Rationally designed helix-turn-helix proteins and their conformational changes upon DNA binding

Piergiorgio Percipalle, A. Simoncsits, S. Zakhariev, C. Guarnaccia, R. Sanchez, S. Pongor

    Research output: Contribution to journalArticle

    Abstract

    Circular dichroism and electrophoretic mobility shift studies were performed to confirm that dimerized N-terminal domains of bacterial repressors containing helix-turn-helix moths are capable of high-affinity and specific DNA recognition as opposed to the monomeric N-terminal domains. Specific, high-affinity DNA binding proteins were designed and produced in which two copies of the N-terminal 1-62 domain of the bacteriophage 434 repressor are connected either in a dyad-symmetric fashion, with a synthetic linker attached to the C-termini, or as direct sequence repeats. Both molecules bound to their presumptive cognate nearly as tightly as does the natural (full-length and non-covalently dimerized) 434 repressor, showing that covalent dimerization can be used to greatly enhance the binding activity of individual protein segments. Circular dichroism spectroscopy showed a pronounced increase in the α-helix content when these new proteins interacted with their cognate DNA and a similar, although 30% lower, increase was also seen upon their interaction with non-cognate DNA. These results imply that a gradual conformational change may occur when helix-turn-helix motifs bind to DNA, and that a scanning mechanism is just as plausible for this motif class as that which is proposed for the more flexible basic-leucine zipper and basic-helix-loop-helix motifs.

    Original languageEnglish (US)
    Pages (from-to)3200-3205
    Number of pages6
    JournalEMBO Journal
    Volume14
    Issue number13
    StatePublished - Jan 1 1995

    Fingerprint

    DNA
    Circular Dichroism
    Helix-Loop-Helix Motifs
    Proteins
    Helix-Turn-Helix Motifs
    Circular dichroism spectroscopy
    Leucine Zippers
    Electrophoretic mobility
    Bacteriophages
    Dimerization
    Moths
    Nucleic Acid Repetitive Sequences
    DNA-Binding Proteins
    Spectrum Analysis
    Scanning
    Molecules

    Keywords

    • Bacterial repressors
    • Circular dichroism
    • Conformational charges
    • DNA-binding proteins
    • Helix-turn-helix proteins

    ASJC Scopus subject areas

    • Neuroscience(all)
    • Molecular Biology
    • Biochemistry, Genetics and Molecular Biology(all)
    • Immunology and Microbiology(all)

    Cite this

    Percipalle, P., Simoncsits, A., Zakhariev, S., Guarnaccia, C., Sanchez, R., & Pongor, S. (1995). Rationally designed helix-turn-helix proteins and their conformational changes upon DNA binding. EMBO Journal, 14(13), 3200-3205.

    Rationally designed helix-turn-helix proteins and their conformational changes upon DNA binding. / Percipalle, Piergiorgio; Simoncsits, A.; Zakhariev, S.; Guarnaccia, C.; Sanchez, R.; Pongor, S.

    In: EMBO Journal, Vol. 14, No. 13, 01.01.1995, p. 3200-3205.

    Research output: Contribution to journalArticle

    Percipalle, P, Simoncsits, A, Zakhariev, S, Guarnaccia, C, Sanchez, R & Pongor, S 1995, 'Rationally designed helix-turn-helix proteins and their conformational changes upon DNA binding', EMBO Journal, vol. 14, no. 13, pp. 3200-3205.
    Percipalle P, Simoncsits A, Zakhariev S, Guarnaccia C, Sanchez R, Pongor S. Rationally designed helix-turn-helix proteins and their conformational changes upon DNA binding. EMBO Journal. 1995 Jan 1;14(13):3200-3205.
    Percipalle, Piergiorgio ; Simoncsits, A. ; Zakhariev, S. ; Guarnaccia, C. ; Sanchez, R. ; Pongor, S. / Rationally designed helix-turn-helix proteins and their conformational changes upon DNA binding. In: EMBO Journal. 1995 ; Vol. 14, No. 13. pp. 3200-3205.
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