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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.

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

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.

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.

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 ∼2.4 meV. At temperatures above ∼160 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.

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.

We studied the effects of temperature and hydrostatic pressure on the dynamical properties and folding stability of highly concentrated lysozyme solutions in the absence and presence of the osmolytes trimethylamine-N-oxide (TMAO) and urea. Elastic incoherent neutron scattering (EINS) was applied to determine the mean-squared displacement (MSD) of the protein's hydrogen atoms to yield insights into the effects of these cosolvents on the averaged sub-nanosecond dynamics in the pressure range from ambient up to 4000 bar. To evaluate the additional effect of self-crowding, two protein concentrations (80 and 160 mg mL-1) were used. We observed a distinct effect of TMAO on the internal hydrogen dynamics, namely a reduced mobility. Urea, on the other hand, revealed no marked effect and consequently, no counteracting effect in an urea-TMAO mixture was observed. Different from the less concentrated protein solution, no significant effect of pressure on the MSD was observed for 160 mg mL-1 lysozyme. The EINS experiments were complemented by Fourier-transform infrared (FTIR) spectroscopy measurements, which led to additional insights into the folding stability of lysozyme under the various environmental conditions. We observed a stabilization of the protein in the presence of the compatible osmolyte TMAO and a destabilization in the presence of urea against temperature and pressure for both protein concentrations. Additionally, we noticed a slight destabilizing effect upon self-crowding at very high protein concentration (160 mg mL-1), which is attributable to transient destabilizing intermolecular interactions. Furthermore, a pressure-temperature diagram could be obtained for lysozyme at these high protein concentrations that mimics densely packed intracellular conditions.

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.

Low-density lipoprotein (LDL) is the principal cholesterol transporter in the human blood circulation. The quasi-spherical LDL particles (∼20 nm) are made up of a complex combination of various lipids and a large single amphipathic protein moiety, named apolipoprotein B-100. LDL has a hydrophobic core and an amphiphilic shell. Each particle has a specific phase transition temperature (Tm) corresponding to the melting of the core lipids from an ordered liquid crystalline to a disordered fluid phase.The structural impact of high hydrostatic pressure (HHP) was studied with different types of LDL (native, oxidized and triglyceride rich) with SANS (PSI, Switzerland) and SAXS (Elettra, Italy). The HHP ranged from 50 to 3000 bar. Temperature points were chosen below, on and above Tm.The pair distance distribution functions p(r) of the SAS curves revealed pressure dependent changes of the particle structure seen in the low q-region (∼0.25 nm−1), also reflected by a decrease of Radii of Gyration (Rg) with increasing pressure.Especially the p(r) functions from the SAXS data do not only show an overall particle change but also a highly pressure sensitive inner organization. A triple-peak feature indicating the lamellar lipid organization below Tm was induced by raising the pressure up to 3000 bar. These pressure sensitive lipid layers were observed by scattering intensity changes in SAXS curves at q=1.7 nm−1.The shape alterations could be evidenced by fitting an ellipsoidal model to the SANS curves resulting in a decrease of the ellipsoidal radii under pressure. A new LDL model considering the cross-sectional electron density profile is developed and applied to the SAS data to get a more detailed insight into pressure dependent behavior.