Search

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-β-d-glucoside (OG) and n-dodecyl-β-D-maltoside (β-DM). The data reveal that the LHCII-OG complex is equivalent to the trimeric crystal structure. Remarkably, however, we observe─for the first time─a stable oligomer composed of three LHCII trimers in the case of the LHCII-β-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.

Was war 2021 eigentlich für ein Jahr für die Verpackungsbranche? Was wird bleiben von diesen zwölf Monaten? Auf was darf man sich im Jahr 2022 freuen? Was war super in 2021 – und was vielleicht weniger schön? Im letzten packREPORT des Jahres lesen Sie exklusiv die Einschätzungen von fünf bekannten Entscheidungsträgern der Branche.

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.

Trimeric photosystem I from the cyanobacterium Thermosynechococcus elongatus (TePSI) is an intrinsic membrane protein, which converts solar energy into electrical energy by oxidizing the soluble redox mediator cytochrome c6 (Cyt c6) and reducing ferredoxin. Here, we use cryo-electron microscopy and small angle neutron scattering (SANS) to characterize the transient binding of Cyt c6 to TePSI. The structure of TePSI cross-linked to Cyt c6 was solved at a resolution of 2.9 A and shows additional cofactors as well as side chain density for 84% of the peptide chain of subunit PsaK, revealing a hydrophobic, membrane intrinsic loop that enables binding of associated proteins. Due to the poor binding specificity, Cyt c6 could not be localized with certainty in our cryo-EM analysis. SANS measurements confirm that Cyt c6 does not bind to TePSI at protein concentrations comparable to those for cross-linking. However, SANS data indicate a complex formation between TePSI and the non-native mitochondrial cytochrome from horse heart (Cyt cHH). Our study pinpoints the difficulty of identifying very small binding partners (less than 5% of the overall size) in EM structures when binding affinities are poor. We relate our results to well resolved co-structures with known binding affinities and recommend confirmatory methods for complexes with KM values higher than 20 μM.

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.

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

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.

High-resolution structures of photosynthetic pigment–protein complexes are often determined using crystallography or cryo-electron microscopy (cryo-EM), which are restricted to the use of protein crystals or to low temperatures, respectively. However, functional studies and biotechnological applications of photosystems necessitate the use of proteins isolated in aqueous solution, so that the relevance of high-resolution structures has to be independently verified. In this regard, small-angle neutron and X-ray scattering (SANS and SAXS, respectively) can serve as the missing link because of their capability to provide structural information for proteins in aqueous solution at physiological temperatures. In the present review, we discuss the principles and prototypical applications of SANS and SAXS using the photosynthetic pigment–protein complexes phycocyanin (PC) and Photosystem I (PSI) as model systems for a water-soluble and for a membrane protein, respectively. For example, the solution structure of PSI was studied using SAXS and SANS with contrast matching. A Guinier analysis reveals that PSI in solution is virtually free of aggregation and characterized by a radius of gyration of about 75 Å. The latter value is about 10% larger than expected from the crystal structure. This is corroborated by an ab initio structure reconstitution, which also shows a slight expansion of Photosystem I in buffer solution at room temperature. In part, this may be due to conformational states accessible by thermally activated protein dynamics in solution at physiological temperatures. The size of the detergent belt is derived by comparison with SANS measurements without detergent match, revealing a monolayer of detergent molecules under proper solubilization conditions. Eesti Teadusfondi

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-s-d-maltoside (sDM) 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 A. 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 A, i.e., it is about 6 A 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 sDM-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 A. 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 sDM-dPSIIcc.

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.

The structure of phycobiliproteins of the cyanobacterium Acaryochloris marina was investigated in buffer solution at physiological temperatures, i.e. under the same conditions applied in spectroscopic experiments, using small angle neutron scattering. The scattering data of intact phycobiliproteins in buffer solution containing phosphate can be well described using a cylindrical shape with a length of about 225 A and a diameter of approximately 100 A. This finding is qualitatively consistent with earlier electron microscopy studies reporting a rod-like shape of the phycobiliproteins with a length of about 250 (M. Chen et al., FEBS Letters 583, 2009, 2535) or 300 A (J. Marquart et al., FEBS Letters 410, 1997, 428). In contrast, phycobiliproteins dissolved in buffer lacking phosphate revealed a splitting of the rods into cylindrical subunits with a height of 28 A only, but also a pronounced sample aggregation. Complementary small angle neutron and X-ray scattering experiments on phycocyanin suggest that the cylindrical subunits may represent either trimeric phycocyanin or trimeric allophycocyanin. Our findings are in agreement with the assumption that a phycobiliprotein rod with a total height of about 225 A can accommodate seven trimeric phycocyanin subunits and one trimeric allophycocyanin subunit, each of which having a height of about 28 A. The structural information obtained by small angle neutron and X-ray scattering can be used to interpret variations in the low-energy region of the 4.5 K absorption spectra of phycobiliproteins dissolved in buffer solutions containing and lacking phosphate, respectively.

