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  • 1
    Electronic Resource
    Electronic Resource
    Determination of macroion diffusion coefficients using an analytical electrophoresis apparatus (1997)
    Springer
    European biophysics journal 25 (1997), S. 481-487 
    Details
    ISSN: 1432-1017
    Keywords: Key words Analytical electrophoresis ; Diffusion ; Macromolecules ; Simulations
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract Methods are presented to determine the effective macroion diffusion coefficient in the presence or absence of an electric field, using a unique analytical electrophoresis apparatus in which both electrophoretic mobility and steady-state electrophoresis may be studied. Approximate analytic solutions to the differential equations and boundary conditions describing diffusion are derived. These solutions are shown to be good approximations to numerial simulations of the differential equations, and provide a good phenomenological description of experimental data for the oligonucleotide p(dA)20⋅p(dT)20 in 100 mM KCl, 20 mM Tris-HCl, pH 8.0 buffer. Diffusion and electrophoresis measurements are made on a single sample without changing the buffer or the macroion concentration, thus the data are directly comparable.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002490050064
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  • 2
    Electronic Resource
    Electronic Resource
    Effector-induced self-association and conformational changes in the enhancer-binding protein NTRC (1996)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 22 (1996), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Klebsiella pneumoniae nitrogen regulatory protein NTRC is a response regulator which activates transcription in response to nitrogen limitation, and is a member of the family of σN-dependent enhancer-binding proteins. Using limited trypsin digestion, two domains of NTRC were detected and conformational changes within the protein in response to the binding of ligands were also observed. In the absence of ligands, the major digestion products were 42, 36 and 12.5 kDa bands corresponding to the central plus C-terminal domain, the central domain, and the N-terminal domains, respectively. Upon binding of purine but not pyrimidine nucleotides, the 36 kDa band was insensitive to further proteolysis, indicative of a conformational change in the central domain. Analysis of the dependence of this insensitivity on ATPγS concentration suggested an apparent dissociation constant (Kd) for ATPγS of 150 μM. In the presence of DNA, both the central and C-terminal domains of NTRC were insensitive to proteolytic cleavage, indicative of a further conformational change. NTRC S160F, a mutant form of NTRC that is active in the absence of phosphorylation, was more stable to proteolysis than the wild-type protein. This mutant protein is apparently locked in a conformation resembling the DNA-bound form of wild-type NTRC. The involvement of ligands in self-association was studied using sedimentation equilibrium analysis. In the absence of ligand, wild-type NTRC displayed a monomer–dimer equilibrium with a Kd of 6 μM. In the presence of ATPγS the equilibrium was shifted towards the dimer form (Kd = 0.8 μM). A similar dissociation constant for the monomer–dimer interaction was observed with NTRC S160F in the absence of ATPγS (Kd = 0.5 μM). The addition of ATPγS induced a significant association of NTRC S160F to higher-order states with a dimer–octamer model producing a slightly, but not significantly better fit to the data than a dimer–hexamer model. We propose that ligand-mediated self-association provides a common mechanism for activation of this class of transcriptional regulatory proteins.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1996.01530.x
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  • 3
    Electronic Resource
    Electronic Resource
    Analysis of an Escherichia coli mutant TyrR protein with impaired capacity for tyrosine-mediated repression, but still able to activate atσ70 promoters (1995)
    Osney Mead, Oxford OX2 0EL, UK : Blackwell Science Ltd
    Molecular microbiology 17 (1995), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In Escherichia coli, TyrR represses and activates transcription of operons required for tyrosine, phenylalanine and tryptophan biosynthesis and uptake. The TyrR central domain is homologous with NtrC and some other bacterial regulatory proteins, although TyrR regulates σ70, not σ54, promoters. We isolated a central domain TyrR mutant (TyrR E274Q) by substitution of a normally conserved amino acid. The mutant was unable to bring about tyrosine-mediated repression of aroF, aroL, tyrB, and tyrP and had diminished capability for tyrosine- and phenylalanine-mediated repression of aroP. In contrast, it was able to effect wild-type levels of phenylalanine-mediated repression of aroG, tryptophan-mediated repression of aroP and transcriptional activation of mtr and tyrP. The binding of purified TyrR E274Q to ATP (a requirement for tyrosine binding) and to the strong TyrR box of tyrP operator DNA were normal, but tyrosine binding and tyrosine-dependent hexamerization were significantly impaired. These properties are consistent with the proposal that self association is essential for tyrosine-mediated repression by TyrR but not for tyrosine- or phenylalanine-mediated activation. E274 of TyrR must participate in either the binding of tyrosine, or the coupling of ATP binding with a conformational change that alters the affinity of the ATP-dependent aromatic amino acid-binding site.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1995.mmi_17030471.x
