Structural characterization suggests models for monomeric and dimeric forms of full-length ezrin.

Ezrin is a member of the ERM (ezrin-radixin-moesin) family of proteins that have been conserved through metazoan evolution. These proteins have dormant and active forms, where the latter links the actin cytoskeleton to membranes. ERM proteins have three domains: an N-terminal FERM [band Four-point-one (4.1) ERM] domain comprising three subdomains (F1, F2, and F3); a helical domain; and a C-terminal actin-binding domain. In the dormant form, FERM and C-terminal domains form a stable complex. We have determined crystal structures of the active FERM domain and the dormant FERM:C-terminal domain complex of human ezrin. We observe a bistable array of phenylalanine residues in the core of subdomain F3 that is mobile in the active form and locked in the dormant form. As subdomain F3 is pivotal in binding membrane proteins and phospholipids, these transitions may facilitate activation and signaling. Full-length ezrin forms stable monomers and dimers. We used small-angle X-ray scattering to determine the solution structures of these species. As expected, the monomer shows a globular domain with a protruding helical coiled coil. The dimer shows an elongated dumbbell structure that is twice as long as the monomer. By aligning ERM sequences spanning metazoan evolution, we show that the central helical region is conserved, preserving the heptad repeat. Using this, we have built a dimer model where each monomer forms half of an elongated antiparallel coiled coil with domain-swapped FERM:C-terminal domain complexes at each end. The model suggests that ERM dimers may bind to actin in a parallel fashion.

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

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 significant. 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 αß subunits in which the structure of the αß 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 αß monomers are rotated by ∼73° to an "open" configuration in contrast to the "closed" configuration 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.

Cooperative Subunit Refolding of a Light-Harvesting Protein through a Self-Chaperone Mechanism.

The fold of a protein is encoded by its amino acid sequence, but how complex multimeric proteins fold and assemble into functional quaternary structures remains unclear. Here we show that two structurally different phycobiliproteins refold and reassemble in a cooperative manner from their unfolded polypeptide subunits, without biological chaperones. Refolding was confirmed by ultrafast broadband transient absorption and two-dimensional electronic spectroscopy to probe internal chromophores as a marker of quaternary structure. Our results demonstrate a cooperative, self-chaperone refolding mechanism, whereby the ß-subunits independently refold, thereby templating the folding of the α-subunits, which then chaperone the assembly of the native complex, quantitatively returning all coherences. Our results indicate that subunit self-chaperoning is a robust mechanism for heteromeric protein folding and assembly that could also be applied in self-assembled synthetic hierarchical systems.

Coherently wired light-harvesting in photosynthetic marine algae at ambient temperature.

Photosynthesis makes use of sunlight to convert carbon dioxide into useful biomass and is vital for life on Earth. Crucial components for the photosynthetic process are antenna proteins, which absorb light and transmit the resultant excitation energy between molecules to a reaction centre. The efficiency of these electronic energy transfers has inspired much work on antenna proteins isolated from photosynthetic organisms to uncover the basic mechanisms at play. Intriguingly, recent work has documented that light-absorbing molecules in some photosynthetic proteins capture and transfer energy according to quantum-mechanical probability laws instead of classical laws at temperatures up to 180 K. This contrasts with the long-held view that long-range quantum coherence between molecules cannot be sustained in complex biological systems, even at low temperatures. Here we present two-dimensional photon echo spectroscopy measurements on two evolutionarily related light-harvesting proteins isolated from marine cryptophyte algae, which reveal exceptionally long-lasting excitation oscillations with distinct correlations and anti-correlations even at ambient temperature. These observations provide compelling evidence for quantum-coherent sharing of electronic excitation across the 5-nm-wide proteins under biologically relevant conditions, suggesting that distant molecules within the photosynthetic proteins are 'wired' together by quantum coherence for more efficient light-harvesting in cryptophyte marine algae.

CLIC proteins, ezrin, radixin, moesin and the coupling of membranes to the actin cytoskeleton: a smoking gun?

The CLIC proteins are a highly conserved family of metazoan proteins with the unusual ability to adopt both soluble and integral membrane forms. The physiological functions of CLIC proteins may include enzymatic activity in the soluble form and anion channel activity in the integral membrane form. CLIC proteins are associated with the ERM proteins: ezrin, radixin and moesin. ERM proteins act as cross-linkers between membranes and the cortical actin cytoskeleton. Both CLIC and ERM proteins are controlled by Rho family small GTPases. CLIC proteins, ERM and Rho GTPases act in a concerted manner to control active membrane processes including the maintenance of microvillar structures, phagocytosis and vesicle trafficking. All of these processes involve the interaction of membranes with the underlying cortical actin cytoskeleton. The relationships between Rho GTPases, CLIC proteins, ERM proteins and the membrane:actin cytoskeleton interface are reviewed. Speculative models are proposed involving the formation of localised multi-protein complexes on the membrane surface that assemble via multiple weak interactions. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

Molecular structures reveal the origin of spectral variation in cryptophyte light harvesting antenna proteins.

