Energy and Structure of the M2 Helix in Acetylcholine Receptor-Channel Gating

We studied single-channel currents from neuromuscular acetylcholine receptor-channels with mutations in the pore-lining, M2 helix of the ɛ-subunit. Three parameters were quantified: 1), the diliganded gating equilibrium constant (E₂), which reflects the energy difference between C(losed) and O(pen) conformations; 2), the correlation between the opening rate constant and E₂ on a log-log scale (Φ), which illuminates the energy character of the residue (C- versus O-like) within the C↔O isomerization process; and 3), the open-channel current amplitude (i₀), which reports whether a mutation alters the energetics of ion permeation. The largest E₂ changes were observed in the cytoplasmic half of ɛM2 (5′, 9′, 12′, 13′, and 16′), with smaller changes apparent for residues ≥17′. Φ was ∼0.54 for most ɛM2 residues, but was ∼0.32 at the positions that had largest E₂ changes. An arginine substitution reduced i₀ significantly at six positions, with the magnitude of the reduction increasing, 16′→2′. The measurements suggest that the 9′, 12′, and 13′ residues experience large and late free-energy changes in the channel-opening process. We speculate that in the gating isomerization the pore-facing residues >6′ and <16′ experience multiple energy perturbations associated with changes in protein structure and, perhaps, hydration.     

The roots of the halophyte Salicornia brachiata are a source of new halotolerant diazotrophic bacteria with plant growth-promoting potential

Soil salinity is the major cause limiting plant productivity worldwide. Nitrogen-fixing bacteria were enriched and characterised from roots of Salicornia brachiata, an extreme halophyte which has substantial economic value as a bioresource of diverse and valuable products. Nitrogen-free semisolid NFb medium with malate as carbon source and up to 4% NaCl were used for enrichment and isolation of diazotrophic bacteria. The isolates were tested for plant growth-promoting traits and 16S rRNA, nifH and acdS genes were analysed. For selected strains, plant growth-promoting activities were tested in axenically grown Salicornia seedlings at different NaCl concentrations (0–0.5M). New halotolerant diazotrophic bacteria were isolated from roots of S. brachiata. The isolates were identified as Brachybacterium saurashtrense sp. nov., Zhihengliuella sp., Brevibacterium casei, Haererehalobacter sp., Halomonas sp., Vibrio sp., Cronobacter sakazakii, Pseudomonas spp., Rhizobium radiobacter, and Mesorhizobium sp. Nitrogen fixation as well as plant growth-promoting traits such as indole acetic acid (IAA) production, phosphate solubilisation, and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity were demonstrated. For Brachybacterium saurashtrense and Pseudomonas sp., significant plant growth-promoting activities were observed in Salicornia in salt stress conditions. Salicornia brachiata is a useful source of new halotolerant diazotrophic bacteria with plant growth-promoting potential.     

Malabarase,a serine protease with anticoagulant activity from Trimeresurus malabaricus venom

In the present study we describe the purification and characterization of Malabarase, a serine protease from Trimeresurus malabaricus venom. Purification was achieved by gel-permeation chromatography on Sephadex G-75 followed by ion-exchange chromatography on CM Sephadex C-25. Homogeneity of Malabarase was confirmed by RP-HPLC. Malabarase is a monomer that migrated as a single protein band on SDS-PAGE under both reducing and non-reducing conditions. The molecular mass of Malabarase was determined to be 23.4 kDa using MALDI-TOF mass spectrometry. Malabarase is the first serine protease purified from T. malabaricus venom and is selective for fibrinogen. Malabarase hydrolyzes Aα and Bβ but not γ-chains of fibrinogen similar to the metalloproteases, Malabarin and Trimarin, isolated from the same venom. However, the action of Malabarase on plasma coagulation is opposite than those of Malabarin, Trimarin and the whole venom. Malabarase significantly prolonged plasma coagulation time from 152–341 s; whereas Malabarin, Trimarin, and whole venom, greatly reduce plasma clotting time from 152 to 12, 48, and 14 s, respectively. Malabarase did not show hemorrhagic or myotoxic activity. In contrast, Malabarin, Trimarin and whole venom are highly hemorrhagic and myotoxic. These observations support the specificity of Malabarase towards fibrinogen and its non-toxic nature. In conclusion, Malabarase is a fibrinogen-specific, anti-coagulant, and non-toxic serine protease. Its selective action and non-toxic nature might make it useful for treating thrombotic disorders.     

Covalent modification of cysteine 193 impairs ATPase function of nucleotide-binding domain of a Candida drug efflux pump

N-ethylmaleimide (NEM) impairs the ATPase function of N-terminal NBD of Candida drug resistance gene product Cdr1p. To identify the reactive cysteine(s) for such a contribution, we adopted a three-arm approach that included covalent modification, cysteine mutagenesis, and structure homology modeling. The covalent modification results clearly indicate the ability of NEM and iodoacetic acid (IAA) to potently inhibit the ATPase activity of N-terminal NBD. Since this domain contains five cysteine residues in its sequence, we mutated each and found four of these (C325A, C363A, C402A, and C462A) to stay sensitive to NEM/IAA modification and influence ATPase activity, while C193A mutation completely abrogated the catalytic function. The structural homology modeling data further validate these biochemical findings by ruling out any plausible interactions within the cysteine residues, and deriving the importance of Cys-193 in lying at a bond length clearly feasible to interact with ATP and divalent cation to critically influence ATP hydrolysis.     

