Biomedical subjects
Publications and source records attributed to K R Easwaran.
Aluminum (Al) transport across yeast cells was studied using Dy(NO3)3 as a shift reagent by 27Al-NMR spectroscopy. The results showed that (a) Al enters the yeast cells at 15 min and over a period of time, within 4 h, an equilibrium sets in between outside and inside Al; (b) citrate does not favor Al going into the yeast cells at pH 5.0; and (c) EDTA brings out all the Al that has entered the yeast cells.
We report on spectroscopic studies of the chiral structure in phospholipid tubules formed in mixtures of alcohol and water. Synthetic phospholipids containing diacetylenic moieties in the acyl chains self-assemble into hollow, cylindrical tubules in appropriate conditions. Circular dichroism provides a direct measure of chirality of the molecular structure. We find that the CD spectra of tubules formed in mixtures of alcohol and water depends strongly on the alcohol used and the lipid concentration. The relative spectral intensity of different circular dichroism bands correlates with the number of bilayers observed using microscopy. The results provide experimental evidence that tubule formation is based on chiral packing of the lipid molecules and that interbilayer interactions are important to the tubule structure.
Aluminium exposure has been shown to result in aggregation of microtubule-associated protein tau in vitro. In the light of recent observations that the native random structure of tau protein is maintained in its monomeric and dimeric states as well as in the paired helical filaments characteristic of Alzheimer's disease, it is likely that factors playing a causative role in neurofibrillary pathology would not drastically alter the native conformation of tau protein. We have studied the interaction of tau protein with aluminium using circular dichroism (CD) and 27Al NMR spectroscopy. The CD studies revealed a five-fold increase in the observed elipticity of the tau-aluminium assembly. The increase in elipticity was not associated with a change in the general conformation of the protein and was most likely due to an aggregation of the tau protein induced by aluminium. 27Al NMR spectroscopy confirmed the binding of aluminium to tau protein. Hyperphosphorylation of tau in Alzheimer's disease is known to be associated with defective microtubule assembly in this condition. Abnormally phosphorylated tau exists in a polymerized form in the paired helical filaments (PHF) which constitute the neurofibrillary tangles found in Alzheimer's disease. While it is hypothesized that its altered biophysical characteristics render abnormally phosphorylated tau resistant to proteolysis, causing the formation of stable deposits, the sequence of events resulting in the polymerization of tau are little understood, as are the additional factors or modifications required for this process. Based on the results of our spectroscopic studies, a model for the sequence of events occurring in neurofibrillary pathology is proposed.
The conformation of the synthetic cyclic tetrapeptide cyclo(D-Phe-Pro-Sar-Gly) has been determined in solution using the nuclear magnetic resonance technique and in the crystal state by X-ray crystallography. Results showed that the peptide exhibited two different conformations in solution, conformer 1 having cis-trans-cis-trans peptide bonds and conformer 2 having trans-cis-trans-cis peptide bonds. No intramolecular hydrogen bonds were observed in the structures. The X-ray diffraction studies showed the crystals to be orthorhombic with space group P2(1)2(1)2(1) with unit-cell dimensions, a = 5.790, b = 10.344, c = 31.446 A, Z = 4, R = 0.104 for 2301 observed reflections. The crystal structure showed only one type of conformer having cis-trans-cis-trans peptide bonds similar to the conformer 1 in solution.
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The circular dichroism, fluorescence, Nuclear Magnetic Resonance and BLM conductance studies indicate that A23187 forms a stable complex with amino acids at low ionophore concentrations (< 10(-4)M). However, A23187 prefers to be in a dimeric structure with no significant binding to amino acids, at concentrations higher than 10(-4)M. It was also observed that at lower concentrations, at which the amino acids bind to the ionophore, the affinity for calcium ions was several orders of magnitude lower than that at higher ionophore concentrations. We have also conducted molecular modeling studies to examine the structure of the A23187 dimer and its amino acid complexes. The results of these modeling studies strongly support our experimental results and validate the formation of a hydrogen bonded and energetically stable A23187 dimer and its amino acid complexes.
We report in this paper the aggregation properties of amphotericin-B (amp-B) in solution using CD and 1H-NMR techniques. Our results indicate that the preferred structure of amp-B in dimethylsulfoxide is a monomer at low concentrations (10(-4) M and below) and a stable dimer at higher concentrations (range 5.10(-3) M to 10(-2) M). In a DMSO/ethanol mixture (1:1 (v/v)), the antibiotic is monomeric, irrespective of the concentration within the range studied. We propose a head-to-tail model based on NMR data. An understanding of the head-to-tail dimer, is, we believe important, particularly in view of the recent report wherein it is proposed that the drug inserts into bilayers as head-to-tail oligomers.
