The nucleobase-cation-symport-1 (NCS1) transporters are essential components of salvage pathways for nucleobases and related metabolites. Here, we report the 2.85-angstrom resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, 10 of which are arranged in two inverted repeats of five helices. The structures of the outward-facing open and substrate-bound occluded conformations were solved, showing how the outward-facing cavity closes upon binding of substrate. Comparisons with the leucine transporter LeuT(Aa) and the galactose transporter vSGLT reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite sides of the membrane. The reciprocal opening and closing of these cavities is synchronized by the inverted repeat helices 3 and 8, providing the structural basis of the alternating access model for membrane transport.

Photosystem II (PSII) is a large homodimeric protein-cofactor complex that acts as light-driven water:plastoquinone oxidoreductase and is located in the photosynthetic thylakoid membrane of plants, green algae and cyanobacteria. The principal function of PSII is to oxidize two water molecules at the unique Mn 4 Ca cluster to molecular (atmospheric) oxygen, 4 protons and 4 electrons. The protons serve to drive ATP synthetase and the electrons reduce plastoquinone (Q B ) to plastoquinol (Q B H 2 ) that is exported and delivers the electrons (through the cytochrome b 6 f complex) to photosystem I. Here the electrons gain a high reducing potential and serve at NADP reductase to generate NADPH that together with ATP reduces CO 2 to carbohydrates in the Calvin cycle. The crystal structure of PSII from Thermosynechococcus elongatus at 2.9-Å resolution [1] allowed the unambiguous assignment of all 20 protein subunits and complete modeling of all 35 chlorophyll a, 2 pheophytin, 2 cytochrome, 2 plastoquinone, and 12 carotenoid molecules, 25 integral lipids, 1 chloride ion and the Mn 4 Ca cluster per PSII monomer. The presence of a third plastoquinone Q C and a second plastoquinone-transfer channel, which were not observed before, suggest mechanisms for plastoquinolplastoquinone exchange, and we calculated other possible water or dioxygen and proton channels. Putative oxygen positions obtained from Xenon derivative crystals indicate a role for lipids in oxygen diffusion to the cytoplasmic side of PSII. The chloride position suggests a role in protontransfer reactions because it is bound through a putative water molecule to the Mn 4 Ca cluster at a distance of 6.5 Å and is close to two possible proton transfer channels.

A method for performing high-throughput in situ serial X-ray crystallography with soluble and membrane proteins in the lipid cubic phase is described. It works with microgram quantities of protein and lipid (and ligand when present) and is compatible with the most demanding sulfur SAD phasing.

Two-component signal transduction systems are the main mechanism by which bacteria sense and respond to their environment, and their membrane-located histidine protein kinases generally constitute the sensory components of these systems. Relatively little is known about their fundamental mechanisms and precise nature of the molecular signals sensed, because of the technical challenges of producing sufficient quantities of these hydrophobic membrane proteins. This study evaluated the heterologous production, purification and activities of the 16 intact membrane sensor kinases of Enterococcus faecalis. Following the cloning of the genes into expression plasmid pTTQ18His, all but one kinase was expressed successfully in Escherichia coli inner membranes. Purification of the hexa-histidine 'tagged' recombinant proteins was achieved for 13, and all but one were verified as intact. Thirteen intact kinases possessed autophosphorylation activity with no added signal when assayed in membrane vesicles or as purified proteins. Signal testing of two functionally-characterized kinases, FsrC and VicK, was successful examplifying the potential use of in vitro activity assays of intact proteins for systematic signal identification. Intact FsrC exhibited an approximately 10-fold increase in activity in response to a two-fold molar excess of synthetic GBAP pheromone, whilst glutathione, and possibly redox potential, were identified for the first time as direct modulators of VicK activity in vitro. The impact of DTT on VicK phosphorylation resulted in increased levels of phosphorylated VicR, the downstream response regulator, thereby confirming the potential of this in vitro approach for investigations of modulator effects on the entire signal transduction process of two-component systems.

A method for performing high-throughput in situ serial X-ray crystallography with soluble and membrane proteins in the lipid cubic phase at cryogenic temperatures (100 K) is described. It works with nanogram to single-digit microgram quantities of protein and lipid (and ligand when present), and is compatible with both high-resolution native data collection and experimental phasing without the need for crystal harvesting.