RESUMO
Photosynthetic organisms have established photoprotective mechanisms in order to dissipate excess light energy into heat, which is commonly known as non-photochemical quenching. Cyanobacteria utilize the orange carotenoid protein (OCP) as a high-light sensor and quencher to regulate the energy flow in the photosynthetic apparatus. Triggered by strong light, OCP undergoes conformational changes to form the active red state (OCPR). In many cyanobacteria, the back conversion of OCP to the dark-adapted state is assisted by the fluorescence recovery protein (FRP). However, the exact molecular events involving OCP and its interaction with FRP remain largely unraveled so far due to their metastability. Here, we use small-angle neutron scattering combined with size exclusion chromatography (SEC-SANS) to unravel the solution structures of FRP-OCP complexes using a compact mutant of OCP lacking the N-terminal extension (∆NTEOCPO) and wild-type FRP. The results are consistent with the simultaneous presence of stable 2:2 and 2:1 FRP-∆NTEOCPO complexes in solution, where the former complex type is observed for the first time. For both complex types, we provide ab initio low-resolution shape reconstructions and compare them to homology models based on available crystal structures. It is likely that both complexes represent intermediate states of the back conversion of OCP to its dark-adapted state in the presence of FRP, which are of transient nature in the photocycle of wild-type OCP. This study demonstrates the large potential of SEC-SANS in revealing the solution structures of protein complexes in polydisperse solutions that would otherwise be averaged, leading to unspecific results.
Assuntos
Cianobactérias , Synechocystis , Luz , Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , Fotossíntese , Cromatografia em Gel , Synechocystis/metabolismoRESUMO
Utilized for gaining structural insights, small-angle neutron and X-ray scattering techniques (SANS and SAXS, respectively) enable an examination of biomolecules, including photosynthetic pigment-protein complexes, in solution at physiological temperatures. These methods can be seen as instrumental bridges between the high-resolution structural information achieved by crystallography or cryo-electron microscopy and functional explorations conducted in a solution state. The review starts with a comprehensive overview about the fundamental principles and applications of SANS and SAXS, with a particular focus on the recent advancements permitting to enhance the efficiency of these techniques in photosynthesis research. Among the recent developments discussed are: (i) the advent of novel modeling tools whereby a direct connection between SANS and SAXS data and high-resolution structures is created; (ii) the employment of selective deuteration, which is utilized to enhance spatial selectivity and contrast matching; (iii) the potential symbioses with molecular dynamics simulations; and (iv) the amalgamations with functional studies that are conducted to unearth structure-function relationships. Finally, reference is made to time-resolved SANS/SAXS experiments, which enable the monitoring of large-scale structural transformations of proteins in a real-time framework.
Assuntos
Fotossíntese , Proteínas , Espalhamento a Baixo Ângulo , Microscopia Crioeletrônica , Difração de Raios X , Proteínas/químicaRESUMO
Most cyanobacteria utilize a water-soluble Orange Carotenoid Protein (OCP) to protect their light-harvesting complexes from photodamage. The Fluorescence Recovery Protein (FRP) is used to restore photosynthetic activity by inactivating OCP via dynamic OCP-FRP interactions, a multistage process that remains underexplored. In this work, applying time-resolved spectroscopy, we demonstrate that the interaction of FRP with the photoactivated OCP begins early in the photocycle. Interacting with the compact OCP state, FRP completely prevents the possibility of OCP domain separation and formation of the signaling state capable of interacting with the antenna. The structural element that prevents FRP binding and formation of the complex is the short α-helix at the beginning of the N-terminal domain of OCP, which masks the primary site in the C-terminal domain of OCP. We determined the rate of opening of this site and show that it remains exposed long after the relaxation of the red OCP states. Observations of the OCP transitions on the ms time scale revealed that the relaxation of the orange photocycle intermediates is accompanied by an increase in the interaction of the carotenoid keto group with the hydrogen bond donor tyrosine-201. Our data refine the current model of photoinduced OCP transitions and the interaction of its intermediates with FRP.