In addition to their membrane-bound chlorophyll a/c light-harvesting antenna, the cryptophyte algae have evolved a unique phycobiliprotein antenna system located in the thylakoid lumen. The basic unit of this antenna consists of two copies of an αß protomer where the α and ß subunits scaffold different combinations of a limited number of linear tetrapyrrole chromophores. While the ß subunit is highly conserved, encoded by a single plastid gene, the nuclear-encoded α subunits have evolved diversified multigene families. It is still unclear how this sequence diversity results in the spectral diversity of the mature proteins. By careful examination of three newly determined crystal structures in comparison with three previously obtained, we show how the α subunit amino acid sequences control chromophore conformations and hence spectral properties even when the chromophores are identical. Previously we have shown that α subunits control the quaternary structure of the mature αß.αß complex (either open or closed), however, each species appeared to only harbor a single quaternary form. Here we show that species of the Hemiselmis genus contain expressed α subunit genes that encode both distinct quaternary structures. Finally, we have discovered a common single-copy gene (expressed into protein) consisting of tandem copies of a small α subunit that could potentially scaffold pairs of light harvesting units. Together, our results show how the diversity of the multigene α subunit family produces a range of mature cryptophyte antenna proteins with differing spectral properties, and the potential for minor forms that could contribute to acclimation to varying light regimes.

Quantitative investigations of quantum coherence for a light-harvesting protein at conditions simulating photosynthesis.

Recent measurements using two-dimensional electronic spectroscopy (2D ES) have shown that the initial dynamic response of photosynthetic proteins can involve quantum coherence. We show how electronic coherence can be differentiated from vibrational coherence in 2D ES. On that basis we conclude that both electronic and vibrational coherences are observed in the phycobiliprotein light-harvesting complex PC645 from Chroomonas sp. CCMP270 at ambient temperature. These light-harvesting antenna proteins of the cryptophyte algae are suspended in the lumen, where the pH drops significantly under sustained illumination by sunlight. Here we measured 2D ES of PC645 at increasing levels of acidity to determine if the change in pH affects the quantum coherence; quantitative analysis reveals that the dynamics are insensitive to the pH change.

Flow of excitation energy in the cryptophyte light-harvesting antenna phycocyanin 645.

We report a detailed description of the energy migration dynamics in the phycocyanin 645 (PC645) antenna complex from the photosynthetic alga Chroomonas CCMP270. Many of the cryptophyceae are known to populate greater depths than most other algal families, having developed a 99.5% efficient light-harvesting system. In this study, we used femtosecond time-resolved spectroscopy and global analysis to characterize the excited-state dynamics of PC645. Several different pump colors were selected to excite different fractions of the four phycobiliprotein pairs present in the complex. Measurements were also performed at cryogenic temperature to enhance spectral resolution and selectively promote downhill energy transfers. Upon excitation of the highest-energy bilins (dihydrobiliverdins), energy is transferred from the core of the complex to the periphery within 0.82 ps. Four bilins (mesobiliverdin (MBV) A/B and phycocyanobilins (PCB) 158C/D), which are responsible for the central band of the absorption spectrum, show concerted spectral dynamics. These chromophores show a biphasic decay with lifetimes of 0.6 ps (MBV) and 5-7 ps (PCB 158) to the lowest bilin pair (PCB 82C/D) absorbing around 650-657 nm. Within this lifetime of several picoseconds, the excitations reach the PCB 82 bilins on the two poles at the smaller sides of PC645. A slow 44-46 ps energy transfer step to the lowest-energy PCB 82 bilin concludes the dynamics.

Phycobiliprotein diffusion in chloroplasts of cryptophyte Rhodomonas CS24.

Unicellular cryptophyte algae employ antenna proteins with phycobilin chromophores in their photosynthetic machinery. The mechanism of light harvesting in these organisms is significantly different than the energy funneling processes in phycobilisomes utilized by cyanobacteria and red algae. One of the most striking features of cryptophytes is the location of the water-soluble phycobiliproteins, which are contained within the intrathylakoid spaces and are not on the stromal side of the lamellae as in the red algae and cyanobacteria. Studies of mobility of phycobiliproteins at the lumenal side of the thylakoid membranes and how their diffusional behavior may influence the energy funneling steps in light harvesting are reported. Confocal microscopy and fluorescence recovery after photobleaching (FRAP) are used to measure the diffusion coefficient of phycoerythrin 545 (PE545), the primary light harvesting protein of Rhodomonas CS24, in vivo. It is concluded that the diffusion of PE545 in the lumen is inhibited, suggesting possible membrane association or aggregation as a potential source of mobility hindrance.