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.

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.

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-β-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.

Vibrational and conformational protein dynamics of photosystem II (PS II) membrane fragments from spinach were investigated by elastic and inelastic incoherent neutron scattering (EINS and IINS). As to the EINS experiments, the average atomic mean square displacement values of PS II membrane fragments hydrated at a relative humidity of 57% exhibit a dynamical transition at ~230K. In contrast, the dynamical transition was absent at a relative humidity of 44%. These findings are in agreement with previous studies which reported a “freezing” of protein mobility due to dehydration (Pieper et al. (2008) Eur. Biophys. J. 37: 657–663) and its correlation with an inhibition of electron transfer from QA− to QB (Kaminskaya et al. (2003) Biochemistry 42, 8119–8132). IINS spectra of a sample hydrated at a relative humidity of 57% show a distinct Boson peak at ~7.5meV at 20K, which shifts towards lower energy values upon temperature increase to 250K. This unexpected effect is interpreted in terms of a “softening” of the protein matrix along with the onset of conformational protein dynamics as revealed by the EINS experiments. Information on the density of vibrational states of pigment–protein complexes is important for a realistic calculation of excitation energy transfer kinetics and spectral lineshapes and is often routinely obtained by optical line-narrowing spectroscopy at liquid helium temperature. The data presented here demonstrate that IINS is a valuable experimental tool in determining the density of vibrational states not only at cryogenic, but also at nearly physiological temperatures up to 250K. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Elastic incoherent neutron scattering (EINS), a non-invasive technique which is capable of measuring the mean square displacement of atoms in the sample, has been widely used in biology for exploring the dynamics of proteins and lipid membranes but studies on photosynthetic systems are scarce. In this study we investigated the dynamic characteristics of Photosystem II (PSII) membrane fragments between 280 and 340 K, i.e., in the physiological temperature range and in the range of thermal denaturation of some of the protein complexes. The mean square displacement values revealed the presence of a hydration-sensitive transition in the sample between 310 and 320 K, suggesting that the oxygen evolving complex (OEC) plays an important role in the transition. Indeed, in samples in which the OEC had been removed by TRIS- or heat-treatments (323 and 333 K) no such transition was found. Further support on the main role of OEC in these reorganizations is provided by data obtained from differential scanning calorimetry experiments, showing marked differences between the untreated and TRIS-treated samples. In contrast, circular dichroism spectra exhibited only minor changes in the excitonic interactions below 323 K, showing that the molecular organization of the pigment-protein complexes remains essentially unaffected. Our data, along with earlier incoherent neutron scattering data on PSII membranes at cryogenic temperatures (Pieper et al., Biochemistry 46:11398-11409, 2007), demonstrate that this technique can be applied to characterize the dynamic features of PSII membranes, and can be used to investigate photosynthetic membranes under physiologically relevant experimental conditions.

This short review paper describes spectroscopic studies on pigment–pigment and pigment–protein interactions of chlorophyll (Chl) a and b bound to the recombinant protein of class IIa water soluble chlorophyll protein (WSCP) from cauliflower. Two Chls form a strongly excitonically coupled open sandwich dimer within the tetrameric protein matrix. In marked contrast to the mode of excitonic coupling of Chl and bacterio-Chl molecules in light harvesting complexes and reaction centers of all photosynthetic organisms, the unique structural pigment array in the Chl dimer of WSCP gives rise to an upper excitonic state with a large oscillator strength. This property opens the way for thorough investigations on exciton relaxation processes in Chl–protein complexes. Lifetime measurements of excited singlet states show that the unusual stability towards photodamage of Chls bound to WSCP, which lack any protective carotenoid molecule, originates from a high diffusion barrier to interaction of molecular dioxygen with Chl triplets. Site selective spectroscopic methods provide a wealth of information on the interactions of the Chls with the protein matrix and on the vibronic structure of the pigments. The presented data and discussions illustrate the great potential of WSCP as a model system for systematic experimental and theoretical studies on the functionalizing of Chls by the protein matrix. It opens the way for further detailed analyses and a deeper understanding of the properties of pigment protein complexes.

