Covalent tethering of the dimer interface annuls aggregation in thymidylate synthase

Thymidylate synthase (TS), a dimeric enzyme, forms large soluble aggregates at concentrations of urea (3.3-5 M), well below that required for complete denaturation, as established by fluorescence and size-exclusion chromatography. In contrast to the wild-type enzyme, an engineered mutant of TS (T155C/E188C/C244T), TSMox, in which two subunits are crosslinked by disulfide bridges between residues 155-188' and 188-155', does not show this behavior. Aggregation behavior is restored upon disulfide bond reduction in the mutant protein, indicating the involvement of interface segments in forming soluble associated species. Intermolecular disulfide crosslinking has been used as a probe to investigate the formation of larger non-native aggregates. The studies argue for the formation of large multimeric species via a sticky patch of polypeptide from the dimer interface region that becomes exposed on partial unfolding. Covalent reinforcement of relatively fragile protein-protein interfaces may be a useful strategy in minimizing aggregation of non-native structures in multimeric proteins.

Structure formation in short designed peptides probed by proteolytic cleavage

The formation of local structure, in short peptides has been probed by examining cleavage patterns and rates of proteolysis of designed sequences with a high tendency to form β-hairpin structures. Three model sequences which bear fluorescence donor and acceptor groups have been investigated: Dab-Gaba-Lys-Pro-Leu-Gly-Lys-Val-Xxx-Yyy-Glu-Val-Ala-Ala-Cys-Lys-NH2 ï EDANS Xxx-Yyy: Peptide 1=DPro-LPro, Peptide 2=DPro-Gly, Peptide 3=Leu-Ala Fluorescence resonance energy transfer (FRET) provides a convenient probe for peptide cleavage. MALDI mass spectrometry has been used to probe sites of cleavage and CD spectroscopy to access the overall backbone conformation using analog sequences, which lack strongly absorbing donor and acceptor groups. The proteases trypsin, subtilisin, collagenase, elastase, proteinase K and thermolysin were used for proteolysis and the rates of cleavage determined. Peptide 3 is the most susceptible to cleavage by all the enzymes except thermolysin, which cleaves all three peptides at comparable rates. Peptides 1 and 2 are completely resistant to the action of trypsin, suggesting that β-turn formation acts as a deterrent to proteolytic cleavage.

N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yI)colcemid, a probe for different classes of colchincine-binding site on tubulin

The nature of binding of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-colcemid (NBD-colcemid), an environment-sensitive fluorescent analogue of colchicine, to tubulin was tested. This article reports the first fluorometric study where two types of binding site of colchincine analogue on tubulin were detected. Binding of NBD-colcemid to one of these sites equilibrates slsowly. NBD-colcemid competes with colchicine for this site. Binding of NBD-colcemid to this site also causes inhibition of tubulin self-assembly. In contrast, NBD-colcemid binding to the other site is characterised by rapid equilibration and lack of competition with colchicine. Nevertheless, binding to this site is highly specific for the cholchicine nucleus, as alkyl-NBD analogues have no significant binding activity. Fast-reaction-kinetic studies gave 1.76 × 105 M–1 s–1 for the association and 0.79 s–1 for the dissociation rate constants for the binding of NBD-colcemid to the fast site of tubulin. The association rate constants for the two phases of the slow site are 0.016 × 10–4 M–1 s–1 and 3.5 × 10–4 M–1 respectively. These two sites may be related to the two sites of colchicine reported earlier, with binding characteristics altered by the increased hydrophobic nature of NBD-colcemid.

Differential fluorescence of the chromocenters and nucleolar equivalents of the yeast nucleus in acridine orange

The nucleus with its limiting membrane and organelles was visible in the majority of the yeast cells stained vitally with the fluorochrome, acridine orange, at a dilution of 1 in 40,000. The intra-nuclear structures could be distinguished by their differential fluorescence. The chromocenters were green while the nucleolar equivalents were orange. The vacuole showed no fluorescence.

Note: Dynamic point spread function for single and multiphoton fluorescence microscopy

We propose and demonstrate a dynamic point spread function (PSF) for single and multiphoton fluorescence microscopy. The goal is to generate a PSF whose shape and size can be maneuvered from highly localized to elongated one, thereby allowing shallow-to-depth excitation capability during active imaging. The PSF is obtained by utilizing specially designed spatial filter and dynamically altering the filter parameters. We predict potential applications in nanobioimaging and fluorescence microscopy.

