D. DiFiore

One Protein, Two Probes A central challenge in the use of x-ray diffraction to characterize macromolecular structure is the propensity of the high-energy radiation to damage the sample during data collection. Recently, a powerful accelerator-based, ultrafast x-ray laser source has been used to determine the geometric structures of small protein crystals too fragile for conventional diffraction techniques. Kern et al. (p. 491 , published online 14 February) now pair this method with concurrent x-ray emission spectroscopy to probe electronic structure, as well as geometry, and were able to characterize the metal oxidation states in the oxygen-evolving complex within photosystem II crystals, while simultaneously verifying the surrounding protein structure.

The dioxygen we breathe is formed by light-induced oxidation of water in photosystem II. O2 formation takes place at a catalytic manganese cluster within milliseconds after the photosystem II reaction centre is excited by three single-turnover flashes. Here we present combined X-ray emission spectra and diffraction data of 2-flash (2F) and 3-flash (3F) photosystem II samples, and of a transient 3F' state (250 μs after the third flash), collected under functional conditions using an X-ray free electron laser. The spectra show that the initial O-O bond formation, coupled to Mn reduction, does not yet occur within 250 μs after the third flash. Diffraction data of all states studied exhibit an anomalous scattering signal from Mn but show no significant structural changes at the present resolution of 4.5 Å. This study represents the initial frames in a molecular movie of the structural changes during the catalytic reaction in photosystem II.

The thermophilic cyanobacterium Thermosynechococcus elongatus was cultivated under controlled growth conditions using a new type of photobioreactor, allowing us to optimise growth conditions and the biomass yield. A fast large-scale purification method for monomeric and dimeric photosystem II (PSII) solubilized from thylakoid membranes of this cyanobacterium was developed using fast protein liquid chromatography (FPLC). The obtained PSII core complexes (PSIIcc) were analysed for their pigment stoichiometry, photochemical and oxygen evolution activities, as well as lipid and detergent composition. Thirty-six chlorophyll a (Chla), 2 pheophytin a (Pheoa), 9+/- 1 beta-carotene (Car), 2.9+/-0.8 plastoquinone 9 (PQ9) and 3.8+/-0.5 Mn were found per active centre. For the monomeric and dimeric PSIIcc, 18 and 20 lipid as well as 145 and 220 detergent molecules were found in the detergent shell, respectively. The monomeric and dimeric complexes showed high oxygen evolution activity with 1/4 O(2) released per 37-38 Chla and flash in the best cases. Crystals were obtained from dimeric PSIIcc by a micro-batch method. They diffract synchrotron X-rays to a maximum resolution of 2.9-A, resulting in complete data sets of 3.2 A resolution.

Most of the dioxygen on earth is generated by the oxidation of water by photosystem II (PS II) using light from the sun. This light-driven, four-photon reaction is catalyzed by the Mn(4)CaO(5) cluster located at the lumenal side of PS II. Various X-ray studies have been carried out at cryogenic temperatures to understand the intermediate steps involved in the water oxidation mechanism. However, the necessity for collecting data at room temperature, especially for studying the transient steps during the O-O bond formation, requires the development of new methodologies. In this paper we report room temperature X-ray diffraction data of PS II microcrystals obtained using ultrashort (< 50 fs) 9 keV X-ray pulses from a hard X-ray free electron laser, namely the Linac Coherent Light Source. The results presented here demonstrate that the "probe before destroy" approach using an X-ray free electron laser works even for the highly-sensitive Mn(4)CaO(5) cluster in PS II at room temperature. We show that these data are comparable to those obtained in synchrotron radiation studies as seen by the similarities in the overall structure of the helices, the protein subunits and the location of the various cofactors. This work is, therefore, an important step toward future studies for resolving the structure of the Mn(4)CaO(5) cluster without any damage at room temperature, and of the reaction intermediates of PS II during O-O bond formation.

X-ray free-electron laser (XFEL) sources enable the use of crystallography to solve three-dimensional macromolecular structures under native conditions and without radiation damage. Results to date, however, have been limited by the challenge of deriving accurate Bragg intensities from a heterogeneous population of microcrystals, while at the same time modeling the X-ray spectrum and detector geometry. Here we present a computational approach designed to extract meaningful high-resolution signals from fewer diffraction measurements.