Assuntos
Proteínas de Bactérias , Cianobactérias , Proteínas de Bactérias/química , Cianobactérias/metabolismo , Análise Espectral , Transdução de Sinais , Carotenoides/química , Ficobilissomas/químicaRESUMO
We used small-angle neutron scattering partially coupled with size-exclusion chromatography to unravel the solution structures of two variants of the Orange Carotenoid Protein (OCP) lacking the N-terminal extension (OCP-ΔNTE) and its complex formation with the Fluorescence Recovery Protein (FRP). The dark-adapted, orange form OCP-ΔNTEO is fully photoswitchable and preferentially binds the pigment echinenone. Its complex with FRP consists of a monomeric OCP component, which closely resembles the compact structure expected for the OCP ground state, OCPO. In contrast, the pink form OCP-ΔNTEP, preferentially binding the pigment canthaxanthin, is mostly nonswitchable. The pink OCP form appears to occur as a dimer and is characterized by a separation of the N- and C-terminal domains, with the canthaxanthin embedded only into the N-terminal domain. Therefore, OCP-ΔNTEP can be viewed as a prototypical model system for the active, spectrally red-shifted state of OCP, OCPR. The dimeric structure of OCP-ΔNTEP is retained in its complex with FRP. Small-angle neutron scattering using partially deuterated OCP-FRP complexes reveals that FRP undergoes significant structural changes upon complex formation with OCP. The observed structures are assigned to individual intermediates of the OCP photocycle in the presence of FRP.
Assuntos
Proteínas de Bactérias , Cianobactérias , Proteínas de Bactérias/química , Cantaxantina , Espalhamento a Baixo Ângulo , Cianobactérias/metabolismo , Modelos BiológicosRESUMO
The orange carotenoid protein plays a vital role in the photoprotection of cyanobacteria and exhibits a significant structural change upon photoactivation. A rarely considered aspect is the importance of internal protein dynamics in facilitating the structural transition to the active state. In this study, we use quasielastic neutron scattering under (in situ) blue light illumination for the first time to directly probe the protein dynamics of the orange carotenoid protein in the dark-adapted and active states. This shows that the localized internal dynamics of amino acid residues is significantly enhanced upon photoactivation. This is attributed to the photoinduced structural changes exposing larger areas of the protein surface to the solvent, thus resulting in a higher degree of motional freedom. However, the flexibility of the W288A mutant assumed to mimic the active state structure is found to be different, thus highlighting the importance of in situ experiments.
Assuntos
Proteínas de Bactérias , Iluminação , Proteínas de Bactérias/química , Conformação Proteica , Luz , NêutronsRESUMO
The high-resolution crystal structure of the trimeric major light-harvesting complex of photosystem II (LHCII) is often perceived as the basis for understanding its light-harvesting and photoprotective functions. However, the LHCII solution structure and its oligomerization or aggregation state may generally differ from the crystal structure and, moreover, also depend on its functional state. In this regard, small-angle scattering experiments provide the missing link by offering structural information in aqueous solution at physiological temperatures. Herein, we use small-angle scattering to investigate the solution structures of two different preparations of solubilized LHCII employing the nonionic detergents n-octyl-ß-d-glucoside (OG) and n-dodecyl-ß-D-maltoside (ß-DM). The data reveal that the LHCII-OG complex is equivalent to the trimeric crystal structure. Remarkably, however, we observeâfor the first timeâa stable oligomer composed of three LHCII trimers in the case of the LHCII-ß-DM preparation, implying additional pigment-pigment interactions. The latter complex is assumed to mimic trimer-trimer interactions which play an important role in the context of photoprotective nonphotochemical quenching.
