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  • 1
    Electronic Resource
    Electronic Resource
    Measuring protein self-association using pulsed-field-gradient NMR spectroscopy: Application to myosin light chain 2 (1995)
    Springer
    Journal of biomolecular NMR 6 (1995), S. 321-328 
    Details
    ISSN: 1573-5001
    Keywords: Pulsed-field-gradient NMR ; Translational diffusion coefficient ; Self-association ; Myosin light chain ; CHAPS
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary At the millimolar concentrations required for structural studies, NMR spectra of the calcium-binding protein myosin light chain 2 (MLC2) showed resonance line widths indicative of extensive self-association. Pulsed-field-gradient (PFG) NMR spectroscopy was used to examine whether MLC2 aggregation could be prevented by the zwitterionic bile salt derivative 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). PFG NMR measurements indicated that CHAPS was capable of preventing MLC2 self-association, but only at concentrations well above the critical micelle concentration of ∼7.5 mM. CHAPS was most effective at a concentration of 22.5 mM, where the apparent molecular mass of MLC2 correponded to a protein monomer plus seven molecules of bound detergent. The resolution and sensitivity of 2D 15N-1H HSQC spectra of MLC2 were markedly improved by the addition of 25 mM CHAPS, consistent with a reduction in aggregation following addition of the detergent. The average amide nitrogen T2 value for MLC2 increased from ∼30 ms in the absence of CHAPS to ∼56 ms in the presence of 25 mM CHAPS. The results of this study lead us to propose that PFG NMR spectroscopy can be used as a facile alternative to conventional techniques such as analytical ultracentrifugation for examining the self-association of biological macromolecules.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00197813
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  • 2
    Electronic Resource
    Electronic Resource
    Measuring macromolecular diffusion using heteronuclear multiple-quantum pulsed-field-gradient NMR (1997)
    Springer
    Journal of biomolecular NMR 10 (1997), S. 1-8 
    Details
    ISSN: 1573-5001
    Keywords: Pulsed-field-gradient NMR ; Translational diffusion coefficient ; Self-association ; Macromolecules ; Solvent suppression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract We have previously shown that 1H pulsed-field-gradient(PFG) NMR spectroscopy provides a facile method for monitoring proteinself-association and can be used, albeit with some caveats, to measure theapparent molecular mass of the diffusant [Dingley et al. (1995) J. Biomol.NMR, 6, 321–328]. In this paper we show that, for15N-labelled proteins, selection of1H-15N multiple-quantum (MQ) coherences in PFGdiffusion experiments provides several advantages over monitoring1H single-quantum (SQ) magnetization. First, the use of agradient-selected MQ filter provides a convenient means of suppressingresonances from both the solvent and unlabelled solutes. Second,1H-15N zero-quantum coherence dephases morerapidly than 1H SQ coherence under the influence of a PFG.This allows the diffusion coefficients of larger proteins to be measuredmore readily. Alternatively, the gradient length and/or the diffusion delaymay be decreased, thereby reducing signal losses from relaxation. In orderto extend the size of macromolecules to which these experiments can beapplied, we have developed a new MQ PFG diffusion experiment in which themagnetization is stored as longitudinal two-spin order for most of thediffusion period, thus minimizing sensitivity losses due to transverserelaxation and J-coupling evolution.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1018339526108
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  • 3
    Electronic Resource
    Electronic Resource
    Zipping up transcription factors: Rational design of anti-Jun and anti-Fos peptides (1997)
    Springer
    International journal of peptide research and therapeutics 4 (1997), S. 67-77 
    Details
    ISSN: 1573-3904
    Keywords: Peptide drug ; Peptide delivery ; Leucine zipper ; Anticancer drugs ; Transcription factors ; Jun ; Fos
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Various members of the bZip and bHLH-Zip families of eukaryotic transcription factors,including Jun, Fos, and Myc, have been identified as oncoproteins; mutation or deregulatedexpression of these proteins leads to certain types of cancer. These proteins can only bind totheir cognate DNA enhancer sites following homodimerization, or heterodimerization withanother family member, via their leucine zipper domain. Thus, a novel anticancer strategywould be to inhibit dimerization of these proteins, thereby blocking their DNA binding andtransactivation functions. In this paper we show that it is possible to rationally design leucinezipper peptides that bind with high affinity to the leucine zipper dimerization domains of c-Jun and c-Fos, thus preventing the formation of functional c-Jun homodimers and c-Jun:c-Fosheterodimers; we refer to such peptides as superzippers (SZs). In vivo, c-Jun:SZ and c-Fos:SZheterodimers should be nonfunctional as they lack one of the two basic domains that areessential for DNA binding. While the transport of a peptidic agent into cells often poses asevere obstacle to its therapeutic use, we show that a 46-residue leucine zipper peptide canbe transported into HeLa cells by coupling it to a 17-residue carrier peptide from theAntennapedia homeodomain, thus paving the way for detailed studies of the therapeuticpotential of superzipper peptides.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008883300477
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    Paper (German National Licenses)
    Fulltext
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