Rhizogenesis fromNigella sativa protoplasts

Summary Protoplasts were isolated for the first time from cell suspensions ofNigella sativa. These were then cultured in media and observed at regular intervals. Different concentrations of auxin and kinetin were tried with success to obtain root from the callus tissues of the protoplasts.     

Structure–activity relationships of carbocyclic 6-benzylthioinosine analogues as subversive substrates of Toxoplasma gondii adenosine kinase

Carbocyclic 6-benzylthioinosine analogues were synthesized and evaluated for their binding affinity against Toxoplasma gondii adenosine kinase [EC.2.7.1.20]. Various substituents on the aromatic ring of the 6-benzylthio group resulted in increased binding affinity to the enzyme as compared to the unsubstituted compound. Carbocyclic 6-(p-methylbenzylthio)inosine 9n exhibited the most potent binding affinity. Docking simulations were performed to position compound 9n into the T. gondii adenosine kinase active site to determine the probable binding mode. Experimental investigations and theoretical calculations further support that an oxygen atom of the sugar is not critical for the ligand-binding. In agreement with its binding affinity, carbocyclic 6-(p-methylbenzylthio)inosine 9n demonstrated significant anti-toxoplasma activity (IC₅₀ = 11.9 μM) in cell culture without any apparent host-toxicity.     

Comparative study of catalase-peroxidases (KatGs)

Catalase-peroxidases or KatGs from seven different organisms, including Archaeoglobus fulgidus,Bacillus stearothermophilus, Burkholderia pseudomallei, Escherichia coli, Mycobacterium tuberculosis, Rhodobacter capsulatus and Synechocystis PCC 6803, have been characterized to provide a comparative picture of their respective properties. Collectively, the enzymes exhibit similar turnover rates with the catalase and peroxidase reactions varying between 4900 and 15,900 s⁻¹ and 8-25 s⁻¹, respectively. The seven enzymes also exhibited similar pH optima for the peroxidase (4.25-5.0) and catalase reactions (5.75), and high sensitivity to azide and cyanide with IC₅₀ values of 0.2-20 μM and 50-170 μM, respectively. The K_Ms of the enzymes for H₂O₂ in the catalase reaction were relatively invariant between 3 and 5 mM at pH 7.0, but increased to values ranging from 20 to 225 mM at pH 5, consistent with protonation of the distal histidine (pKa approximately 6.2) interfering with H₂O₂ binding to Cpd I. The catalatic k_cat was 2- to 3-fold higher at pH 5 compared to pH 7, consistent with the uptake of a proton being involved in the reduction of Cpd I. The turnover rates for the INH lyase and isonicotinoyl-NAD synthase reactions, responsible for the activation of isoniazid as an anti-tubercular drug, were also similar across the seven enzymes, but considerably slower, at 0.5 and 0.002 s⁻¹, respectively. Only the NADH oxidase reaction varied more widely between 10⁻⁴ and 10⁻² s⁻¹ with the fastest rate being exhibited by the enzyme from B. pseudomallei.     

Electrostatics of the intracellular vestibule of K+ channels

Previous calculations using continuum electrostatic calculations showed that a fully hydrated monovalent cation is electrostatically stabilized at the center of the cavity of the KcsA potassium channel. Further analysis demonstrated that this cavity stabilization was controlled by a balance between the unfavorable reaction field due to the finite size of the cavity and the favorable electrostatic field arising from the pore helices. In the present study, continuum electrostatic calculations are used to investigate how the stability of an ion in the intracellular vestibular cavity common to known potassium channels is affected as the inner channel gate opens and the cavity becomes larger and contiguous with the intracellular solution. The X-ray structure of the calcium-activated potassium channel MthK, which was crystallized in the open state, is used to construct models of the KcsA channel in the open state. It is found that, as the channel opens, the barrier at the helix bundle crossing decreases to approximately 0 kcal/mol, but that the ion in the cavity is also significantly destabilized. The results are compared and contrasted with additional calculations performed on the KvAP (voltage-activated) and KirBac1.1 (inward rectifier) channels, as well as models of the pore domain of Shaker in the open and closed state. In conclusion, electrostatic factors give rise to energetic constraints on ion permeation that have important functional consequences on the various K+ channels, and partly explain the presence or absence of charged residues near the inner vestibular entry.     

Characterization of a glutathione metabolic mutant of Mycobacterium tuberculosis and its resistance to glutathione and nitrosoglutathione

Glutathione is a tripeptide and antioxidant, synthesized at high levels by cells during the production of reactive oxygen and nitrogen intermediates. Glutathione also serves as a carrier molecule for nitric oxide in the form of S-nitrosoglutathione. Previous studies from this laboratory have shown that glutathione and S-nitrosoglutathione are directly toxic to mycobacteria. Glutathione is not transported into the cells as a tripeptide. Extracellular glutathione is converted to a dipeptide due to the action of transpeptidase, and the dipeptide is then transported into the bacterial cells. The processing of glutathione and S-nitrosoglutathione is brought about by the action of the enzyme gamma-glutamyl transpeptidase. The function of gamma-glutamyl transpeptidase is to cleave glutathione and S-nitrosoglutathione to the dipeptide (Cys-Gly), which is then transported into the bacterium by the multicomponent ABC transporter dipeptide permease. We have created a mutant strain of Mycobacterium tuberculosis lacking this metabolic enzyme. We investigated the sensitivity of this strain to glutathione and S-nitrosoglutathione compared to that of the wild-type bacteria. In addition, we examined the role of glutathione and/or S-nitrosoglutathione in controlling the growth of intracellular M. tuberculosis inside mouse macrophages.