The apo-form of the 24.4 kDa AA9 family lytic polysaccharide monooxygenase TaLPMO9A from Thermoascus aurantiacus has been isotopically labeled and recombinantly expressed in Pichia pastoris. In this paper, we report the 1H, 13C, and 15N chemical shift assignments, as well as an analysis of the secondary structure of the protein based on the secondary chemical shifts.

Mutations in Cu/Zn-superoxide dismutase (SOD1) gene are linked to 10-20% of familial amyotrophic lateral sclerosis (fALS) cases. The mutations cause misfolding and self-assembly of SOD1 into toxic oligomers and aggregates, resulting in motor neuron degeneration. The molecular mechanisms underlying SOD1 aggregation and toxicity are unclear. Characterization of misfolded SOD1 is particularly challenging due to its metastable nature. Antibodies against misfolded SOD1 are useful tools for this purpose, provided their specificity and selectivity are well characterized. Here, we characterized three recently introduced anti-misfolded SOD1 antibodies and compared them with two commercial, anti-misfolded SOD1 antibodies raised against the fALS-linked variant G93A-SOD1. As controls, we compared the reactivity of these antibodies to two polyclonal anti-SOD1 antibodies expected to be insensitive to misfolding. We asked to what extent the antibodies could distinguish between WT and variant SOD1 and between native and misfolded conformations. WT, G93A-SOD1, or E100K-SOD1 were incubated under aggregation-promoting conditions and monitored using thioflavin-T fluorescence, electron microscopy, and dot blots. WT and G93A-SOD1 also were analyzed using native-PAGE/Western blot. The new anti-misfolded SOD1 and the commercial antibody B8H10 showed variable reactivity using dot blots, but generally showed maximum reactivity at the time misfolded SOD1 oligomers were expected to be most abundant. In contrast, only B8H10 and the control antibodies were reactive in Western blots. Unexpectedly, the polyclonal antibodies showed strong preference for the misfolded form of G93A-SOD1 in dot blots. Surprisingly, anti-misfolded SOD1 antibody C4F6 was specific for the apo form of G93A-SOD1 but insensitive to misfolding. Antibody 10C12 showed preference for early misfolded structures, whereas 3H1 bound preferentially to late structures. These new antibodies allow distinction between putative early- and late-forming pre-fibrillar SOD1 oligomers.

The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane transport protein, catalyzes galactoside/H+ symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar-binding. Camelid nanobodies were made against a double-mutant Gly46 → Trp/Gly262 → Trp (LacYWW) that produces an outward-open conformation, as opposed to the cytoplasmic open-state crystal structure of WT LacY. Nanobody 9047 (Nb9047) stabilizes WT LacY in a periplasmic-open conformation. Here, we describe the X-ray crystal structure of a complex between LacYWW, the high-affinity substrate analog 4-nitrophenyl-α-d-galactoside (NPG), and Nb9047 at 3-Å resolution. The present crystal structure demonstrates that Nb9047 binds to the periplasmic face of LacY, primarily to the C-terminal six-helical bundle, while a flexible loop of the Nb forms a bridge between the N- and C-terminal halves of LacY across the periplasmic vestibule. The bound Nb partially covers the vestibule, yet does not affect the on-rates or off-rates for the substrate binding to LacYWW, which implicates dynamic flexibility of the Nb-LacYWW complex. Nb9047-binding neither changes the overall structure of LacYWW with bound NPG, nor the positions of side chains comprising the galactoside-binding site. The current NPG-bound structure exhibits a more occluded periplasmic vestibule than seen in a previous structure of a (different Nb) apo-LacYWW/Nb9039 complex that we argue is caused by sugar-binding, with major differences located at the periplasmic ends of transmembrane helices in the N-terminal half of LacY.

