Cryogenic analysis of frozen hydrated biological tissue at the Hard X-ray Micro-Probe Beamline at PETRA III

C

Vibrio natriegens as Host for Expression of Multisubunit Membrane Protein Complexes

Escherichia coli is a convenient host for the expression of proteins, but the heterologous production of large membrane protein complexes often is hampered by the lack of specific accessory genes required for membrane insertion or cofactor assembly. In this study we introduce the non-pathogenic and fast-growing Vibrio natriegens as a suitable expression host for membrane-bound proteins from Vibrio cholerae. We achieved production of the primary Na+ pump, the NADH:quinone oxidoreductase (NQR), from V. cholerae in an active state, as indicated by increased overall NADH:quinone oxidoreduction activity of membranes from the transformed V. natriegens, and the sensitivity toward Ag+, a specific inhibitor of the NQR. Complete assembly of V. cholerae NQR expressed in V. natriegens was demonstrated by BN PAGE followed by activity staining. The secondary transport system Mrp from V. cholerae, another membrane-bound multisubunit complex, was also produced in V. natriegens in a functional state, as demonstrated by in vivo Li+ transport. V. natriegens is a promising expression host for the production of membrane protein complexes from Gram-negative pathogens

Light-Dependent Phosphorylation of the <i>Drosophila</i> Inactivation No Afterpotential D (INAD) Scaffolding Protein at Thr170 and Ser174 by Eye-Specific Protein Kinase C

<div><p><i>Drosophila</i> inactivation no afterpotential D (INAD) is a PDZ domain-containing scaffolding protein that tethers components of the phototransduction cascade to form a supramolecular signaling complex. Here, we report the identification of eight INAD phosphorylation sites using a mass spectrometry approach. PDZ1, PDZ2, and PDZ4 each harbor one phosphorylation site, three phosphorylation sites are located in the linker region between PDZ1 and 2, one site is located between PDZ2 and PDZ3, and one site is located in the N-terminal region. Using a phosphospecific antibody, we found that INAD phosphorylated at Thr170/Ser174 was located within the rhabdomeres of the photoreceptor cells, suggesting that INAD becomes phosphorylated in this cellular compartment. INAD phosphorylation at Thr170/Ser174 depends on light, the phototransduction cascade, and on eye-Protein kinase C that is attached to INAD via one of its PDZ domains.</p></div

Crystallization and preliminary analysis of the NqrA and NqrC subunits of the Na⁺-translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae

The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio cholerae is a membrane protein complex consisting of six different subunits NqrA-NqrF. The major domains of the NqrA and NqrC subunits were heterologously expressed in Escherichia coli and crystallized. The structure of NqrA1-377 was solved in space groups C2221 and P21 by SAD phasing and molecular replacement at 1.9 and 2.1 Å resolution, respectively. NqrC devoid of the transmembrane helix was co-expressed with ApbE to insert the flavin mononucleotide group covalently attached to Thr225. The structure was determined by molecular replacement using apo-NqrC of Parabacteroides distasonis as search model at 1.8 Å resolution

Light-Dependent Isoelectric Point Shift of the INAD Scaffolding Protein.

<p><i>Drosophila</i> head extracts were two-dimensionally separated by isoelectric focusing and SDS gel electrophoresis, blotted onto PVDF membranes and probed with a pan-specific α-INAD antibody. A, Wild type flies were light or dark adapted for 12–18 h prior to protein extraction from heads as indicated by the bars beside the blots (white bar, 12–18 h light; black bar, 12–18 h dark). B, Wild type flies were dark adapted for 12–18 h and then illuminated for 5 seconds prior to protein extraction from heads (white bar, 5 seconds light). C, Wild type flies were light adapted for 12–18 h prior to protein extraction from heads. For λ-phosphatase treatment, protein extracts from fly heads were incubated with λ-phosphatase (+λ-PP) for 1 h at 30°C (lower panel). A sample without λ-phosphatase (-λ-PP) was carried along as a control (upper panel). D, <i>norpA</i>^<i>P24</i> mutant flies lacking phospholipase Cβ were light (white bar) or dark (black bar) adapted for 12–18 h prior to protein extraction from heads. Molecular mass markers (in kDa) are indicated on the left, pH on the top.</p