: (570, 600), PC612: (550, 600), and PE555 (540, 560). Error bars indicate one SD asdetermined from three trials for PC645 and PC612 and six trials for PE555.

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map, whereas the last two residues of α2 (Lys69–Lys70) are disordered. TheC-terminal residue of each β subunit (Ala177) is disordered. The first 14 res-idues of the β subunit (helix hX) are absent in the electron density, theirposition being occupied by an ordered PEG molecule that extends acrossa crystallographic twofold axis. The only modified residue is Asn72 in the βsubunit where the side-chain nitrogen is methylated (5).

Structure of PC612. The PC612 structure contains a single (αβ)2 homodimer inthe asymmetric unit. Electron density is seen for the complete α subunitsand one of the β subunits. In the second β subunit, electron density starts atAsp3 and continues to the C terminus.

Structure of PE555. The PE555 structure has three copies of the (α1β).(α2β)heterodimer in the asymmetric unit. The first heterodimer shows clearelectron density for the full α2 subunit and all but the last residue (Val67) ofα1. The β chain associated with α2 is complete, whereas the remaining β chainstarts at Asp3. The heterodimer structure is nearly symmetric.

The other two copies of the heterodimer appear to be superpositions of an(α1β).(α2β) dimer with an (α2β).(α1β) dimer (i.e., rotation of 180° about thepseudo-twofold axis). In each α subunit, electron density is seen for bothpossible C-terminal regions [as seen in the asymmetric (α1β).(α2β) structure].There are five residues within the first 60 that distinguish α1 from α2. Theseresidues show density (or lack of density) for both possible α chains, inparticular α1Phe45/α2Met45.

Sequence Determination for H. virescens PC612, Chroomonas sp. CCMP270, andH. andersenii CCMP644. Algal cells were grown to exponential stage at 20°,20 μmol photons·m−2·s−1 on a 12-h light/12-h dark cycle. RNA was isolatedusing RNAqueous 4 PCR (Ambion) or Total RNA Isolation Reagent (AdvancedBiotechnologies). cDNA was generated using SuperScript III Reverse Tran-scriptase (Invitrogen Life Technologies) at 50 °C with random hexamer/nonamer or oligo (dT) as primers and was used for degenerate PCR. The βsubunit degenerate primers were designed based on the alignment of DNAsequences from cryptophytes Guillardia theta and Rhodomonas salina andall the red algal β subunit sequences in GenBank. The α subunit degenerateprimer pairs were based on Edman N-terminal sequencing (CCMP270) or onthe best partial amino acid sequences derived from electron-density maps.PCR products were cloned into T-vectors, and isolated colonies were selectedrandomly for sequencing. The resulting sequences were used to designoutward-directed PCR primer pairs for cDNA-based inverse PCR according toHuang and Chen (38). For the complete α subunit sequences, 5′ RACE wasdone with the FirstChoice RLM -RACE kit (Ambion) or ExactSTART kit (Epi-centre). For the 3′ end of the β subunit, genomic DNA-based inverse PCR wasperformed. The assembled sequences were confirmed by PCR from the startcodon to beyond the stop codon using specific nondegenerate primers.

Quantum Chemical Calculations. The initial conformations of the phycobilinswere extracted from the Protein Data Bank file with the covalently boundcysteine residue. The cysteine residues were capped with an acetyl andN-methyl amino group. Each of the tetrapyrrole nitrogens was protonated(resulting in a +1 charge), and the two solvent-exposed carbocyclic acidchains were deprotonated (resulting in a −2 charge) with an overall mo-lecular charge of −1 on all phycobilins. The molecular charge also is consis-tent with polarizable continuum pKa calculations implemented using theuniversal solvation model designated for solvation model density (39). Hy-drogen atoms were added and optimized using b3lyp/cc-pvtz, followedby a bond-length optimization with dihedral angles restrained using theGaussian 09 software package (Gaussian, Inc.).

The phycobilin transition density from the S0 to the S1 state was obtainedfrom a configuration interaction singles (CIS)/cc-pvtz calculation, again usingthe Gaussian 09 software package. Transition density cubes were inspectedvisually to ensure the proper excited state was probed. The gas-phase cou-plings were computed from the transition densities using the methodologyoutlined by Krueger et al. (40). No scaling of the couplings was used tocorrect for the overall overestimation of the transition dipole moments bythe CIS because they were overestimated by only ∼9% compared with theexperimental value of 2.34 eÅ (41).