RESUMO
Albeit achieving the X-ray diffraction structure of dimeric photosystem II core complexes (dPSIIcc) at the atomic resolution, the nature of the detergent belt surrounding dPSIIcc remains ambiguous. Therefore, the solution structure of the whole detergent-protein complex of dPSIIcc of Thermosynechococcus elongatus (T. elongatus) solubilized in n-dodecyl-ß-d-maltoside (ßDM) was investigated by a combination of small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) with contrast variation. First, the structure of dPSIIcc was studied separately in SANS experiments using a contrast of 5% D2O. Guinier analysis reveals that the dPSIIcc solution is virtually free of aggregation in the studied concentration range of 2-10 mg/mL dPSIIcc, and characterized by a radius of gyration of 62 Å. A structure reconstitution shows that dPSIIcc in buffer solution widely retains the crystal structure reported by X-ray free electron laser studies at room temperature with a slight expansion of the entire protein. Additional SANS experiments on dPSIIcc samples in a buffer solution containing 75% D2O provide information about the size and shape of the whole detergent-dPSIIcc. The maximum position of P(r) function increases to 68 Å, i.e., it is about 6 Å larger than that of dPSIIcc only, thus indicating the presence of an additional structure. Thus, it can be concluded that dPSIIcc is surrounded by a monomolecular belt of detergent molecules under appropriate solubilization conditions. The homogeneity of the ßDM-dPSIIcc solutions was also verified using dynamic light scattering. Complementary SAXS experiments indicate the presence of unbound detergent micelles by a separate peak consistent with a spherical shape possessing a radius of about 40 Å. The latter structure also contributes to the SANS data but rather broadens the SANS curve artificially. Without the simultaneous inspection of SANS and SAXS data, this effect may lead to an apparent underestimation of the size of the PS II-detergent complex. The formation of larger unbound detergent aggregates in solution prior to crystallization may have a significant effect on the crystal formation or quality of the ßDM-dPSIIcc.
Assuntos
Detergentes , Complexo de Proteína do Fotossistema II , Cristalização , Difração de Nêutrons , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Persistent non-photochemical hole burning at 4.2 K is an efficient experimental tool to unravel position and nature of low-energy excitonic states in pigment-protein complexes. This is demonstrated here for the case of the trimeric chlorophyll (Chl) a/b light-harvesting complexes of Photosystem II (LHC II) of green plants, where previous work (Pieper et al. J Phys Chem B 103:2412, 1999a) reported a highly localized lowest energy state at 680 nm. At that time, this finding appeared to be consistent with the contemporary knowledge about the LHC II structure, which mainly suggested the presence of weakly coupled Chl heterodimers. Currently, however, it is widely accepted that the lowest state is associated with an excitonically coupled trimer of Chl molecules at physiological temperatures. This raises the question, why an excitonically coupled state has not been identified by spectral hole burning. A re-inspection of the hole burning data reveals a remarkable dependence of satellite hole structure on burn fluence, which is indicative of the excitonic coupling of the low-energy states of trimeric LHC II. At low fluence, the satellite hole structure of the lowest/fluorescing ~ 680 nm state is weak with only one shallow satellite hole at 649 nm in the Chl b spectral range. These findings suggest that the lowest energy state at ~ 680 nm is essentially localized on a Chl a molecule, which may belong to a Chl a/b heterodimer. At high fluence, however, the lowest energy hole shifts blue to ~ 677 nm and is accompanied by two satellite holes at ~ 673 and 663 nm, respectively, indicating that this state is excitonically coupled to other Chl a molecules. In conclusion, LHC II seems to possess two different, but very closely spaced lowest energy states at cryogenic temperatures of 4.2 K.
Assuntos
Transferência de Energia , Complexo de Proteína do Fotossistema II/metabolismo , Viridiplantae/fisiologia , Clorofila/metabolismo , TemperaturaRESUMO
A novel ultrasonic instrumentation was successfully implemented in a compaction simulator. A through-transmission set-up was realised with longitudinal and transverse transducers being alternately positioned inside Euro-D-modified punches. Key features of the data acquisition are described. Considerable attention was paid to an accurate displacement measurement and a synchronic acquisition of the ultrasonic signal. Vivapur 102 and Di-Cafos A150 were chosen for evaluation. In contrast to other published instrumentations, production-relevant powder densification speeds were feasible whilst featuring outstanding measurement precision. Maximum ultrasonic speed was achieved at maximum density. Materials differed considerably regarding the slope of the decompression phase, which might be suitable for assessing elasticity and speed sensitivity of powders or formulations without compressing twice. The developed set-up furthermore enables in-die measurements of apparent Young's modulus and apparent Poisson's ratio (i.e. their change throughout the course of the tableting process). Young's modulus increased upon densification and values match with literature data. Poisson's ratio increased linearly as a function of solid fraction for plastically deforming Vivapur 102, whereas it was practically constant for brittle Di-Cafos A150. Increased mechanistic understanding of deformation factors (e.g. rearrangement, fragmentation, elasticity) and estimation of mechanical compatibility of mixtures, is feasible. Moreover, in-die Young's modulus and Poisson's ratio are valuable for compression simulations based on finite or discrete element method.