The interactions between the polyene antibiotic amphotericin B with dipalmitoylphosphatidylcholine were investigated in vesicles (using circular dichroism) and in chloroform solution (using circular dichroism and 1H, 13C, and 31P nuclear magnetic resonance). The results show that amphotericin B readily aggregates in vesicles and that the extent of aggregation depends on the lipid:drug concentration ratio. Introduction of sterol molecules into the membrane hastens the process of aggregation of amphotericin B. In chloroform solutions amphotericin B strongly interacts with phospholipid molecules to form a stoichiometric complex. The results suggest that there are interactions between the conjugated heptene stretch of amphotericin B and the methylene groups of lipid acyl chains, while the sugar moiety interacts with the phosphate head group by the formation of a hydrogen bond. A model is proposed for the lipid-amphotericin B complex, in which amphotericin B interacts equally well with the two lipid acyl chains, forming a 1:1 complex.
Detailed studies on the solution conformation of polyene antibiotic, filipin III using circular dichroism (CD) and proton nuclear magnetic resonance techniques have been carried out. In dimethyl sulfoxide (DMSO), filipin III exhibits concentration dependent aggregation-monomeric at lower and oligomeric at higher concentrations of the antibiotic. At concentrations used for 1H NMR studies (6 x 10(-3)M) the molecule coexists as monomeric and oligomeric species. However, titration experiments indicated that, in a mixed solvent system of DMSO:methanol (2:3 v/v) the antibiotic exists only as a monomer. Complete 1H NMR assignments and the conformation of the monomer filipin III have been determined by the combined use of DQFCOSY and ROESY experiments in DMSO:methanol solvent system.
Crystal structures of two different forms of the calcium perchlorate complex of cyclo(Ala-Leu-Pro-Gly)2 have been determined and refined using X-ray crystallographic techniques. Orthorhombic form: C32H52N8O8.Ca(ClO4)2.7H2O.2CH3OH, space group C222(1), a = 14.366, b = 18.653, c = 19.824 A, Z = 4, R = 0.068 for 2208 observed reflections. Monoclinic form: C32H52N8O8.Ca(ClO4)2.4H2O, space group C2, a = 21.096, b = 10.182, c = 11.256 A, beta = 103.33 degrees, Z = 2, R = 0.075 for 2165 observed reflections. The cyclic peptide molecule in both the structures has the form of a twofold symmetric, slightly elongated bowl. Type II' beta-turns, involving Gly and Ala at the corners, exist at the two ends of the molecule. The interior of the molecule is substantially hydrophilic, and the external surface of the bowl is largely hydrophobic. The calcium ion is located at the centre of the mouth of the bowl-like molecule. In both crystal forms, four peptide carbonyl oxygens from the cyclic peptide and two solvent oxygens coordinate to the metal ion. The mode of complexation may be described as incomplete encapsulation as, for example, in the case of metal complexes of antamanide. In the crystal structures the complex ions are held together by hydrogen bonds involving perchlorate ions and water molecules. The molecular structure observed in the crystals is entirely consistent with the results of solution studies, which also indicate the conformation of the cyclic peptide in the complex to be similar to that of the uncomplexed molecule.
For the first time, based on bilayer membrane conductance experiments, it has been shown that A23187, a carboxylic calcium ionophore, incorporated in lipid bilayers gives single channel currents similar to the well known gramicidin channel. The current characteristics indicate the possibility that the transmembrane ion transport by this important calcium ionophore is initially by a carrier mechanism but with time is by a channel or pore mechanism due to the aggregation of the molecule in a lipid matrix.
The conformation and ion-binding characteristics of a cyclic octapeptide, cyclo (Ala-Leu-Pro-Gly)2, in a liphophilic solvent, acetonitrile, have been studied using CD and nmr spectroscopy. The peptide binds preferentially to divalent cations such as calcium, magnesium, and barium. The conformations of the free cyclic peptide and its calcium complex are very similar with well-defined beta- and gamma-turns. The cyclic peptide readily forms equimolar and possibly 2:1 (peptide:cation) complexes with divalent cations.
The circular dichroism studies on calcium ionophore, A23187, incorporated in Dipalmitoyl phosphatidyl choline (DPPC) vesicle showed interesting time dependent changes in the CD spectra. Analysis of the data indicated the possible aggregation of the observed dimeric structure of this molecule in non-polar solvents into a stacked dimeric pore in the phospholipid vesicle.
