It is well established that Escherichia coli represents a powerful tool for the over-expression of human proteins for structure/function studies. In many cases, such as for membrane transporters, the bacterial toxicity or the aggregation of the target protein hamper the expression limiting the application of this tool. The aim of this study was finding the appropriate conditions for the expression of reluctant proteins that is the human neutral amino acid transporters ASCT2 and B0AT1, that have great relevance to human health in cancer therapy and in COVID-19 research, respectively. The cDNAs coding for the proteins of interest were cloned in the pCOLD I vector and different E. coli strains (BL21 codon plus RIL, and RosettaGami2) were cultured in absence or in presence of glucose (0.5–1%), at low temperature (15 °C), and low inducer concentrations (10–100 µM). Cell growth and protein production were monitored by optical density measurements and western blotting assay, respectively. Even though in different conditions, the expression of both amino acid transporters was obtained.Reducing the growth rate of specific E. coli strains by lowering the temperature and the IPTG concentration, together with the addition of glucose, two reluctant human neutral amino acid transporters have been expressed in E. coli . The results have a potentially great interest in drug discovery since ASCT2 is an acknowledged target of anticancer therapy, and B0AT1 together with ACE2 is part of a receptor for the SARS-Cov-2 RBD proteins.

to H V 1 gating. [3] Its structure is homologous to classical voltage-gated channels' voltage-sensing domain (VSD). [4] Experimentally, exclusively closed structures are available -a crystal structure of a chimera between mouse H V 1 and Ciona intestinalis voltage-sensing phosphatase [5] and an NMR structure of the human H V 1. [6] Also, electron paramagnetic resonance (EPR) spectroscopy data decipher the resting structure of H V 1. [7] An AlphaFold structure has been recently added (Figure 1). AlphaFold uses a machine-learning approach. [8] Its deep learning algorithm exploits physical and biological knowledge about protein structure -mainly gained by X-ray crystallography, cryo-electron microscopy, or NMR. None of these technical approaches allows the application of membrane voltage, a prerequisite to attain the open channel configuration of voltage-sensitive channels. Since Hv1 belongs to this class of channels, the AlphaFold structure reflects, in all likelihood, the closed channel structure.The analysis of the AlphaFold structure corroborates the conclusion (Figure 1A,B). It shows three positively charged arginine residues in the membrane-spanning part of the 4th helix (S4) responsible for Hv1's voltage sensitivity. They may interact with three negatively charged aspartate residues on the first (S1) and third helices (S3). The AlphaFold structure suggests the following Coulombic interactions: R205 -D112 (on S1), R211 -D185 (on S3) and R208 -D174 (on S3). There is consensus that i) D174 is part of the intracellular electrostatic network [9,10] and ii) D174's salt bridge with R208 stabilizes the closed, resting-state conformation. [11] The open channel's structure has solely been computed as homology models. [12][13][14][15][16][17][18] One of the most recent models reflects the experimentally estimated gating charge suggesting that the transition from the closed to the open conformation may be correctly captured. [17] Yet, the closed conformations of the homology model (Figure S1, Supporting Information) and AlphaFold (Figure 1A,B) differ in pore diameter (Figure 1C). While the former is large enough to allow water molecules to pass, the latter is much too narrow. The observation suggests that an assessment of H V 1's unitary water permeability, p f , may be used as a diagnostic tool. By undertaking water flux measurements through purified and reconstituted H V 1 channels in The voltage-gated proton channel, H V 1, is crucial for innate immune responses. According to alternative hypotheses, protons either hop on top of an uninterrupted water wire or bypass titratable amino acids, interrupting the water wire halfway across the membrane. To distinguish between both hypotheses, the water mobility for the putative case of an uninterrupted wire is estimated. The predicted single-channel water permeability 2.3 × 10 −12 cm 3 s −1 reflects the permeability-governing number of hydrogen bonds between water molecules in single-file configuration and pore residues. However, the measured unitary water permeability does no...

The voltage-gated proton channel, HV1, is crucial for innate immune responses. According to alternative hypotheses, protons either hop on top of an uninterrupted water wire or bypass titratable amino acids, interrupting the water wire halfway across the membrane. To distinguish between both hypotheses, we estimate the water mobility for the putative case of an uninterrupted wire. The predicted single-channel water permeability 3x10-12cm3s-1 reflects the permeability-governing number of hydrogen bonds between water molecules in single-file configuration and pore residues. However, the measured unitary water permeability does not confirm the prediction, i.e., it is negligible. Osmotic deflation of reconstituted lipid vesicles reveals trapped water inside the HV1 wild-type channel and D174A mutant open at 0 mV. The conductance of 1400 H+s-1 per wild-type channel agrees with the calculated diffusion limit for a ~0.2nm capture radius for protons. Removal of a charged amino acid (D174) at the pore mouth decreases H+ conductance, conceivably by reducing the capture radius. At least one intervening amino acid contributes to H+ conductance while blocking water flow.

No abstract

No abstract