Conformational analysis of d-tubocurarine: Implications for minimal dimensions of its binding site within ion channels

All the minimum-energy conformations of d-tubocurarine were calculated by the method of molecular mechanics. The energy was minimized from 413 closed forms of the 18-member ring. The set of minimum-energy conformations includes 10 forms with energies less than 6 kcal/mol from the most stable one. Among the four lowest minimum-energy conformations, two forms correspond to those known from X-ray studies, whereas two conformations were not detected experimentally earlier. The flexibility of d-tubocurarine was estimated by calculating six paths of interconversion between the four lowest minimum-energy conformations. Using a molecular graphics technique, it was found that the most extended minimum-energy conformation of d-tubocurarine may fit in an ion channel of a rectangular profile of 8.7 × 11.2 Å, while one tetrahydroisoquinoline head may fit a profile as small as 6.9 × 11.0 Å. A possible model of d-tubocurarine location within the ion channel of the neuronal nicotinic acetylcholine receptor is suggested.     

A theoretical conformational analysis of several substrates of cholinesterase having a cyclic ammonium group structure]

Conformational possibilities of pirrolidine analogues of acetylcholine beta-(N-methyl pirrolidinium)-ethyl ester of acetic acid and beta-(N-ethyl pirrolidinium)-ethyl ester of acetic acid and beta-(N-ethyl pirrolidinium)-ethyl ester of acetic acid were investigated by the method of atomic potentials. The conformational energy was considered as a sum of non-bonded and electrostatical interactions, torsional energy and distortions of bond angles. It has been shown that the replacement of the nitrogen methyl group to ethyl group results in decrease of the average barrier height between two gauche conformations of the O--C--C--N fragment. Comparison of conformational properties of some cholinesterase substrates permit to draw a suggestion that the barrier height influences the rate of the enzymatic hydrolysis.     

Theoretical conformational analysis of methylamide N-acetyl-L-arginine]

The spatial structure of methylamide N-acetyl-L-argine was studied taking into account the non-valent and electrostati interactions, the torsion energy, and the distorsion of valency angles. Calculation of the favourable conformations of the molecule was carried out with the use of all the combinations of angles phi, psi, chi1 divided by chi4 as an intital approximation. These correspond to the low energy forms of the main chain and to the minima of the torsion potentials of the side chain. Conformational possibilities of arginine and lysine were compared. The calculated stable conformation of N-acetyl-L-arginine-methylamide are compared with the geometry of arginine residues in the proteins with known structure.     

Evaluation of GABA Production and Probiotic Activities of Enterococcus faecium BS5

Probiotics and Antimicrobial Proteins - Gamma-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in the central nervous system and is produced by irreversible decarboxylation of...     

Irreversible block of cardiac mutant Na+ channels by batrachotoxin

Batrachotoxin (BTX) not only keeps the voltage-gated Na(+) channel open persistently but also reduces its single-channel conductance. Although a BTX receptor has been delimited within the inner cavity of Na(+) channels, how Na(+) ions flow through the BTX-bound permeation pathway remains unclear. In this report we tested a hypothesis that Na(+) ions traverse a narrow gap between bound BTX and residue N927 at D2S6 of cardiac hNa(v)1.5 Na(+) channels. We found that BTX at 5 microM indeed elicited a strong block of hNa(v)1.5-N927K currents (approximately 70%) after 1000 repetitive pulses (+50 mV/20 ms at 2 Hz) without any effects on Na(+) channel gating. Once occurred, this unique use-dependent block of hNa(v)1.5-N927K Na(+) channels recovered little at holding potential (-140 mV), demonstrating that BTX block is irreversible under our experimental conditions. Such an irreversible effect likewise developed in fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels albeit with a faster on-rate; approximately 90% of peak Na(+) currents were abolished by BTX after 200 repetitive pulses (+50 mV/20 ms). This use-dependent block of fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels by BTX was duration dependent. The longer the pulse duration the larger the block developed. Among N927K/W/R/H/D/S/Q/G/E substitutions in fast inactivation-deficient hNa(v)1.5 Na(+) channels, only N927K/R Na(+) currents were highly sensitive to BTX block. We conclude that (a) BTX binds within the inner cavity and partly occludes the permeation pathway and (b) residue hNa(v)1.5-N927 is critical for ion permeation between bound BTX and D2S6, probably because the side-chain of N927 helps coordinate permeating Na(+) ions.     