Two related homodetic bicyclic nonapeptides (cyclo Glu-X-Pro-Gly-Lys-X-Pro-Gly)-cyclo (l gamma----5 epsilon), X = Ala(BCP2), X = Leu(BCP3) have been synthesized using conventional solution phase methods involving mixed anhydride coupling reactions starting with appropriately protected naturally occurring amino acids. The conformation and ion binding properties of BCP2 have been studied by nuclear magnetic resonance and circular dichroism techniques. The results of these studies have been compared to those of BCP3. The presence of Ala caused both Ala-Pro bonds to be trans in free BCP2. This characteristic imparted subtle differences to the ion-binding properties of BCP2 as compared to free BCP3 which has one cis Leu-Pro bond and one trans Leu-Pro bond.
The transport kinetics of three lanthanide ions (viz., Pr3+, Nd3+, and Eu3+) across dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine unilamellar vesicles mediated by the two carboxylic ionophores lasalocid A and A23187 have been studied by proton nuclear magnetic resonance spectroscopy. Time-dependent changes in the chemical shifts of head group choline signals have been measured to calculate apparent rate constants of transport. These experiments have been done at different ionophore concentrations to determine the stoichiometry of the transporting species. The rates of transport have been found to be faster in the absence of intravesicular La3+ compared to those observed in its presence. The stoichiometry of the transporting species has been found to be 2:1 (ionophore:cation) for both lasalocid A and A23187 in dimyristoylphosphatidylcholine vesicles. However, stoichiometries of greater than 2 have been obtained for lasalocid A mediated lanthanide ion transport across dipalmitoylphosphatidylcholine vesicles. Possible reasons for the observations of such noninteger stoichiometries are discussed. Our results also indicated that A23187 is a more efficient carrier ionophore than lasalocid A.
The interaction of lanthanide(III) nitrates (La3+ to Lu3+) with the carboxylic ionophore lasalocid A (LS) has been studied by circular dichroism (CD) and fluorescence spectroscopic techniques in acetonitrile and in methanol. Analysis of the CD data in acetonitrile has revealed the coexistence of both 1:1 (ionophore:cation) and 2:1 complexes in solution. For 1.22 A greater than ionic radius greater than 1.13 A, 1:1 complexes are preferred, and for 1.13 A greater than ionic radius greater than 1.03 A, 2:1 complexes are preferred. Induced CD bands for Ln3+ ions have been observed upon binding to LS in acetonitrile. The LS-Ln3+ complexes are less stable in methanol than in acetonitrile. CD spectral changes showed that the conformations of the complexes in methanol are different from those in acetonitrile. The complexes have rather open conformations in methanol compared to those in acetonitrile. The results underscore the importance of ionic radius, solvent environment, and ionization state of LS in determining the conformations of the ionophore-cation complexes.
The interactions of carrier ionophores, nonactin, A23187, and lasalocid A with liposomes formed from the synthetic lipids dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine are investigated by differential scanning calorimetry and 1H and 31P nuclear magnetic resonance techniques. The results indicate that the mode of interaction of these ionophores is dependent on the fluidity of the bilayer and on the chemical nature of these ionophores. The 31P NMR studies are suggestive of the formation of small particles that are probably intervesicular lipid-ionophore aggregates in multilamellar vesicles when they are incorporated with these ionophores at high concentrations. The results are interpreted on the basis of the chemical structure and conformations of the ionophores in membrane mimetic media. The 1H NMR line-width measurements indicate that the aromatic rings containing the carboxyl groups of lasalocid A and A23187 are located near the membrane interface while the rest of the molecule is buried in the membrane interior.
The interaction of cupric isonicotinohydrazide (CuIIINH), an antiviral compound, with calf thymus DNA was investigated by circular dichroism (CD) and nuclear magnetic resonance (NMR). Gel electrophoresis of DNA incubated with CuIIINH showed cleavage of DNA to various extents. This cleavage was found to be time and concentration dependent. In the presence of CuIIINH the positive CD band at 274 nm disappeared and the negative band at 246 nm showed a decrease in the mean residual ellipticity value, indicating binding of CuIIINH to DNA. 31P NMR studies indicated that the binding of copper in CuIIINH is to the phosphate oxygen of the DNA backbone. The binding of CuIIINH was also found to be reversible. Addition of ethylenediaminetetraacetic acid to the CuIIINH-DNA complex resulted in breaking of the complex and restoring the original structural features of the B family of DNA in the resulting fragments. At the concentration level of CuIIINH employed, both CuSO4 and INH independently did not show any interaction with DNA.