Purification and Reconstitution of the Glutamate Carrier GltT of the Thermophilic Bacterium Bacillus stearothermophilus

An affinity tag consisting of six adjacent histidine residues followed by an enterokinase cleavage site was genetically engineered at the N-terminus of the glutamate transport protein GltT of the thermophilic bacterium Bacillus stearothermophilus. The fusion protein was expressed in Escherichia coli and shown to transport glutamate. The highest levels of expression were observed in E. coli strain DH5α grown on rich medium. The protein could be purified in a single step by Ni2+-NTA affinity chromatography after solubilization of the cytoplasmic membranes with the detergent Triton X100. Purified GltT was reconstituted in an active state in liposomes prepared from E. coli phospholipids. The protein was reconstituted in detergent-treated preformed liposomes, followed by removal of the detergent with polystyrene beads. Active reconstitution was realized with a wide range of Triton X100 concentrations. Neither the presence of glycerol, phospholipids, nor substrates of the transporter was necessary during the purification and reconstitution procedure to keep the enzyme in an active state. In B. stearothermophilus, GltT translocates glutamate in symport with protons or sodium ions. In membrane vesicles derived from E. coli cells expressing GltT, the Na+ ion dependency seems to be lost, suggesting a role for the lipid environment in the cation specificity. In agreement with the last observation, glutamate transport catalyzed by purified GltT reconstituted in E. coli phospholipid is driven by an electrochemical gradient of H+ but not of Na+.

The plasma resonance in the response and in the rf impedance of a capacitively shunted Josephson junction in the presence of thermal noise

The rf impedance and the wide‐band response to radiation of a resistively shunted junction (RSJ) model Josephson junction have been measured in the presence of thermal noise, using a phase‐locked‐loop analog of the RSJ model. In the conditions for which analytical calculations are valid there is good agreement between the theory and the analog. When the RSJ model is shunted with a capacitance, a plasma type resonance can occur in the rf impedance when the junction is biased in the supercurrent (in‐lock). When thermal noise is present, this plasma resonance can also occur when a nonzero average voltage is generated across the junction. We found that in this case a resonance also occurs in the wide‐band response to radiation. This resonance takes place at a frequency that can be identified as the attempt frequency, with which the junction attempts to escape the phase locked condition in which it exists for a fraction of the time even though the average voltage across the junction is nonzero. The average lifetimes of the junction in lock τin (?=0) and out of lock τout (?=?out) were also measured in the presence of thermal noise. τin is in good agreement with existing theory and τout is derived from the measurements

Cloning, expression, and isolation of the mannitol transport protein from the thermophilic bacterium Bacillus stearothermophilus

A mannitol phosphotransferase system (PTS) was identified in Bacillus stearothermophilus by in vitro complementation with Escherichia coli EI, HPr, and IIAMtl. Degenerate primers based on regions of high amino acid similarity in the E. coli and Staphylococcus carnosus EIIMtl were used to develop a digoxigenin-labeled probe by PCR. Using this probe, we isolated three overlapping DNA fragments totaling 7.2 kb which contain the genesmtlA,mtlR,mtlF, andmtlD, encoding the mannitol IICB, a regulator, IIA, and a mannitol-1-phosphate dehydrogenase, respectively. The mtlA gene consists of 1,413 bp coding for a 471-amino-acid protein with a calculated mass of 50.1 kDa. The amino acid sequence shows high similarity with the sequence of IICBMtl of S. carnosus and the IICB part of the IICBAMtls of E. coli and B. subtilis. The enzyme could be functionally expressed in E. coli by placing it behind the strong tac promoter. The rate of thermal inactivation at 60&C of B. stearothermophilus IICBMtl expressed in E. coli was two times lower than that of E. coli IICBMtl. IICBMtl in B. stearothermophilus is maximally active at 85&C and thus very thermostable. The enzyme was purified on Ni-nitrilotriacetic acid resin to greater than 95 % purity after six histidines were fused to the C-terminal part of the transporter. Many bacteria transport D-mannitol and other carbohy-drates via a phosphoenolpyruvate (PEP)-dependent phospho

Aryloxymaleimides for cysteine modification, disulfide bridging and the dual functionalization of disulfide bonds

Tuning the properties of maleimide reagents holds significant promise in expanding the toolbox of available methods for bioconjugation. Herein we describe aryloxymaleimides which represent 'next generation maleimides' of attenuated reactivity, and demonstrate their ability to enable new methods for protein modification at disulfide bonds

EEG-based visual deviance detection in freely behaving mice

The mouse is widely used as an experimental model to study visual processing. To probe how the visual system detects changes in the environment, functional paradigms in freely behaving mice are strongly needed. We developed and validated the first EEG-based method to investigate visual deviance detection in freely behaving mice. Mice with EEG implants were exposed to a visual deviant detection paradigm that involved changes in light intensity as standard and deviant stimuli. By subtracting the standard from the deviant evoked waveform, deviant detection was evident as bi-phasic negativity (starting around 70 ms) in the difference waveform. Additionally, deviance-associated evoked (beta/gamma) and induced (gamma) oscillatory responses were found. We showed that the results were stimulus-independent by applying a "flip-flop " design and the results showed good repeatability in an independent measurement. Together, we put forward a validated, easy-to-use paradigm to measure visual deviance processing in freely behaving mice.Functional Genomics of Muscle, Nerve and Brain Disorder