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  • 4
    Electronic Resource
    Electronic Resource
    Evidence for two aromatic amino acid-binding sites, one ATP-dependent and the other ATP-independent, in the Escherichia coli regulatory protein TyrR (1995)
    Osney Mead, Oxford OX2 0EL, UK : Blackwell Science Ltd
    Molecular microbiology 17 (1995), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In Escherichia coli, genetic regulation of aromatic amino acid biosynthesis and uptake is effected by the protein TyrR, which acts via ligand-mediated repression and activation. Characterization of the interactions of tyrosine, phenylalanine and tryptophan with TyrR revealed the presence of two separate aromatic amino acid-binding sites, one ATP-dependent, the other ATP-independent. Binding to the ATP-dependent site induces the self-association of TyrR. Using sedimentation equilibrium analyses, dissociation constants for this site in the dimeric and hexameric forms of TyrR were determined to be 330 μM and 24 μM, respectively, for tyrosine, and 55 mM and 3.7 mM, respectively, for phenylalanine. Tryptophan bound with a strength similar to that of phenylalanine, and both phenylalanine and tryptophan competed with the binding of tyrosine. The ATP-independent site, which has not been observed previously, was characterized by ultraviolet (u.v.) difference spectroscopy and a sedimentation-velocity meniscus-depletion method. Phenylalanine bound co-operatively to this site, exhibiting half-saturation at 260 µM. Tryptophan competed weakly with phenylalanine, half-saturation occurring at 1.2 mM. No binding of tyrosine to this site could be detected. We propose that the binding of phenylalanine or tryptophan to this ATP-independent site is responsible for phenylalanine- and tryptophan-mediated regulation by TyrR.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1995.mmi_17030483.x
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  • 5
    Electronic Resource
    Electronic Resource
    Observation of internal cleavage and ligation reactions of a ribozyme (2004)
    [s.l.] : Nature Publishing Group
    Nature structural & molecular biology 11 (2004), S. 1107-1113 
    Details
    ISSN: 1545-9985
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Medicine
    Notes: [Auszug] We have used single-molecule spectroscopy to untangle conformational dynamics and internal chemistry in the hairpin ribozyme. The active site of the ribozyme is stably formed by docking two internal loops, but upon cleavage undocking is accelerated by two orders of magnitude. The markedly different ...
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1038/nsmb842
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  • 6
    Electronic Resource
    Electronic Resource
    An apparatus for membrane-confined analytical electrophoresis (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 1611-1619 
    Details
    ISSN: 0173-0835
    Keywords: Steady state electrophoresis ; Analytical electrophoresis ; Mobility determination ; Membrane-confined apparatus ; Solution charge ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A membrane-confined analytical electrophoresis apparatus for measuring the solution charge of macromolecules has been described previously (T. M. Laue et al., Anal. Biochem. 1989, 182, 377-382). Presented here is a design for this apparatus, which permits the on-line acquisition and display of absorbance data from up to 512 positions along an analysis chamber. Concentration distributions of macromolecules in solution can be monitored in the chamber to provide steady-state electrophoresis, electrophoretic mobility and diffusion measurements. Buffer chambers press semipermeable membranes against the open ends of a fused-silica cuvette to form the analysis chamber. This configuration permits both the flow of buffer and the establishment of an electric field across the cuvette, while retaining macromolecules in the field of view. Though a gel may be included in the analysis chamber, none is required for gradient stabilization. The volume of sample required for analysis is 8 μL, most of which is recoverable. Experimental conditions can be varied during study by simply changing the circulating buffer and/or the electric field. The analysis and buffer chambers are held in an aluminum housing that sits in an aluminum water jacket. The water jacket provides temperature control, shielding from external electrical noise and also serves as an optical mask. Plans for the cell assembly, optical system and the computer interface for data acquisition are provided. The assembly and operation of the apparatus and the analysis of data are described.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150191016
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