The cyanobacterium Acaryochloris marina is unique because it mainly contains Chlorophyll d (Chl d) in the core complexes of PS I and PS II instead of the usually dominant Chl a. Furthermore, its light harvesting system has a structure also different from other cyanobacteria. It has both, a membrane-internal chlorophyll containing antenna and a membrane-external phycobiliprotein (PBP) complex. The first one binds Chl d and is structurally analogous to CP43. The latter one has a rod-like structure consisting of three phycocyanin (PC) homohexamers and one heterohexamer containing PC and allophycocyanin (APC). In this paper, we give an overview on the investigations of excitation energy transfer (EET) in this PBP-light-harvesting system and of charge separation in the photosystem II (PS II) reaction center of A. marina performed at the Technische Universität Berlin. Due to the unique structure of the PBP antenna in A. marina, this EET occurs on a much shorter overall time scale than in other cyanobacteria. We also briefly discuss the question of the pigment composition in the reaction center (RC) of PS II and the nature of the primary donor of the PS II RC.

Difference fluorescence line-narrowing spectroscopy at 4.5 K was employed to investigate electron-phonon and electron-vibrational coupling strengths of the lower exciton level of water-soluble chlorophyll-binding protein (WSCP) from cauliflower reconstituted with chlorophyll a or chlorophyll b, respectively. The electron-phonon coupling is found to be moderate with integral Huang-Rhys factors S in the order of 0.81-0.85. A weak dependence of S on excitation wavelength within the inhomogeneously broadened fluorescence origin band is attributed to a sizable contribution of nonresonant excitation that varies with excitation wavelength. The strongly asymmetric and highly structured one-phonon profile is characterized by a peak phonon frequency (ω(m)) of ~24 cm(-1) and further discernible peaks at 48 and 88 cm(-1), respectively. A structural assignment of this unusual one-phonon profile is proposed. As will be shown in the accompanying paper (part II) (DOI 10.1021/jp111457t), the parameters of electron-phonon coupling readily account for shape and position of the fluorescence origin bands at 666.1 and 683.8 nm for chlorophyll b- and chlorophyll a-WSCP, respectively. A rich structure of S(1)→S(0) vibrational frequencies was resolved in the wavenumber range between 180 and 1665 cm(-1) for both chlorophyll a- and chlorophyll b-WSCP. The corresponding individual Huang-Rhys factors fall in the range between 0.0011 and 0.0500. To the best of our knowledge, this is the first report of S-factors for vibrational modes of chlorophyll b. Most remarkable is the presence of two additional modes at 228 and 327 cm(-1) compared with the vibrational spectrum of chlorophyll in solution. The additional modes can most likely be attributed to H-bond formation in the vicinity of the chlorophyll molecule bound by WSCP.

44 Pag., 2 Tabl. The definitive version is available at: http://pubs.acs.org/journal/jpcbfk, The parameters of barrier distributions on the protein energy landscape in the excited electronic state of the pigment/protein system have been determined by means of spectral hole burning for the lowest-energy pigments of CP43 core antenna complex and CP29 minor antenna complex of spinach Photosystem II (PS II) as well as of trimeric and monomeric LHCII complexes transiently associated with the pea Photosystem I (PS I) pool. All of these complexes exhibit sixty to several hundred times lower spectral hole burning yields as compared with molecular glassy solids previously probed by means of the hole growth kinetics measurements. Therefore, the entities (groups of atoms), which participate in conformational changes in protein, appear to be significantly larger and heavier than those in molecular glasses. No evidence of a small (1 cm−1) spectral shift tier of the spectral diffusion dynamics has been observed. Therefore, our data most likely reflect the true barrier distributions of the intact protein and not those related to the interface or surrounding host. Possible applications of the barrier distributions as well as the assignments of low-energy states of CP29 and LHCII are discussed in light of the above results., Research at Concordia University is supported by NSERC and CFI. R.P. would like to thank Spanish MICINN (grant AGL2008-00377). M.S. acknowledges the contribution of the Photosynthetic Systems Program, Chemical Sciences, Geosciences, and Biosciences Division, Basic Energy Sciences, USDOE. J.P. and K.-D.I. gratefully acknowledge support from Deutsche Forschungsgemeinschaft (SFB 429, TP A1, and TP A3, respectively).