Note: Dynamic point spread function for single and multiphoton fluorescence microscopy

We propose and demonstrate a dynamic point spread function (PSF) for single and multiphoton fluorescence microscopy. The goal is to generate a PSF whose shape and size can be maneuvered from highly localized to elongated one, thereby allowing shallow-to-depth excitation capability during active imaging. The PSF is obtained by utilizing specially designed spatial filter and dynamically altering the filter parameters. We predict potential applications in nanobioimaging and fluorescence microscopy.

Saccharide binding to three Gal/GalNAc specific lectins: Fluorescence, spectroscopic and stopped-flow kinetic studies

Fluorescence and stopped-flow spectrophotometric studies on three plant lectins fromPsophocarpus tetragonolobus (winged bean),Glycine max (soybean) andArtocarpus integrifolia (jack fruit) have been studied usingN-dansylgalactosamine as a fluorescent ligand. The best monosaccharide for the winged bean agglutinin I (WBA I) and soybean (SBA) is Me-agrGalNAc and for jack fruit agglutinin (JFA) is Me-agrGal. Examination of the percentage enhancement and association constants (1.51×106, 6.56×106 and 4.17×105 M–1 for SBA, WBA I and JFA, respectively) suggests that the combining regions of the lectins SBA and WBA I are apolar whereas that of JFA is polar. Thermodynamic parameters obtained for the binding of several monosaccharides to these lectins are enthalpically favourable. The binding of monosaccharides to these lectins suggests that the-OH groups at C-1, C-2, C-4 and C-6 in thed-galactose configuration are important loci for interaction with these lectins. An important finding is that the JFA binds specifically to Galß1-3GaINAc with much higher affinity than the other disaccharides which are structurally and topographically similar.The results of stopped-flow spectrometry on the binding ofN-dansylgalactosamine to these lectins are consistent with a bimolecular single step mechanism. The association rate constants (2.4×105, 1.3×104, and 11.7×105 M–1 sec–1 for SBA, WBA I and JFA, respectively) obtained are several orders of magnitude slower than the ones expected for diffusion controlled reactions. The dissociation rate constants (0.2, 3.2×10–2, 83.3 sec–1 for SBA, WBA I and JFA, respectively) obtained for the dissociation ofN-dansylgalactosamine from its lectin complex are slowest for SBA and WBA I when compared with any other lectin-ligand dissociation process.

Coplanar Capacitively Coupled Probe Fed Microstrip Antennas for Wideband Applications

The design and analysis of a coplanar capacitive fed microstrip antenna suspended above the ground plane is presented. It is demonstrated that the proposed approach can be used for designing antennas with impedance bandwidth of about 50% and a good gain to operate in various microwave bands. The model of the antenna incorporates the capacitive feed strip which is fed by a coaxial probe using equivalent circuit approach, and matches simulation and experimental results. The capacitive feed strip used here is basically a rectangular microstrip capacitor formed from a truncated microstrip transmission line and all its open ends are represented by terminal or edge capacitances. The error analysis was carried out for validity of the model for different design parameters. The antenna configuration can be used where unidirectional radiation patterns are required over a wide bandwidth.

A fluorescent probe study of the solubilisation of cholesterol by biie salt-phospholipid micelles

The fluorescent probe dansyl cadaverine has been shown to bind strongly to mixed bile salt-phospholipid micelles containing unsaturation in the fatty acyl chains. Incorporation of cholesterol into the mixed micelles reduces the number of molecules of bound dansyl cadaverine without altering the binding affinity. These results suggest a tighter packing of the hydrocarbon matrix of the micelles in the presence of cholesterol.

Top polarization as a probe of new physics

We investigate the effects of new physics scenarios containing a high mass vector resonance on top pair production at the LHC, using the polarization of the produced top. In particular we use kinematic distributions of the secondary lepton coming from top decay, which depends on top polarization, as it has been shown that the angular distribution of the decay lepton is insensitive to the anomalous tbW vertex and hence is a pure probe of new physics in top quark production. Spin sensitive variables involving the decay lepton are used to reconstruct the top polarization. Some sensitivity is found for the new couplings of the top.

Photobleaching reduced fluorescence correlation spectroscopy

A dual beam excitation-depletion pulse technique is proposed for photobleaching reduced fluorescence correlation spectroscopy (FCS). Excitation pulse promote the molecules to the excited singlet state (S-1), a fraction of that population goes to energetically favorable metastable triplet state (T-1) due to strong intersystem crossing. The depletion pulse followed by excitation pulse instantaneously depletes the triplet states thereby recycling the bleached molecules back to the ground state (S-0). FCS study on diffusing Fluorescein and Rh6G molecules show more than 95% reduction in triplet state population and the associated photobleaching. (c) 2010 American Institute of Physics.