Assuntos
Força Compressiva , Composição de Medicamentos/instrumentação , Modelos Químicos , Comprimidos/química , Ondas Ultrassônicas , Química Farmacêutica , Composição de Medicamentos/métodos , Módulo de Elasticidade , Estudos de Viabilidade , Análise de Elementos Finitos , PósRESUMO
Orange carotenoid proteins (OCPs) are photoswitchable macromolecules playing an important role in nonphotochemical quenching of excess energy in cyanobacterial light harvesting. Upon absorption of a blue photon (450-500 nm), OCPs undergo a structural change from the ground state OCPO to the active state OCPR, but high-resolution structures of the active state OCPR are not yet available. Here, we use small-angle scattering methods combined with simulation tools to determine low-resolution structures of the active state at low protein concentrations via two approaches: first, directly by in situ illumination of wild-type OCP achieving a turnover to the active state of >90% and second, by using the mutant OCPW288A anticipated to mimic the active state structure. Data fits assuming the shape of an ellipsoid yield three ellipsoidal radii of about 9, 29, and 51 ± 1 Å, in the case of the ground state OCPO. In the active state, however, the molecule becomes somewhat narrower with the two smaller radii being 9 and only 19 ± 3 Å, while the third dimension of the ellipsoid is significantly elongated to 85-92 ± 5 Å. Reconstitutions of the active state structure corroborate that OCPR is significantly elongated compared to the ground state OCPO and characterized by a separation of the N-terminal and C-terminal domains with unfolded N-terminal extension. By direct comparison of small-angle scattering data, we directly show that the mutant OCPW288A can be used as a structural analogue of the active state OCPR. The small-angle experiments are repeated for OCPO and the mutant OCPW288A at high protein concentrations of 50-65 mg/mL required for neutron spectroscopy investigating the molecular dynamics of OCP (see accompanying paper). The results reveal that the OCPO and OCPW288A samples for dynamics experiments are preferentially dimeric and widely resemble the structures of the ground and active states of OCP, respectively. This enables us to properly characterize the molecular dynamics of both states of OCP in the accompanying paper.
Assuntos
Proteínas de Bactérias/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/efeitos da radiação , Luz , Mutação , Difração de Nêutrons , Maleabilidade , Conformação Proteica , Espalhamento a Baixo Ângulo , Soluções/química , Synechocystis/química , Difração de Raios XRESUMO
Orange carotenoid proteins (OCPs), which are protecting cyanobacterial light-harvesting antennae from photodamage, undergo a pronounced structural change upon light absorption. In addition, the active state is anticipated to boost a significantly higher molecular flexibility similar to a "molten globule" state. Here, we used quasielastic neutron scattering to directly characterize the vibrational and conformational molecular dynamics of OCP in its ground and active states, respectively, on the picosecond time scale. At a temperature of 100 K, we observe mainly (vibronic) inelastic features with peak energies at 5 and 6 meV (40 and 48 cm-1, respectively). At physiological temperatures, however, two (Lorentzian) quasielastic components represent localized protein motions, that is, stochastic structural fluctuations of protein side chains between various conformational substates of the protein. Global diffusion of OCP is not observed on the given time scale. The slower Lorentzian component is affected by illumination and can be well-characterized by a jump-diffusion model. While the jump diffusion constant D is (2.82 ± 0.01) × 10-5 cm2/s at 300 K in the ground state, it is increased by â¼20% to (3.48 ± 0.01) × 10-5 cm2/s in the active