Haemaphysalis longicornis, the cattle tick or bush tick, has an extended distribution throughout Asia and the Pacific region, including China, Russia, the Republic of Korea (ROK), Japan, Australia, New Zealand, and the South Pacific islands. It is an obligate ectoparasite found commonly on medium to large sized wild and domestic animals, with humans as an accidental host. Haemaphysalis longicornis transmits a number of pathogens, including severe fever with thrombocytopenia syndrome and tick-borne encephalitis viruses, bacteria, helminths, and protozoans, that impact on veterinary (wild and domestic animals) and human health. Surveys of rickettsial pathogens associated with H. longicornis from China, the ROK, and Japan have resulted in the discovery of more than 35 incompletely characterized molecular isolates of Rickettsia. In response to the increased global threat of tick-borne rickettsial diseases, H. longicornis collected in the ROK and China were assessed in our laboratory and two additional Rickettsia spp. isolates (ROK-HL727 and XinXian HL9) were identified. These agents were fully characterized by multilocus sequence typing using partial gene fragment sequences of rrs, gltA, ompA, ompB, and sca4. Phylogenetic comparisons of these Rickettsia isolates with known Rickettsia species and other molecular isolates identified from H. longicornis were performed to better understand their interrelationships. Phylogenetic analysis of the sequences from these 5 gene fragments showed that ROK-HL727 was closely related to rickettsial isolates of H. longicornis previously reported from China, the ROK and Japan, but distinct from any currently recognized Rickettsia species. It therefore qualifies genetically as a new species, introduced herein as Candidatus Rickettsia longicornii. The XinXian-HL9 isolate detected from China was determined to be genetically similar to the human pathogen Rickettsia heilongjiangensis. People living and working in areas where H. longicornis is endemic should be aware of the potential for rickettsial diseases.

Soluble guanylyl cyclase (sGC, EC 4.6.1.2) is a key enzyme in the regulation of vascular tone. In view of the therapeutic interest of the NO/cGMP pathway, drugs were developed that either increase the NO sensitivity of the enzyme or activate heme-free apo-sGC. However, modulation of sGC activity by endogenous agents is poorly understood. In the present study we show that the maximal activity of NO-stimulated purified sGC is significantly increased by cytosolic preparations of porcine coronary arteries. Purification of the active principle by several chromatographic steps resulted in a protein mixture consisting of 100, 70, and 40 kDa bands on SDS polyacrylamide gel electrophoresis. The respective proteins were identified by LC-MS/MS as gelsolin, annexin A6, and actin, respectively. Further purification resulted in loss of activity, indicating an interaction of sGC with a protein complex rather than a single protein. The partially purified preparation had no effect on basal sGC activity or enzyme activation by the heme mimetic BAY 60-2770, suggesting a specific effect on the conformation of the NO-bound heterodimeric holoenzyme. Since the three proteins identified are all related to contractile elements of smooth muscle, our data suggest that regulation of vascular tone involves a modulatory interaction of sGC with the cytoskeleton.

Development of resistance against existing anti-epileptic drugs has alarmed the scientific innovators to find novel potential chemical starting points for the treatment of epilepsy and GABAA inhibition is a promising drug target strategy against epilepsy. The crystal structure of a subtype-selective β3-homopentameric ligand-gated ion channel of GABAA receptor has been used for the first time for screening the Asinex library for discovery of GABAA agonists as potential anti-epileptic agents. Co-crystallized ligand established the involvement of part of the β7-β8 loop (Glu155 and Tyr157) and β9-β10 loop (Phe200 and Tyr205) residues as the crucial amino acids in effective binding, an essential feature, being hydrogen bond or ionic interaction with Glu155 residue. Top ranked hits were further subjected to binding energy estimation, ADMET analysis and ligand efficiency matric calculations as consecutive filters. About 19 compounds qualifying all parameters possessed interaction of one positively charged group with Glu155 with good CNS drug-like properties. Simulation studies were performed on the apo protein, its complex with co-crystallized ligand and the best hit qualifying all screening parameters. The best hit was also analyzed using Quantum mechanical studies, off-target analysis and hit modification. The off-target analysis emphasized that these agents did not have any other predicted side-effects.

Our understanding on the folding of membrane proteins lags behind that of soluble proteins due to challenges posed by the exposure of hydrophobic regions during in vitro chemical denaturation and refolding experiments. While different folding models are accepted for soluble proteins, only the two-stage model and the long-range interactions model have been proposed so far for helical membrane proteins. To address our knowledge gap on how different membrane proteins traverse their folding pathways, we have systematically investigated the structural features of SDS-denatured states and the kinetics for reversible unfolding of sensory rhodopsin II (pSRII), a retinal-binding photophobic receptor from Natronomonas pharaonis. pSRII is difficult to denature, and only SDS can dislodge the retinal chromophore without rapid aggregation. Even in 30% SDS (0.998 ΧSDS) pSRII retains the equivalent of six out of seven transmembrane helices, while the retinal binding pocket is disrupted, with transmembrane residues becoming more solvent-exposed. Folding of pSRII from an SDS-denatured state harbouring a covalently-bound retinal chromophore shows deviations from an apparent two-state behaviour. SDS-denaturation to form the sensory opsin apo-protein is reversible. We report pSRII as a new model protein which is suitable for membrane protein folding studies, and has a unique folding mechanism that differs from those of bacteriorhodopsin and bovine rhodopsin.