Phycocyanin sensitizes both photosystem I and photosystem II in cryptophyte Chroomonas CCMP270 cells.

This article presents an investigation of the energy migration dynamics in intact cells of the unicellular photosynthetic cryptophyte Chroomonas CCMP270 by steady-state and time-resolved fluorescence measurements. By kinetic modeling of the fluorescence data on chlorophyll and phycocyanin 645 excitation (at 400 and 582 nm respectively), it has been possible to show the excited state energy distribution in the photosynthetic antenna of this alga. Excitation energy from phycocyanin 645 is distributed nearly equally between photosystem I and photosystem II with very high efficiency on a 100-ps timescale. The excitation energy trapping times for both photosystem I ( approximately 30 ps) and photosystem I (200 and approximately 540 ps) correspond well to those obtained from experiments on isolated photosystems. The results are compared with previous results for another cryptophyte species, Rhodomonas CS24, and suggest a similar membrane organization for the cryptophytes with the phycobiliproteins tightly packed in the thylakoid lumen around the periphery of the photosystems.

How energy funnels from the phycoerythrin antenna complex to photosystem I and photosystem II in cryptophyte Rhodomonas CS24 cells.

We report an investigation of energy migration dynamics in intact cells of the photosynthetic cryptophyte Rhodomonas CS24 using analyses of steady-state and time-resolved fluorescence anisotropy measurements. By fitting a specific model to the fluorescence data, we obtain three time scales (17, 58, and 113 ps) by which the energy is transferred from phycoerythrin 545 (PE545) to the membrane-associated chlorophylls (Chls). We propose that these time scales reflect both an angular distribution of PE545 around the photosystems and the relative orientations of the donor dihydrobiliverdin (DBV) bilin and the acceptor Chl. Contrary to investigations of the isolated antenna complex, it is demonstrated that energy transfer from PE545 does not occur from a single-emitting bilin, but rather both the peripheral dihydrobiliverdin (DBV) chromophores in PE545 appear to be viable donors of excitation energy to the membrane-bound proteins. The model shows an almost equal distribution of excitation energy from PE545 to both photosystem I (PSI) and photosystem II (PSII), whose trap times correspond well to those obtained from experiments on isolated photosystems.

Developing a structure-function model for the cryptophyte phycoerythrin 545 using ultrahigh resolution crystallography and ultrafast laser spectroscopy.

Cryptophyte algae differ from cyanobacteria and red algae in the architecture of their photosynthetic light harvesting systems, even though all three are evolutionarily related. Central to cryptophyte light harvesting is the soluble antenna protein phycoerythrin 545 (PE545). The ultrahigh resolution crystal structure of PE545, isolated from a unicellular cryptophyte Rhodomonas CS24, is reported at both 1.1A and 0.97A resolution, revealing details of the conformation and environments of the chromophores. Absorption, emission and polarized steady state spectroscopy (298K, 77K), as well as ultrafast (20fs time resolution) measurements of population dynamics are reported. Coupled with complementary quantum chemical calculations of electronic transitions of the bilins, these enable assignment of spectral absorption characteristics to each chromophore in the structure. Spectral differences between the tetrapyrrole pigments due to chemical differences between bilins, as well as their binding and interaction with the local protein environment are described. Based on these assignments, and considering customized optical properties such as strong coupling, a model for light harvesting by PE545 is developed which explains the fast, directional harvesting of excitation energy. The excitation energy is funnelled from four peripheral pigments (beta158,beta82) into a central chromophore dimer (beta50/beta61) in approximately 1ps. Those chromophores, in turn, transfer the excitation energy to the red absorbing molecules located at the periphery of the complex in approximately 4ps. A final resonance energy transfer step sensitizes just one of the alpha19 bilins on a time scale of 22ps. Furthermore, it is concluded that binding of PE545 to the thylakoid membrane is not essential for efficient energy transfer to the integral membrane chlorophyll a-containing complexes associated with PS-II.

Mediation of ultrafast light-harvesting by a central dimer in phycoerythrin 545 studied by transient absorption and global analysis.

We report ultrafast femtosecond transient absorption measurements of energy-transfer dynamics for the antenna protein phycoerythrin 545, PE545, isolated from a unicellular cryptophyte Rhodomonas CS24. The phycoerythrobilins are excited at both 485 and 530 nm, and the spectral response is probed between 400 and 700 nm. Room-temperature measurements are contrasted with measurements at 77 K. An evolution-associated difference spectra (EADS) analysis is combined with estimations of bilin spectral positions and energy-transfer rates to obtain a detailed kinetic model for PE545. It is found that sub pulse-width dynamics include relaxation between the exciton states of a chromophore dimer (beta 50/60) located in the core of the protein. Energy transfer from the lowest exciton state of the phycoerythrobilin (PEB) dimer to one of the periphery 15,16-dihydrobiliverdin (DBV) bilins is found to occur on a time scale of 250 fs at room temperature and 960 fs at 77 K. A host of energy-transfer dynamics involving the beta 158, beta 82, and alpha 19 bilins occur on a time scale of 2 ps at room temperature and 3 ps at 77 K. A final energy transfer occurs between the red-most DBV bilins with a time scale estimated to be approximately 30 ps. The role of the centrally located phycoerythrobilin dimer is seen as crucial-spectrally as it expands the cross-section of absorption of the protein; structurally as it sits in the middle of the protein acting as an intermediary trap; and kinetically, as the internal conversion and subsequent red-shift of the excitation is extremely fast.