The internal molecular dynamics of proteins plays an important role in a number of functional processes in native photosystems. Prominent examples include the photocycle of bacteriorhodopsin and electron transfer in the reaction center of plant photosystem II. In this regard, the recently developed technique of time-resolved quasielastic neutron scattering with laser excitation opens up new perspectives for the study of protein/membrane dynamics in specific functional states of even complex systems. The first direct observation of a functionally modulated protein dynamics has just recently been reported for the model system bacteriorhodopsin (Pieper et al., Phys. Rev. Lett. 100, 2008, 228103.), where a transient softening of the protein was observed on a timescale of approximately 1 ms along with the large-scale structural change in the M-intermediate of bacteriorhodopsin. In contrast, photosystem II membrane fragments with inhibited electron transfer show a suppression of protein dynamics approximately 160 mus after the actinic laser flash (Pieper and Renger, Biochemistry 48, 2009, 6111). This effect may reflect aggregation-like conformational changes capable of dissipation of excess excitation energy to prevent photodamage in the absence of Q(A)--Q(B) electron transfer. These findings indicate that proteins exhibit a remarkable flexibility to accommodate different functional processes. This contribution will discuss methodical aspects, challenges, and recent applications of laser-excited, time-resolved quasielastic neutron scattering.

Incoherent inelastic neutron scattering experiments were performed on Na0.7CoO2 and Na0.28CoO2.1.3H2O in order to understand how the dynamics of the hydrogen-bond network of water is modified in the triangular crystalline lattice NaxCoO2.yH2O. Using quasi-elastic neutron scattering (QENS), we were able to differentiate between two types of proton dynamics: a fast process (due to water strongly bound into the sodium cobalt oxyhydrate structure during the hydration process) and a slow process (likely attributable to a collective motion). High-resolution QENS experiments, carried out on Na0.28CoO2.1.3H2O, show that, at temperatures above 310 K, the water dynamics can be well-described by a random jump diffusion model characterized by a diffusion constant equal to 0.9 x 10(-9)m2/s, which is significantly lower than the rate of diffusion for bulk water. Furthermore, our results indicate that, at room temperature, the sodium ions have no influence on the rotational dynamics of the "fast" water molecules.

Light harvesting and excitation energy transfer in photosynthesis are relatively well understood at cryogenic temperatures up to ∼100 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 ∼100 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 ∼80 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.

A new hydrostatic pressure cell designed for small-angle neutron scattering (SANS) and quasi-elastic neutron scattering (QENS) experiments on biomolecular solutions has been developed at the Laboratoire L?on Brillouin. SANS and QENS experiments on bovine pancreatic trypsin inhibitor (BPTI), a small protein, have been performed and show us that this pressure cell is well adapted to this kind of experiments. Interesting results about the influence of pressure (1?6200?bar) on structure and dynamics of BPTI have been obtained.

This short communication addresses three topics of photosynthetic water cleavage in Photosystem II (PS II): (a) effect of protonation in the acidic range on the extent of the ‘fast’ ns kinetics of P680+· reduction by Y Z , (b) mechanism of O–O bond formation and (c) role of protein flexibility in the functional integrity of PS II. Based on measurements of light-induced absorption changes and quasielastic neutron scattering in combination with mechanistic considerations, evidence is presented for the protein acting as a functionally active constituent of the water cleavage machinery, in particular, for directed local proton transfer. A specific flexibility emerging above a threshold of about 230 K is an indispensable prerequisite for oxygen evolution and plastoquinol formation.

Methyl iodide enclosed in the 51264 cage of cubic II water clathrate is investigated by neutron spectroscopy in the temperature regime from 1.8 to 145?K. The three tunnelling bands observed at the lowest temperature at 497, 372 and 243??eV of relative intensities 2 : 3 : 5 represent three distinct adsorption sites of CH3I. Sites of occurrence probabilities corresponding closely to the observed line intensities are related to the three types of hydrogen bonds of the cage. The band at 372??eV shows fast spin conversion compared to the two others. The corresponding site is facing the shortest O?H..O hydrogen bond with the fastest proton dynamics. Spin conversion may thus reflect hydrogen jumps within the H-bond network of host molecules. Jump rates of the order of 10?4?s?1 can be estimated. Above a temperature T ~ 45?K the methyl dynamics become classical and can be described by rotational diffusion on a circle. A second quasielastic process becomes visible at a higher temperature and is identified as head-to-tail flips of the whole molecule with a characteristic jump distance of about 5 ?. Above T ~ 90?K translational freedom of the CH3I leads to a rattling mode appearing at ~0.95?meV.