state, revealing a strong enhancement of molecular mobility. The increased mobility is also reflected in the average atomic mean square displacement ⟨u2⟩; we determine a ⟨u2⟩ of 1.47 ± 0.05 Å in the ground state, but 1.86 ± 0.05 Å in the active state (at 300 K). This effect is assigned to two factors: (i) the elongated structure of the active state with two widely separated protein domains is characterized by a larger number of surface residues with a concomitantly higher degree of motional freedom and (ii) a larger number of hydration water molecules bound at the surface of the protein. We thus conclude that the active state of the orange carotenoid protein displays an enhanced conformational dynamics. The higher degree of flexibility may provide additional channels for nonradiative decay so that harmful excess energy can be more efficiently converted to heat.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Mutação , Difração de Nêutrons , Maleabilidade , Conformação Proteica , Soluções/química , Synechocystis/química , TemperaturaRESUMO
We used elastic incoherent neutron scattering (EINS) to find out if structural changes accompanying local hydrogen bond rupture are also reflected in global dynamical response of the protein complex. Chromatophore membranes from LH2-only strains of the photosynthetic bacterium Rhodobacter sphaeroides, with spheroidenone or neurosporene as the major carotenoids, were subjected to high hydrostatic pressure at ambient temperature. Optical spectroscopy conducted at high pressure confirmed rupture of tertiary structure hydrogen bonds. In parallel, we used EINS to follow average motions of the hydrogen atoms in LH2, which reflect the flexibility of this complex. A decrease of the average atomic mean square displacements of hydrogen atoms was observed up to a pressure of 5 kbar in both carotenoid samples due to general stiffening of protein structures, while at higher pressures a slight increase of the displacements was detected in the neurosporene mutant LH2 sample only. These data show a correlation between the local pressure-induced breakage of H-bonds, observed in optical spectra, with the altered protein dynamics monitored by EINS. The slightly higher compressibility of the neurosporene mutant sample shows that even subtle alterations of carotenoids are manifested on a larger scale and emphasize a close connection between the local structure and global dynamics of this membrane protein complex.
Assuntos
Complexos de Proteínas Captadores de Luz/química , Bacterioclorofilas/química , Carotenoides/química , Ligação de Hidrogênio , Pressão Hidrostática , Rhodobacter sphaeroides/químicaRESUMO
Spectral hole burning (SHB) and difference fluorescence line narrowing (ΔFLN) are routinely used for investigations of electron-phonon coupling in photosynthetic pigment-protein complexes as well as in other amorphous systems at cryogenic temperatures. Nevertheless, the Huang-Rhys factors S, an integral measure of electron-phonon coupling strength, and the phonon spectral densities obtained by SHB and ΔFLN over the past years have differed significantly in the case of certain photosynthetic pigment-protein complexes. In this work, the specific properties of both types of line-narrowing spectroscopic techniques that may lead to these discrepancies are critically analyzed by a combined experimental and computational approach, using the CP29 antenna complex of green plants as a suitable model system. We confirm that only ΔFLN at low fluence, by providing access to the homogeneously broadened spectrum, is able to deliver correct S values, while SHB may significantly under- or overestimate them, depending on the burn fluence. We also discuss possible other sources of discrepancies in the literature data, e.g., in the case of LHC II aggregates and correct numerical errors found in some previous records.
