Biosensors based on peptide exposure show single molecule conformations in live cells

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Identification of a four copper folding intermediate in mammalian copper metallothionein by electrospray ionization mass spectrometry

 Mammalian metallothioneins (MT) are known to maximally bind 12 copper ions in two six-Cu(I) ion clusters. Using electrospray ionization mass spectrometry of MT at pH 4.5, a four-Cu(I) ion cluster was observed intermediate to a fully formed six Cu(I) in a single domain or a fully formed Cu₁₂MT species. The four-Cu(I) cluster was observed in both MT1 and MT3 isoforms. Addition of increasing amounts of Cu(I) to MT at pH 4.5 resulted in prominent ions whoses masses were consistent with apo-MT, Cu₄MT, Cu₆MT, and Cu₁₂MT. The cooperativity of cluster formation was reduced at pH 2.5. Addition of Cu(I) to apo-MT at a reduced pH resulted in a series of ions consistent with Cu₄ to Cu₁₂MT species. However, formation of the tetracopper MT species remained cooperative at low pH, suggesting that this species is very stable. To determine whether the tetracopper cluster was formed in either the α or β domain, domain peptides of MT3 were used. Addition of Cu(I) to the apo β domain resulted in a peak consistent with the formation of a four-Cu(I) cluster. This is consistent with reports that Cu(I) ions bind preferentially to the β domain of MTs. Received: 2 June 1998 / Accepted: 21 August 1998     

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.     

The quenching of tryptophan fluorescence by protonated and unprotonated imidazole

The fluorescence life-time of N-acetyl-tryptophan-amide (NATA) was measured by multifrequency phase fluorometry, in the presence of increasing concentrations of imidazole. Two pH values were tested, pH 4.5 where imidazole is fully protonated and pH 9.0 where it is fully unprotonated. At both pH values, the inverse life-time increases in a non-linear way with the imidazole concentration, showing that imidazole is not a high efficiency collisional quencher. The data can be analysed in terms of the formation of a complex with a reduced fluorescence life-time. The rate constants for association (at 25°C) are around 5 (±0.2) × 10⁹ M^–1 s^–1 and are thus diffusion controlled. The association equilibrium constant is strongly pH dependent and is much higher than the expected value of 0.4 M^–1 for a collisional complex. The intrinsic fluorescence life-time of the complex is 1.56 (±0.02) ns at pH 9.0 and 1.82 (±0.03) ns at pH 4.5, as compared to 2.37 (±0.03) ns for free NATA at pH 9.0 and 2.83 (±0.05) at pH 4.5 (all atI = 0.34). This means that at both pH values the fluorescence life-time of NATA in the complex is reduced to 61 (±0.5)% of its value in the free state. Despite this, the protonated form of imidazole is a better quencher at low concentrations, owing to a longer residence-time of the complex. At high viscosity the association equilibration is too slow and the system is described by two life-times. The quenching effect ofHis-18 on the fluorescence of the proximalTrp-94 of barnase (Locwenthal et al. 1991, Willaert et al. 1991) is discussed in terms of these findings.     

Functional Casein-Poly saccharide Conjugates Prepared by Controlled Dry Heating

Casein was conjugated with dextran and galactomannan in a controlled dry state at a relative humidity of 79% and at 60°C for 24 hr. The covalent attachment of polysaccharides to casein was confirmed by SDS-PAGE and HPLC. The emulsifying activity of the casein-dextran and casein-galactomannan conjugates was 1.5 times higher than that of casein. The emulsion stability of the casein-dextran and casein-galactomannan conjugates was 10 times higher than that of casein. The improvement in these emulsifying properties reached a steady state when the conjugation of casein with polysaccharide was done for 24 hr in a controlled dry state, suggesting the rapid formation of conjugates through a Maillard reaction in the case of casein. Compared to commercial emulsifiers, the casein-polysaccharide conjugates showed better emulsifying properties in acidic and high-salt concentration systems.     

Antimitotics induced cardiolipin cluster formation possible role in mitochondrial enzyme inactivation

We demonstrate here that drugs which inactivate cytochrome c oxidase are able to segregate cardiolipin essential for the enzyme activity, in a separate phase inaccessible for the enzyme. A molecular explanation of the drug-induced aggregation process is proposed.     