Methyl iodide water clathrates crystallize in the cubic II structure. Methyl tunneling spectra of CH3I identify three adsorption sites. The presence of 3 types of H-bonds in the cage surface are considered as likely origin. The barriers to rotation of methyl groups are low at all sites. Due to finite and different spin conversion times two of the three excited tunnel levels are not in thermal equilibrium. The classical methyl dynamics is almost unaffected by the weak potentials and described as rotational diffusion. A second slower quasielastic process may be interpreted as overall rotation of the molecule.

Results of inelastic neutron scattering (INS) experiments are reported for the solubilized trimeric light-harvesting complex of photosystem II (LHC II) in the temperature range from 5 to 100 K. Two...

Quasielastic neutron scattering has been used to investigate the dynamical behavior of H(2)O in water/tert-butyl alcohol solutions. The measurements were made at fixed temperature (293 K) as a function of tert-butyl alcohol molar fraction, x, in the range 0-0.042. The data have been compared to those of pure water in the temperature range 269-293 K. The effect of tert-butyl alcohol addition on water dynamics is equivalent to that obtained by lowering the temperature of pure water by an amount proportional to the alcohol concentration. The temperature dependence of the diffusivity parameters in pure water and their concentration dependence in tert-butyl alcohol/water solutions can be rescaled to a common curve attributing to each solution a concentration-dependent "structural temperature" lower than the actual thermodynamic one. These results can be understood in terms of Stillinger's picture of water structuring and of other more recent theoretical pictures that emphasize the influence of the geometrical properties of hydrogen bond networks on water mobility.

We present a detailed study of the local dynamics of short polyethylene (PE) chains using molecular dynamics (MD) simulations and quasi-elastic neutron scattering (QENS) data. QENS measurements were carried out on two samples having different chain lengths: C44H90 and PE2K (Mw = 2150 g/mol). The incoherent dynamic structure factors obtained from experiments at T = 450 K and MD simulations carried out at the same temperature are compared in the range 0.5 ≤ Q ≤ 2.0 A-1. Agreement between experimental data and simulations is quantitative. Attempts are made to characterize the momentum transfer and the chain length dependence of the I(Q,t) and S(Q,ω) data on the basis of the Kohlrausch−Williams−Watts (KWW) function and two exponentials relaxation functions in time. As MD simulation data cover a broader frequency window and have a very low statistical noise, a more refined analysis of the intermediate scattering functions in terms of a continuous linear combination of exponentials weighted by a distribution o...

We performed an inelastic and quasielastic neutron scattering investigation of the molecular dynamics of lysozyme solvated in glycerol, at different water contents h (g water/g lysozyme). At low temperature, dry lysozyme solvated with glycerol shows a low-frequency vibrational bump located at around 4 meV. Contrary to the behaviour of strongly hydrated protein powders, this peak is well visible also at room temperature and persists in the experimental spectra at all the hydration degrees. The quasielastic contribution increases remarkably with h, thus suggesting that water molecules perform their plasticizing action by allowing the onset of confined diffusive motions of protein groups. The quasielastic signal could be decomposed in two Lorentzian components, representing motions with characteristic times of, respectively, 15 and 0.8 ps. The Lorentzian curves exhibit constant widths but variable intensities as a function of the hydration degree. The trend of the estimated quasielastic structure factors suggests that a preferential hydration effect may take place in lysozyme solvated with glycerol and water.