Assuntos
Elétrons , Complexos de Proteínas Captadores de Luz/química , Fônons , Complexo de Proteína do Fotossistema II/química , Espectrometria de Fluorescência/métodos , Brassica/química , Chlorobi/química , Transferência de Energia , Spinacia oleracea/químicaRESUMO
Dynamics-function correlations are usually inferred when molecular mobility and protein function are simultaneously impaired at characteristic temperatures or hydration levels. In this sense, excitation energy transfer in the photosynthetic light-harvesting complex II (LHC II) is an untypical example because it remains fully functional even at cryogenic temperatures relying mainly on interactions of electronic states with protein vibrations. Here, we study the vibrational and conformational protein dynamics of monomeric and trimeric LHC II from spinach using inelastic neutron scattering (INS) in the temperature range of 20-305 K. INS spectra of trimeric LHC II reveal a distinct vibrational peak at â¼2.4 meV. At temperatures above â¼160 K, however, the inelastic peak shifts toward lower energies, which is attributed to vibrational anharmonicity. A more drastic shift is observed at about 240 K, which is interpreted in terms of a "softening" of the protein matrix along with the dynamical transition. Monomeric LHC II exhibits a higher degree of conformational mobility at physiological temperatures, which can be attributed to a higher number of solvent-exposed side chains at the protein surface. The effects of the changes in protein dynamics on the spectroscopic properties of LHC II are considered in comparative model calculations. The absorption line shapes of a pigment molecule embedded into LHC II are simulated for the cases of (i) a rigid protein matrix, (ii) a protein matrix with temperature-dependent spectral density of protein vibrations, and (iii) temperature-dependent electron-phonon coupling strength. Our findings indicate that vibrational and conformational protein dynamics affect the spectroscopic (absorption) properties of LHC II at physiological temperatures.
Assuntos
Complexos de Proteínas Captadores de Luz/química , Clorofila/química , Transferência de Energia , Complexos de Proteínas Captadores de Luz/metabolismo , Difração de Nêutrons , Estrutura Terciária de Proteína , Spinacia oleracea/metabolismo , TemperaturaRESUMO
The cyanobacterium Acaryochloris marina developed two types of antenna complexes, which contain chlorophyll-d (Chl d) and phycocyanobilin (PCB) as light-harvesting pigment molecules, respectively. The latter membrane-extrinsic complexes are denoted as phycobiliproteins (PBPs). Spectral hole burning was employed to study excitation energy transfer and electron-phonon coupling in PBPs. The data reveal a rich spectral substructure with a total of four low-energy electronic states whose absorption bands peak at 633, 644, 654, and at about 673 nm. The electronic states at ~633 and 644 nm can be tentatively attributed to phycocyanin (PC) and allophycocyanin (APC), respectively. The remaining low-energy electronic states including the terminal emitter at 673 nm may be associated with different isoforms of PC, APC, or the linker protein. Furthermore, the hole burning data reveal a large number of excited state vibrational frequencies, which are characteristic for the chromophore PCB. In summary, the results are in good agreement with the low-energy level structure of PBPs and electron-phonon coupling parameters reported by Gryliuk et al. (BBA 1837:1490-1499, 2014) based on difference fluorescence line-narrowing experiments.
Assuntos
Cianobactérias/metabolismo , Transferência de Energia , Ficobiliproteínas/metabolismo , Vibração , Ficobiliproteínas/química , Espectrometria de Fluorescência , TemperaturaRESUMO
The structure of monomeric and trimeric photosystem I (PS I) of Thermosynechococcus elongatus BP1 (T. elongatus) was investigated by small-angle X-ray scattering (SAXS). The scattering data reveal that the protein-detergent complexes possess radii of gyration of 58 and 78 Å in the cases of monomeric and trimeric PS I, respectively. The results also show that the samples are monodisperse, virtually free of aggregation, and contain empty detergent micelles. The shape of the protein-detergent complexes can be well approximated by elliptical cylinders with a height of 78 Å. Monomeric PS I in buffer solution exhibits minor and major radii of the elliptical cylinder of about 50 and 85 Å, respectively. In the case of trimeric PS I, both radii are equal to about 110 Å. The latter model can be shown to accommodate three elliptical cylinders equal to those describing monomeric PS I. A structure reconstitution also reveals that the protein-detergent complexes are larger than their respective crystal structures. The reconstituted structures are larger by about 20 Å mainly in the region of the hydrophobic surfaces of the monomeric and trimeric PS I complexes. This seeming contradiction can be resolved by the addition of a detergent belt constituted by a monolayer of dodecyl-ß-D-maltoside molecules. Assuming a closest possible packing, a number of roughly 1024 and 1472 detergent molecules can be determined for monomeric and trimeric PS I, respectively. Taking the monolayer of detergent molecules into account, the solution structure can be almost perfectly modeled by the crystal structures of monomeric and trimeric PS I.