Stress-induced changes in the root architecture of white lupin (Lupinus albus) in response to pH, bicarbonate, and calcium in liquid culture

Current agronomic cultivars of white lupin (Lupinus albus) are intolerant of calcareous or limed soils. In these soils, high pH, bicarbonate (HCO₃^?), and calcium (Ca) concentrations are the major chemical stresses to the root system. To determine the responses of the root system to these factors, evaluate root architecture, and compare genotypes for tolerance, a series of liquid culture experiments was completed using root chambers that allowed the study of the root system in two dimensions. Each stress condition caused changes in different parts of the root system and there was no generalised stress response. HCO₃^? (5 mM) had the greatest effect on cultivars intolerant of calcareous soil; it decreased the dry weight of the shoot and caused the highest percentage of tap root deaths. HCO₃^? also discriminated between short (determinate) and long (indeterminate) roots, as it decreased the number and density of the determinate roots only. Calcium (3 mM) affected all parts of the root system. The tap root was shortened and showed an increased tortuousness in its path compared with 1 mM Ca, although no plants suffered tap root death. The numbers and densities of the two lateral root forms were also decreased, as were the lengths of the indeterminate roots. Stress from alkaline pH (7.5) media caused a lower number and density of determinate lateral roots to be produced than at pH 6.5. The experiments demonstrated that each culture condition elicited a definable stress response. Stress conditions altered the root architecture of genotypes reported to be tolerant of calcareous soil less than in intolerant genotypes. Although soil is more complex than liquid culture, it is possible that in a calcareous or limed soil each stress condition examined may affect the overall stress of the plant, and increased tolerance may result from tolerance to a single stress.     

Binding and precipitating activities ofErythrina lectins with complex type carbohydrates and synthetic cluster glycosides. A comparative study of the lectins fromE. corallodendron,E. cristagalli,E. flabelliformis,andE. indica

Erythrina lectins possess similar structural and carbohydrate binding properties. Recently, tri- and tetra-antennary complex type carbohydrates with non-reducing terminal galactose residues have been shown to be precipitated as tri- and tetravalent ligands, respectively, with certainErythrina lectins [Bhattacharyya L, Haraldsson M, Brewer CF (1988) Biochemistry 271034-41]. The present work describes a comparative study of the binding and precipitating activities of fourErythrina lectins,viz. E. corallodendron, E. cristagalli, E. flabelliformis, andE. indica, with multi-antennary complex type carbohydrates and synthetic cluster glycosides. The results show that though their binding affinities are very similar, theErythrina lectins show large differences in their precipitating activities with the carbohydrates. The results also indicate significant dependence of the precipitating activities of the lectins on the core structure of the carbohydrates. These findings provide a new dimension to the structure-activity relationship of the lectins and their interactions with asparagine-linked carbohydrates.Abbreviations EAL, ECorL, ECL, EFL, and EIL represent the lectins from the seeds ofErythrina arborescens, - E. corallodendron, E. cristagalli, E. flabelliformis, andE. indica respectively - AFOS thetri-antennary complex type oligosaccharide from asialofetuin - AFGP the tri-antennary glycopeptide from asialofetuin - MeGal methyl -d-galactopyranoside Unless stated otherwise all sugars are in thed-configuration.     

pH Dependence of Histidine Affinity for Blood-Brain Barrier Carrier Transport Systems for Neutral and Cationic Amino Acids

The effects of pH (3.5-7.5) on the brain uptake of histidine by the blood-brain barrier (BBB) carriers for neutral and cationic amino acids were tested, in competition with unlabeled histidine, arginine, or phenylalanine, with the single-pass carotid injection technique. Cationic amino acid ( [14C]arginine) uptake was increasingly inhibited by unlabeled histidine as the pH of the injection solution decreased. In contrast, the inhibitory effect of unlabeled histidine on neutral amino acid ( [14C]phenylalanine) uptake decreased with decreasing pH. Brain uptake indices with varying histidine concentrations indicated that the neutral form of histidine inhibited phenylalanine uptake whereas the cationic form competed with arginine uptake. Since phenylalanine decreased [14C]histidine uptake at all pH values whereas arginine did not, it was concluded that the cationic form of histidine had an affinity for the cationic carrier, but was not transported by it. We propose that the saturable entry of histidine into brain is, under normal physiological circumstances, mediated solely by the carrier for neutral amino acids.