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Rationally designed helix‐turn‐helix proteins and their conformational changes upon DNA binding.
Author(s) -
Percipalle P.,
Simoncsits A.,
Zakhariev S.,
Guarnaccia C.,
Sánchez R.,
Pongor S.
Publication year - 1995
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1995.tb07322.x
Subject(s) - biology , helix turn helix , helix (gastropod) , turn (biochemistry) , dna binding protein , dna , alpha helix , biophysics , protein structure , genetics , biochemistry , transcription factor , gene , ecology , snail
Circular dichroism and electrophoretic mobility shift studies were performed to confirm that dimerized N‐terminal domains of bacterial repressors containing helix‐turn‐helix motifs 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 alpha‐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.