The microscopic mechanisms of water diffusion in fully hydrated porcine stratum corneum (SC) have been studied by a combination of incoherent quasielastic neutron scattering (QENS) and pulsed field gradient-nuclear magnetic resonance (PFG-NMR) for two sample orientations. The presence of three types of water in fully hydrated SC is inferred on the basis of water sorption isotherm data, i.e., (a) bound and (b) weakly bound hydration water forming layers between adjacent lipid bilayers of SC, as well as (c) bulk water probably located in the corneocytes and in intercellular regions. Water self-diffusion coefficients for motions parallel and perpendicular to the membrane plane of D ∥ =3.30×10 −10 m 2 /s and D ⊥ =1.56×10 −10 m 2 /s, respectively, were determined by PFG-NMR and assigned to the translational diffusion of weakly bound water. QENS measurements have been carried out using different samples hydrated with H 2 O and D 2 O, respectively, in order to separate the contribution of SC from that of the water. The QENS data for both sample orientations and two different energy resolutions can be fitted by a model which accounts for the microscopic dynamics of all three aforementioned types of water. This analysis establishes rotational diffusion coefficients for bound and weakly bound hydration water of 0.025 and 0.030 meV, respectively. Furthermore, the QENS data prove the presence of bulk water in fully hydrated SC samples.

This short review summarizes our current knowledge about the functional relevance of protein dynamics in photosynthetic reaction centers. In the case of Photosystem II membrane fragments, elastic and quasielastic neutron scattering experiments reveal a dynamical transition at about 240 K corresponding to the activation of picosecond molecular motions. Likewise, a “freezing” of molecular dynamics is observed upon dehydration. Intriguingly, these effects correlate with the pronounced temperature- and hydration-dependence of specific electron transfer steps in Photosystem II indicating that molecular dynamics is an indispensable prerequisite for its function. Thus, electron transfer in Photosystem II appears to be a prototypical example for a dynamics-function correlation. Finally, the laser-neutron pump-probe technique is shown to permit in-situ monitoring of molecular dynamics in specific functional states of a protein in real time.

Vibrational dynamics of the light-harvesting complex II (LHC II) from spinach was investigated by quasi- and inelastic neutron scattering (QENS and INS) at three different temperatures of 80, 160, and 285 K. QENS/INS spectra of solubilised LHC II and of the corresponding buffer solution were obtained separately and exhibit characteristic inelastic features. After subtraction of the buffer contribution, the INS spectrum of LHC II reveals a distinct Boson peak at ∼ 2.5 meV at 80 K that shifts towards lower energies if the temperature is increased to 285 K. This effect is interpreted in terms of a “softening” of the protein matrix along with the dynamical transition at ∼ 240 K. Our findings indicate that INS is a valuable method to obtain the density of vibrational states not only at cryogenic, but also at physiological temperatures.

Properties of biomembranes are modified in presence of additives such as cholesterol (chol) or proteins. Azobenzene-cholesterol (azo-chol) is a photoactive variety of chol since its azo-headgroup exhibits a reversible change of the conformation upon illumination (365nm: trans to cis; 455nm: cis to trans). Quasielastic neutron scattering (QENS) was applied to resolve lipid dynamics within biomimetic membranes consisting of a 1-palmitoyl-2-oleoyl-sn-phosphatidyl-choline (POPC) matrix with embedded azo-chol in either of the two conformational states. A pure POPC membrane and also a POPC membrane with inactive chol were used as control systems. The results report on the fast intramolecular motion of the protons in the POPC chains, a slow hop diffusion and an even slower mode both of the whole POPC molecule. The intramolecular motion and the hop diffusion are modified by the presence of azo-chol in the host membrane as compared with the control systems, but do not depend on the isomerization state. The slowest mode is though sensitive to the isomerization. The findings show the capacity of azo-chol for tuning the dynamics and most probably also the structures of the host lipid membrane though photoactivation.