The influence of poly(ethylene glycol) on the micelle formation of alkyl maltosides under conditions of membrane protein crystallization is investigated.

FEBS LettersVolume 128, Issue 1 p. 149-153 Full-length articleFree Access Generation of large and active membrane layers from thylakoid vesicles Properties and possible applications H.T. Witt, H.T. Witt Max-Volmer-Institut für Biophysikalische und Physikalische Chemie, Technische Universität Berlin, Strasse des 17. Juni 135, 1000 Berlin 12, GermanySearch for more papers by this authorD. DiFiore, D. DiFiore Max-Volmer-Institut für Biophysikalische und Physikalische Chemie, Technische Universität Berlin, Strasse des 17. Juni 135, 1000 Berlin 12, GermanySearch for more papers by this author H.T. Witt, H.T. Witt Max-Volmer-Institut für Biophysikalische und Physikalische Chemie, Technische Universität Berlin, Strasse des 17. Juni 135, 1000 Berlin 12, GermanySearch for more papers by this authorD. DiFiore, D. DiFiore Max-Volmer-Institut für Biophysikalische und Physikalische Chemie, Technische Universität Berlin, Strasse des 17. Juni 135, 1000 Berlin 12, GermanySearch for more papers by this author First published: June 01, 1981 https://doi.org/10.1016/0014-5793(81)81102-7Citations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 P. Mueller, D.O. Rudin, H.T. Tien, W.C. Wescott, Nature, 194, (1962), 979– 980. 2 M. Montal, P. Mueller, Proc. Natl. Acad. Sci. USA, 69, (1972), 3561– 3566. 3 H.G. Schindler, U. Quast, Proc. Natl. Acad. Sci. USA, 77, (1980), 3052– 3056. 4 B.B. Thomas, U. Minnat, P.F. Elbers, Acta Bot. Neerl., 5, (1956), 315– 318. 5 R. Verger, F. Pattus, Chem. Phys. Lipids, 16, (1976), 285– 291. 6 F. Pattus, P. Desnuelle, R. Verger, Biochim. Biophys. Acta, 507, (1978), 62– 70. 7 H.-W. Trissl, P. Gräber, Bioelectrochem. Bioenerg., 7, (1980), 167– 186. 8 H. Rüppel, H.T. Witt, S.P. Colowick N.O. Kaplan Methods in Enzymology (1969), Academic Press London, New York 317– 379. 9 H.T. Witt, Biochim. Biophys. Acta, 505, (1979), 355– 427. 10 R. Reich, R. Scheerer, K.-U. Sewe, H.T. Witt, Biochim. Biophys. Acta, 449, (1976), 285– 294. 11 K.-U. Sewe, R. Reich, Z. Naturforsch., 32c, (1977), 161– 171. 12 W. Arnold, J.R. Azzi, Plant Physiol., 60, (1977), 449– 451. Citing Literature Volume128, Issue1June 01, 1981Pages 149-153 ReferencesRelatedInformation

Nat. Methods 11, 545–548 (2014); published online 16 March 2014; corrected after print 3 June 2015 In the version of this article initially published, the authors claimed that with the tool cctbx.xfel, weak diffraction signals can be measured using fewer crystal specimens than are needed for the previously available program CrystFEL.

Abstract The flash-induced difference spectrum in the range of 450 - 550 nm of protochloroplasts isolated from pea-leaves greened under intermittent illumination (2 min light, 98 min dark) was measured and compared with that of fully developed chloroplasts from pea leaves. Because of the sensitivity of the decay o fthe absorption changes to the ionophore valinomycin they were shown to mainly be due to an electrochromic bandshift of the membrane pigments (chlorophylls-a, -b and carotenoids). The differences in the shape and the amplitude between both spectra are consistently explained within the framework of a recent hypothesis supposed by Sewe and Reich (Z. Naturforsch. 33 c, 161 - 171 (1978)) by the lack of chlorophyll-b in the protochloroplasts. It is concluded, that the transformation of the protochloroplasts into chloroplasts which is accompanied by the incorporation of the light harvesting complex and the formation of grana stacks does not seriously change the orientation of the field indicating pigments within the membrane with respect of the polarity of the light induced vectorial electron transport.