Assuntos
Proteínas de Bactérias/química , Complexo de Proteína do Fotossistema I/química , Multimerização Proteica , Espalhamento a Baixo Ângulo , Synechococcus/metabolismo , Difração de Raios X , Detergentes/química , Modelos Moleculares , Complexo de Proteína do Fotossistema I/metabolismo , Soluções , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Light harvesting and excitation energy transfer in photosynthesis are relatively well understood at cryogenic temperatures up to â¼100 K, where crystal structures of several photosynthetic complexes including the major antenna complex of green plants (LHC II) are available at nearly atomic resolution. The situation is much more complex at higher or even physiological temperatures, because the spectroscopic properties of antenna complexes typically undergo drastic changes above â¼100 K. We have addressed this problem using a combination of quasielastic neutron scattering (QENS) and optical spectroscopy on native LHC II and mutant samples lacking the Chl 2/Chl a 612 pigment molecule. Absorption difference spectra of the Chl 2/Chl a 612 mutant of LHC II reveal pronounced changes of spectral position and their widths above temperatures as low as â¼80 K. The complementary QENS data indicate an onset of conformational protein motions at about the same temperature. This finding suggests that excited state positions in LHC II are affected by protein dynamics on the picosecond time scale. In more detail, this means that at cryogenic temperatures the antenna complex is trapped in certain protein conformations. At higher temperature, however, a variety of conformational substates with different spectral position may be thermally accessible. At the same time, an analysis of the widths of the absorption difference spectra of Chl 2/Chl a 612 reveals three different reorganization energies or Huang-Rhys factors in different temperature ranges, respectively. These findings imply that (dynamic) pigment-protein interactions fine-tune electronic energy levels and electron-phonon coupling of LHC II for efficient excitation energy transfer at physiological temperatures.
Assuntos
Complexos de Proteínas Captadores de Luz/química , Clorofila/química , Transferência de Energia , Complexos de Proteínas Captadores de Luz/genética , Complexos de Proteínas Captadores de Luz/metabolismo , Mutagênese , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , Temperatura , TermodinâmicaRESUMO
Neutron spectroscopy provides experimental data on time-dependent trajectories, which can be directly compared to molecular dynamics simulations. Its importance in helping us to understand biological macromolecules at a molecular level is demonstrated by the results of a literature survey over the last two to three decades. Around 300 articles in refereed journals relate to neutron scattering studies of biological macromolecular dynamics, and the results of the survey are presented here. The scope of the publications ranges from the general physics of protein and solvent dynamics, to the biologically relevant dynamics-function relationships in live cells. As a result of the survey we are currently setting up a neutron Dynamics Data Bank (nDDB) with the aim to make the neutron data on biological systems widely available. This will benefit, in particular, the MD simulation community to validate and improve their force fields. The aim of the database is to expose and give easy access to a body of experimental data to the scientific community. The database will be populated with as much of the existing data as possible. In the future it will give value, as part of a bigger whole, to high throughput data, as well as more detailed studies. A range and volume of experimental data will be of interest in determining how quantitatively MD simulations can reproduce trends across a range of systems and to what extent such trends may depend on sample preparation and data reduction and analysis methods. In this context, we strongly encourage researchers in the field to deposit their data in the nDDB.
