240,249 research outputs found
Twelve-spin "Schrodinger cat"
Pseudopure "cat" state, a superposition of quantum states with all spins up
and all spins down, is experimentally demonstrated for a system of twelve
dipolar-coupled nuclear spins of fully 13C-labeled benzene molecule oriented in
a liquid-crystalline matrix.Comment: Submitted to Applied Physics Letter
Specific fluorescent labeling of chicken myofibril Z-line proteins catalyzed by guinea pig liver transglutaminase
Guinea pig liver transglutaminase has been found to catalyze the covalent incorporation of dansylcadaverine into chicken skeletal muscle myofibril proteins. Epifluorescence microscopy reveals that the incorporated dansylcadaverine is specifically localized at or near the myofibril Z line. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) indicates that actin constitutes a major fraction of the labeled material; the Z-line proteins alpha-actinin and desmin also show significant labeling, as well as tropomyosin, several additional unidentified proteins, and material with an extremely high molecular weight. The Z-line-specific fluorescence can be removed by brief trypsinization, which releases fluorescent alpha-actinin into the supernate. The majority of the fluorescent protein species are resistant to extraction by either 0.6 M KCl or KI. These results, in conjunction with the microscopic localization, suggest that the dansyl- labeled proteins are constituents of the myofibril Z line. A significant amount of fluorescently labeled transglutaminase is also present in labeled myofibrils, which is resistant to extraction with either 0.6 M KCl or KI. This result indicates a strong, noncovalent interaction between the transglutaminase molecule and the myofibril Z line
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Inhibition of a viral enzyme by a small-molecule dimer disruptor.
We identified small-molecule dimer disruptors that inhibit an essential dimeric protease of human Kaposi's sarcoma-associated herpesvirus (KSHV) by screening an alpha-helical mimetic library. Next, we synthesized a second generation of low-micromolar inhibitors with improved potency and solubility. Complementary methods including size exclusion chromatography and 1H-13C HSQC titration using selectively labeled 13C-Met samples revealed that monomeric protease is enriched in the presence of inhibitor. 1H-15N HSQC titration studies mapped the inhibitor binding site to the dimer interface, and mutagenesis studies targeting this region were consistent with a mechanism where inhibitor binding prevents dimerization through the conformational selection of a dynamic intermediate. These results validate the interface of herpesvirus proteases and other similar oligomeric interactions as suitable targets for the development of small-molecule inhibitors
Site-specific isotope labeling of long RNA for structural and mechanistic studies
A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, ‘isotopically labeled guanosine 5′-monophosphate’ (5′-GMP), steadily gave a 5′-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5′-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with 15N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry
Manipulating Protein Conformations By Single-molecule Afm-fret Nanoscopy
Combining atomic force microscopy and fluorescence resonance energy transfer spectroscopy (AFM-FRET), we have developed a single-molecule AFM-FRET nanoscopy approach capable of effectively pinpointing and mechanically manipulating a targeted dye-labeled single protein in a large sampling area and simultaneously monitoring the conformational changes of the targeted protein by recording single-molecule FRET time trajectories. We have further demonstrated an application of using this nanoscopy on manipulation of single-molecule protein conformation and simultaneous single-molecule FRET measurement of a Cy3-Cy5-labeled kinase enzyme, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase). By analyzing time-resolved FRET trajectories and correlated AFM force pulling curves of the targeted single-molecule enzyme, we are able to observe the protein conformational changes of a specific coordination by AFM mechanic force pulling
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