Homology model of dihydropyridine receptor: implications for L-type Ca(2+) channel modulation by agonists and antagonists

L-type calcium channels (LCCs) are transmembrane (TM) proteins that respond to membrane depolarization by selectively permeating Ca(2+) ions. Dihydropyridine (DHP) agonists and antagonist modulate Ca(2+) permeation by stabilizing, respectively, the open and closed states of the channel. The mechanism of action of these drugs remains unclear. Using, as a template, the crystal structure of the KcsA K(+) channel (Doyle et al. (1998) Science 280, 69-77), we have built several homology models of LCC with alternative alignments of TM segments between the proteins. In each model, nifedipine was docked in the pore region and in the interface between repeats III and IV. Several starting structures were generated by constraining the ligand to residues whose mutations reportedly affect DHP binding (DHP-sensing residues). These structures were Monte Carlo-minimized with and without constraints. In the complex with the maximum number of contacts between the ligand and DHP-sensing residues and the lowest ligand-receptor energy, the drug fits snugly in the "water-lake" cavity between segments S6s, which were aligned with M2 segment of KcsA as proposed for Na(+) channel (Lipkind and Fozzard (2000) Biochemistry 39, 8161-8170). In the flattened-boat conformation of DHP ring, the NH group at the stern approaches the DHP-sensing tyrosines in segments IIIS6 and IVS6. Stacking interactions of IVS6 Tyr with the bowsprit aromatic ring stabilize the ligand's orientation in which the starboard COOMe group coordinates Ca(2+) ion chelated by two conserved glutamates in the selectivity filter. In the inverted teepee structure of LCC, the portside COOMe group approaches a bracelet of conserved hydrophobic residues at the helical-bundle crossing, which may function as the activation gate. The dimensions of the gate may readily change upon small rotation of the pore-forming TM segments. The end of the portside group is hydrophobic in nifedipine, (R)-Bay K 8644, and other antagonists. Favorable interactions of this group with the hydrophobic bracelet would stabilize its closed conformation. In contrast, (S)-Bay K 8644 and several other agonists have hydrophilic groups at the portside. Unfavorable interactions of the hydrophilic group with the hydrophobic bracelet would destabilize its closed conformation thereby stabilizing the open conformation. In the agonist-bound channel, Ca(2+) ions would permeate between the hydrophilic face of the ligand and conserved hydrophilic residues in segments IS6 and IIS6. Our model suggests mutational experiments that could further our understanding of the pharmacological modulation of voltage-gated ion channels.     

Two-H-bonded and one-H-bonded structure alternations in collagen

This paper concerns the conformational variability of collagen as related to the concrete tripeptides (GXY)n constituting its primary structure. The previously elaborated model (V.G.Tumanyan, N.G.Esipova, Biophysics 28, 1021-1025, 1983) with two nets of hydrogen bonds is useful for tripeptides where X is an amino acid. If X is an imino acid, the common one-bonded Rich & Crick model is valid. In this work, compound sequences including tripeptides of different types are considered. Molecular mechanics is used to assess the conformations of the junction regions when a structure with two nets of hydrogen bonds precedes the structure with one net, and vice versa. Thus, all types of sequences typical for natural collagen are covered. It is shown that the combined model representing an alternation of the two-H-bonded model and the one-H-bonded Rich & Crick model is satisfactory stereochemically, and provides more favorable energy in comparison with the continuous one-H-bonded model. Besides, a more favorable hydration of the molecule occures in this case. Some conclusions are made about interchain and intrachain ionic bonds. Thus, it is deduced for the concrete fibrillar protein how a one-dimensional structure determines three-dimensional structure. The macromolecular structure thus suggested is in accord with the experimental data on hydrogen exchange.     

Guanidine Derivatives: Conformation, Capability for Chelation, Study as Reversible Inhibitors of Cholinesterases of Different Origin

Study of spatial structure of biologically active guanidine derivatives by the method of molecular mechanics has shown that in an anticoccidial drug, 1,3-bis ( p-chlorobenzylidenamino)guanidine (Cl-BAG) the most preferable are convolute conformations, in which the chlorine atoms that are distant in the valent chain are approached to each other at a distance of 3.7 . This indicates predisposition of the optimal conformations to form chelate complexes with ions of metals, which is confirmed by comparative spectrophotometric studies of the second derivative of differential UV-spectra of Cl-BAG in the presence and absence of calcium ions. Its derivative without chlorine (BAG) is unable to bind Ca²⁺ and has been shown to have no anticoccidial action, which associates the biological potency with the presence of calcium-binding ability of the compounds. The capability of Cl-BAG for chelation depends essentially on nature of the chelated metal ion. The antienzyme testing of inhibiting action of the guanidine derivatives toward cholinesterases of human erythrocytes, horse blood serum, mink brain and serum, optic ganglia of the Pacific squid Todarodes pacificus has revealed difference between the enzymes due to possibility of redistribution of the positive charge between the guanidinium fragment and amino groups and a change of the degree of charge delocalization.     

Monolayer formation of human osteoblastic cells on vertically aligned multiwalled carbon nanotube scaffolds

Monolayer formation of SaOS‐2 (human osteoblast‐like cells) was observed on VACNT (vertically aligned multiwalled carbon nanotubes) scaffolds without purification or functionalization. The VACNT were produced by a microwave plasma chemical vapour deposition on titanium surfaces with nickel or iron as catalyst. Cell viability and morphology studies were evaluated by LDH (lactate dehydrogenase) release assay and SEM (scanning electron microscopy), respectively. The non‐toxicity and the flat spreading with monolayer formation of the SaOs‐2 on VACNT scaffolds surface indicate that they can be used for biomedical applications.