In photosynthetic antenna complexes, apart from defining the positions and orientations of the pigment molecules, the protein matrix plays an important role in excitation energy transfer dynamics. The low-frequency protein vibrations—often referred to as phonons—serve as acceptor modes in nonadiabatic excitation energy transfer between energetically inequivalent electronic or excitonic energy states, assuring a spatially and energetically directed flow of excitation energy within an antenna complex. Due to electron–phonon and electron–vibrational coupling, the purely electronic or excitonic transitions of pigment molecules are usually accompanied by a broad and asymmetric low-frequency sideband spreading a few hundred wavenumbers and, in addition, by a number of distinct lines covering the frequency range between ~200 and 1,700 cm−1. The low-frequency sideband peaking at 20–30 cm−1 is typically identified with a continuous distribution of widely delocalized protein vibrations. The narrow lines at higher frequencies can mostly be attributed to localized pigment vibrations of (bacterio-) chlorophyll molecules, which are only slightly modified by the surrounding protein matrix. In conventional absorption or fluorescence spectra measured at cryogenic temperatures this substructure is usually hidden by significant inhomogeneous broadening due to the heterogeneity of the amorphous protein matrix. Line-narrowing spectroscopies like spectral hole burning and (difference) fluorescence line-narrowing have been proved to be powerful experimental tools for unraveling the hidden homogeneous spectral features from the inhomogeneously broadened spectra. This contribution focuses on electron–phonon and exciton–phonon coupling in photosynthetic pigment–protein complexes. It also discusses the underlying concepts of electron–phonon and electron–vibrational coupling starting from the Franck–Condon principle and the general composition of homogeneously and inhomogeneously broadened spectra of pigment–protein complexes. The advantages and limitations of different spectroscopic techniques in revealing the electron–phonon and electron–vibrational coupling parameters are discussed based on model calculations. The chapter concludes with a discussion of recent results on electron–phonon and electron–vibrational coupling for isolated (bacterio-) chlorophyll molecules as well as for selected photosynthetic pigment–protein complexes.

In adaption to its specific environmental conditions, the cyanobacterium Acaryochloris marina developed two different types of light-harvesting complexes: chlorophyll-d-containing membrane-intrinsic complexes and phycocyanobilin (PCB) — containing phycobiliprotein (PBP) complexes. The latter complexes are believed to form a rod-shaped structure comprising three homo-hexamers of phycocyanin (PC), one hetero-hexamer of phycocyanin and allophycocyanin (APC) and probably a linker protein connecting the PBPs to the reaction centre. Excitation energy transfer and electron-vibrational coupling in PBPs have been investigated by selectively excited fluorescence spectra. The data reveal a rich spectral substructure with a total of five low-energy electronic states with fluorescence bands at 635nm, 645nm, 654nm, 659nm and a terminal emitter at about 673nm. The electronic states at ~635 and 645nm are tentatively attributed to PC and APC, respectively, while an apparent heterogeneity among PC subunits may also play a role. The other fluorescence bands may be associated with three different isoforms of the linker protein. Furthermore, a large number of vibrational features can be identified for each electronic state with intense phonon sidebands peaking at about 31 to 37cm−1, which are among the highest phonon frequencies observed for photosynthetic antenna complexes. The corresponding Huang-Rhys factors S fall in the range between 0.98 (terminal emitter), 1.15 (APC), and 1.42 (PC). Two characteristic vibronic lines at about 1580 and 1634cm−1 appear to reflect CNH+ and CC stretching modes of the PCB chromophore, respectively. The exact phonon and vibrational frequencies vary with electronic state implying that the respective PCB chromophores are bound to different protein environments. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.

International audience; 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.

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 → 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 → 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 ).

The polymer segmental dynamics is investigated in a series of silica-filled rubbers. The presence of inert fillers in polymers greatly affects the mechanical and physical performance of the final materials. For example, silica has been proposed as a reinforcing agent of elastomers in tire production. Results from quasielastic neutron scattering and Dynamic Mechanical Thermal Analysis (DMTA) measurements are presented on styrene–ran-butadiene rubber filled with silica. A clear indication is obtained of the existence of a bimodal dynamics, which can be rationalized in terms of the relaxation of bulk rubber and the much slower relaxation of the rubber adsorbed on the filler surface.

In photosynthetic antenna complexes, the coupling of electronic transitions to low-frequency vibrations of the protein matrix (phonons) plays an essential role in light absorption and ultra-fast excitation energy transfer (EET). The model calculations presented here indicate that inelastic neutron scattering experiments provide invaluable information on the phonon density of states for light-harvesting complex II, which may permit a consistent interpretation of contradictory results from high-resolution optical spectroscopy.

Tert-butyl alcohol (TBA) and trimethyl-amino-N-oxide (TMAO) are organic molecules with the same hydrophobic moiety and a different polar group. They can be used as model systems to study how hydrophobic molecules in aqueous solutions affect water dynamics. We report here first results of a quasi-elastic neutron-scattering experiment on H2O/TBA and H2O/TMAO mixtures, varying the solute concentration in the range 0–2 mol %. In both systems, the effect on the microdiffusivity parameters upon increasing the solute concentration is similar to that observed in pure water on decreasing the temperature: the water dynamics slows down. In the concentration range analysed, no marked differences have been observed between the two systems.