Vibronic resonances facilitate excited-state coherence in light-harvesting proteins at room temperature.

Until recently it was believed that photosynthesis, a fundamental process for life on earth, could be fully understood with semiclassical models. However, puzzling quantum phenomena have been observed in several photosynthetic pigment-protein complexes, prompting questions regarding the nature and role of these effects. Recent attention has focused on discrete vibrational modes that are resonant or quasi-resonant with excitonic energy splittings and strongly coupled to these excitonic states. Here we unambiguously identify excited state coherent superpositions in photosynthetic light-harvesting complexes using a new experimental approach. Decoherence on the time scale of the excited state lifetime allows low energy (56 cm(-1)) oscillations on the signal intensity to be observed. In conjunction with an appropriate model, these oscillations provide clear and direct experimental evidence that the persistent coherences observed originate from quantum superpositions among vibronic excited states.

Spectroscopic Studies of Cryptophyte Light Harvesting Proteins: Vibrations and Coherent Oscillations.

The first step of photosynthesis is the absorption of light by antenna complexes. Recent studies of light-harvesting complexes using two-dimensional electronic spectroscopy have revealed interesting coherent oscillations. Some contributions to those coherences are assigned to electronic coherence and therefore have implications for theories of energy transfer. To assign these femtosecond data and to gain insight into the interplay among electronic and vibrational resonances, we need detailed information on vibrations and coherences in the excited electronic state compared to the ground electronic state. Here, we used broad-band transient absorption and femtosecond stimulated Raman spectroscopies to record ground- and excited-state coherences in four related photosynthetic proteins: PC577 from Hemiselmis pacifica CCMP706, PC612 from Hemiselmis virescens CCAC 1635 B, PC630 from Chroomonas CCAC 1627 B (marine), and PC645 from Chroomonas mesostigmatica CCMP269. Two of those proteins (PC630 and PC645) have strong electronic coupling while the other two proteins (PC577 and PC612) have weak electronic coupling between the chromophores. We report vibrational spectra for the ground and excited electronic states of these complexes as well as an analysis of coherent oscillations observed in the broad-band transient absorption data.

Electronic coherence lineshapes reveal hidden excitonic correlations in photosynthetic light harvesting.

The effective absorption cross-section of a molecule (acceptor) can be greatly increased by associating it with a cluster of molecules that absorb light and transfer the excitation energy to the acceptor molecule. The basic mechanism of such light harvesting by Förster resonance energy transfer (FRET) is well established, but recent experiments have revealed a new feature whereby excitation is coherently shared among donor and acceptor molecules during FRET. In the present study, two-dimensional electronic spectroscopy was used to examine energy transfer at ambient temperature in a naturally occurring light-harvesting protein (PE545 of the marine cryptophyte alga Rhodomonas sp. strain CS24). Quantum beating was observed across a range of excitation frequencies. The shapes of those features in the two-dimensional spectra were examined. Through simulations, we show that two-dimensional electronic spectroscopy provides a probe of the adiabaticity of the free energy landscape underlying light harvesting.

Ultrafast light harvesting dynamics in the cryptophyte phycocyanin 645.

Steady-state and femtosecond time-resolved optical methods have been used to study spectroscopic features and energy transfer dynamics in the soluble antenna protein phycocyanin 645 (PC645), isolated from a unicellular cryptophyte Chroomonas CCMP270. Absorption, emission and polarization measurements as well as one-colour pump-probe traces are reported in combination with complementary quantum chemical calculations of electronic transitions of the bilins. Estimation of bilin spectral positions and energy transfer rates aids in the development of a model for light harvesting by PC645. At higher photon energies light is absorbed by the centrally located dimer (DBV, beta50/beta61) and the excitation is subsequently funneled through a complex interference of pathways to four peripheral pigments (MBV alpha19, PCB beta158). Those chromophores transfer the excitation energy to the red-most bilins (PCB beta82). We suggest that the final resonance energy transfer step occurs between the PCB 82 bilins on a timescale estimated to be approximately 15 ps. Such a rapid final energy transfer step cannot be rationalized by calculations that combine experimental parameters and quantum chemical calculations, which predict the energy transfer time to be 40 ps.