Assuntos
Bases de Dados de Compostos Químicos , Simulação de Dinâmica Molecular , Difração de Nêutrons , Biofísica/métodos , Biofísica/organização & administração , Biofísica/tendências , Carboidratos/química , Ácidos Nucleicos/química , Proteínas/químicaRESUMO
Until recently, it was believed that the CP29 protein from higher plant photosystem II (PSII) contains 8 chlorophylls (Chl's) per complex (Ahn et al. Science 2008, 320, 794-797; Bassi et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10056-10061) in contrast to the 13 Chl's revealed by the recent X-ray structure (Pan et al. Nat. Struct. Mol. Biol. 2011, 18, 309-315). This disagreement presents a constraint on the interpretation of the underlying electronic structure of this complex. To shed more light on the interpretation of various experimental optical spectra discussed in the accompanying paper (part I, DOI 10.1021/jp4004328 ), we report here calculated low-temperature (5 K) absorption, fluorescence, hole-burned (HB), and 300 K circular dichroism (CD) spectra for CP29 complexes with a different number of pigments. We focus on excitonic structure and the nature of the low-energy state using modeling based on the X-ray structure of CP29 and Redfield theory. We show that the lowest energy state is mostly contributed to by a612, a611, and a615 Chl's. We suggest that in the previously studied CP29 complexes from spinach (Pieper et al. Photochem. Photobiol.2000, 71, 574-589) two Chl's could have been lost during the preparation/purification procedure, but it is unlikely that the spinach CP29 protein contains only eight Chl's, as suggested by the sequence homology-based study (Bassi et al. Proc. Natl. Acad. Sci. U.S.A.1999, 96, 10056-10061). The likely Chl's missing in wild-type (WT) CP29 complexes studied previously (Pieper et al. Photochem. Photobiol. 2000, 71, 574-589) include a615 and b607. This is why the nonresonant HB spectra shown in that reference were ~1 nm blue-shifted with the low-energy state mostly localized on about one Chl a (i.e., a612) molecule. Pigment composition of CP29 is discussed in the context of light-harvesting and excitation energy transfer.
Assuntos
Complexos de Proteínas Captadores de Luz/química , Complexo de Proteína do Fotossistema II/química , Clorofila/química , Clorofila/metabolismo , Dicroísmo Circular , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Complexos de Proteínas Captadores de Luz/metabolismo , Modelos Moleculares , Complexo de Proteína do Fotossistema II/isolamento & purificação , Complexo de Proteína do Fotossistema II/metabolismo , Spinacia oleracea/metabolismoRESUMO
Recent structural data revealed that the CP29 protein of higher plant photosystem II (PSII) contains 13 chlorophylls (Chl's) per complex (Pan et al. Nat. Struct. Mol. Biol. 2011, 18, 309), i.e., five Chl's more than in the predicted CP29 homology-based structure model (Bassi et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10056). This lack of consensus presents a constraint on the interpretation of CP29 optical spectra and their underlying electronic structure. To address this problem, we present new low-temperature (5 K) absorption, fluorescence, and hole-burned (HB) spectra for CP29 proteins from spinach, which are compared with the previously reported data. We focus on excitation energy transfer (EET) and the nature of the lowest-energy state(s). We argue that CP29 proteins previously studied by HB spectroscopy lacked at least one Chl a molecule (i.e., a615 or a611), which along with Chl a612 contribute to the lowest energy state in more intact CP29, and one Chl b (most likely b607). This is why the low-energy state and fluorescence maxima reported by Pieper et al. (Photochem. Photobiol.2000, 71, 574) were blue-shifted by ~1 nm, the low-energy state appeared to be highly localized on a single Chl a molecule, and the position of the low-energy state was independent of burning fluence. In contrast, the position of the nonresonant HB spectrum shifts blue with increasing fluence in intact CP29, as this state is strongly contributed to by several pigments (i.e., a611, a612, a615, and a610). Zero-phonon hole widths obtained for the Chl b band at 638.5 nm (5 K) revealed two independent Chl b â Chl a EET times, i.e., 4 ± 0.5 and 0.4 ± 0.1 ps. The latter value is a factor of 2 faster than previously observed by HB spectroscopy and very similar to the one observed by Gradinaru et al. (J. Phys. Chem. B 2000, 104, 9330) in pump-probe experiments. EET time from 650 nm Chl b â Chl a and downward EET from Chl(s) a state(s) at 665 nm occurs in 4.9 ± 0.7 ps. These findings provide important constraints for excitonic calculations that are discussed in the accompanying paper (part II, DOI 10.1021/jp4004278 ).