2D Electronic Spectroscopy. A 5-kHz Ti:sapphire amplified laser system pro-duces 150-fs pulses centered at 800 nm with ∼0.6 mJ. About 0.15 mJ seedsa noncollinear optical parametric amplifier (NOPA), producing pulses witha bandwidth of 60 nm (spectral intensity FWHM) centered in the Vis range,nearly free from angular dispersion. A combination of a folded 4-f gratingcompressor and a single-prism prism compressor is used to compress thepulse from the NOPA. Pulse compression is determined by measuring thenonresonant third-order response from methanol in the sample position.The pulse duration, central wavelength, and bandwidth used for each pro-tein sample are summarized in Table S6. The beam is attenuated by thecombination of a broadband half-waveplate and a 0.7-mm-thick wire-gridpolarizer before entering the four-wave-mixing setup (20).

A spherical mirror with a 50-cm focal length focuses the beam on a 2D cross-hatched phase mask (UV fused silica substrate). The four first-order diffractionbeams, each about 12% of the input power, are arranged in the BOXCARSconfiguration and are directed by a small steering mirror toward the mirrorwith a 50-cm focal length. The steering mirror allows us to use the largespherical mirror at an angle of exactly 0°. The large spherical mirror collimatesand makes parallel the four beams, which then pass above, below, and to thesides of the steering mirror. Three of the beams traverse antiparallel pairs of1°, uncoated UV fused silica glass wedges for pulse delays. One wedge fromeach pair is mounted on a computer-controlled delay stage allowing a stepaccuracy of ∼850 zeptoseconds. The pulse that serves as the final excitationfield interaction is chopped at a frequency of 25 Hz; the chopper also triggersthe detector, which acquires signal for 20 ms (100 laser shots). The fourthbeam, the local oscillator (LO), is attenuated by 104 and interacts with thesample ∼250 fs before the final excitation field, thus limiting its use to a ref-erence field. The four pulses then encounter a second spherical mirror used at0° but with a focal length of 20 cm. The pulses focus and cross in the sampleplane (beam waist diameter ∼50 μm) after encountering another smallsteering mirror. The signal and LO are collimated by a curved mirror with a 20-cm focal length and are directed to an imaging spectrometer (f = 16.3 cm)coupled to a CCD detector with 1,024 pixels in the dispersed dimension. Thespectrometer is calibrated using an Hg/Ar lamp. The phase stability of theapparatus is λ/350 short term (5 min) and λ/200 long term (2 h). All 2D ESexperiments are performed with pulse energies of ≤5 nJ. The coherence time(τ1) was scanned in intervals of 0.2 fs (0.15 fs) from −45 to 45 fs. The waitingtime (τ2) ranged from 0–400 fs in 5-fs steps for PC645 and PC612 measure-ments; the PE555 measurement was monitored at 10-fs intervals. The meas-urements were conducted at 298 K, and the sample was flowed usinga peristaltic pump at a flow rate that guaranteed a fresh spot at each pulse.

Protein samples were stored at −75 °C until required and then werethawed and diluted in a suitable buffer to the appropriate OD for spec-troscopic measurements (ODλ max <0.4).

ACKNOWLEDGMENTS. We thank Michael Melkonian for giving K.H.-E. ac-cess to the Melkonian group’s research facilities. This research was under-taken on the MX1 beamline at the Australian Synchrotron, Victoria,Australia. The access to major research facilities program is supported bythe Commonwealth of Australia under the International Science Linkagesprogram. Portions of this research were carried out at the Stanford Synchro-tron Radiation Lightsource, a Directorate of the Stanford Linear AcceleratorCenter National Accelerator Laboratory and an Office of Science User Facilityoperated for the US Department of Energy (DOE) Office of Science by Stan-ford University. The Stanford Synchrotron Radiation Lightsource StructuralMolecular Biology Program is supported by the DOE Office of Biological andEnvironmental Research and by the National Institutes of Health, NationalInstitute of General Medical Sciences (including P41GM103393). Use of theAdvanced Photon Source, an Office of Science User Facility operated for theUS DOE Office of Science by Argonne National Laboratory, was supported bythe US DOE under Contract DE-AC02-06CH11357. This work was supportedby grants from the Australian Research Council. G.D.S. and B.R.G. receivedsupport from Defense Advanced Research Projects Agency (Quantum Effectsin Biological Environments) and the Natural Sciences and EngineeringResearch Council of Canada. E.C. received financial support from theEuropean Research Council (ERC) under the European Community’s Sev-enth Framework Programme (FP7/2007-2013) with the ERC Starting GrantQUENTRHEL (Grant Agreement 278560).

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Harrop et al. PNAS | Published online June 16, 2014 | E2675

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