Current limits of structural biology: The transient interaction between cytochrome c 6 and photosystem I.

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 c 6 (Cyt c 6 ) and reducing ferredoxin. Here, we use cryo-electron microscopy and small angle neutron scattering (SANS) to characterize the transient binding of Cyt c 6 to TePSI. The structure of TePSI cross-linked to Cyt c 6 was solved at a resolution of 2.9 Å 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 c 6 could not be localized with certainty in our cryo-EM analysis. SANS measurements confirm that Cyt c 6 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 c HH ). 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 K M values higher than 20 µM.

Diversity and productivity in a long-term grassland experiment.

Plant diversity and niche complementarity had progressively stronger effects on ecosystem functioning during a 7-year experiment, with 16-species plots attaining 2.7 times greater biomass than monocultures. Diversity effects were neither transients nor explained solely by a few productive or unviable species. Rather, many higher-diversity plots outperformed the best monoculture. These results help resolve debate over biodiversity and ecosystem functioning, show effects at higher than expected diversity levels, and demonstrate, for these ecosystems, that even the best-chosen monocultures cannot achieve greater productivity or carbon stores than higher-diversity sites.

Kinetics of the light-induced proton translocation associated with the pH-dependent formation of the metarhodopsin I/II equilibrium of bovine rhodopsin.

The kinetics of the formation of the metaII (MII) state of bovine rhodopsin was investigated by time-resolved electrical and absorption measurements with rod outer segment (ROS) fragments. Photoexcitation leads to proton transfer in the direction from the cytosolic to the intradiscal side of the membrane, probably from the Schiff base to the acceptor glutamate 113. Two components of comparable amplitude are required to describe the charge movement with exponential times of 1.1 (45%) and 3.0 ms (55%) (pH 7.8, 22 degreesC, 150 mM KCl). The corresponding activation energies are 86 and 123 kJ/mol, respectively (150 mM KCl). The time constants and amplitudes depend strongly on pH. Between pH 7.1 and 3.8 the kinetics becomes much faster, with the faster and slower components accelerating by factors of about 8 and 2, respectively. Complementary single-flash absorption experiments at 380 nm and 10 degreesC show that the formation of MII also occurs with two components with similar time constants and pH dependence. This suggests that both signals monitor the same molecular events. The pH dependence of the two apparent time constants and amplitudes of the optical data can be described well over the pH range 4-7.5 by two coupled equilibria between MI and two isochromic MII species MIIa and MIIb: MI MIIa(380) MIIb(380), with k0 proportional to the proton concentration. This model implies that deprotonation of the Schiff base and proton uptake are tightly coupled in ROS membranes. Models with k2 proportional to the proton concentration cannot describe the data. Photoreversal of MII by blue flashes (420 nm) leads to proton transfer in a direction opposite to that of the signal associated with MII formation. In this transition the Schiff base is reprotonated, most likely from glutamate 113. At pH 7.3, 150 mM KCl, 22 degreesC, this electrical charge reversal has an exponential time constant of about 30 ms and is about 10 times slower than the forward charge motion.

Functional aspect of a phosphopeptide derived from calf thymus nuclei and DNA.

Phosphopeptides (PPs) isolated from highly purified calf thymus DNA (N-DNA) and extracted from calf thymus nuclei were fractionated, and the effect of one PP fraction on DNA replication has been examined. In the absence of inhibitors, the increasing PP concentration caused a linear decrease of 3H-thymidine uptake in L5178Y cells. If PP fraction was mildly hydrolysed with 1NHCl, the decrease in uptake was much steeper. The studies in which the inhibitors were used revealed that by the addition of the unhydrolysed PP fraction the inhibition of 3H-thymidine uptake by alpha-amanitin could be completely overcome, and that the inhibition by puromycin was reduced to 65-77% of the control. With puromycin, there was a gradual decrease of 3H-thymidine uptake with PP concentration above 3 mg/ml. The PPs gave an increase in incorporation of 3H-thymidine even after removal of alpha-amanitin and puromycin; thus, it is suggested that there is no direct interaction of either inhibitor with PP in the cell. Data on the utilization of 3H-cytidine for the synthesise of new DNA suggest that PP fraction might cause an acceleration of DNA replication.

Cellular and nuclear volume of human cells during the cell cycle.

The volumes of whole cells and nuclei of cultured human cells were studied at different times after synchronization of growth using the Coulter counter and scanning microphotometry. It was found that the increase in cell volume is compatible with both linear or exponential growth during the cell cycle. The growth of the nuclear volume is not correlated with the beginning of the DNA synthesis. The nuclear volume starts to increase already 6 hr prior DNA synthesis. The data also indicate that the nuclear volume growth could proceed in two stages. The relation of this result to radiation sensitivity is discussed.