Biophysics and Physicobiology
Online ISSN : 2189-4779
ISSN-L : 2189-4779
Regular Article
Quantitative analyses of the equilibria among DNA complexes of a blue-light-regulated bZIP module, Photozipper
Yoichi NakataniOsamu Hisatomi
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2018 Volume 15 Pages 8-17

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Abstract

Aureochrome1 is a blue-light-receptor protein identified in a stramenopile alga, Vaucheria frigida. Photozipper (PZ) is an N-terminally truncated, monomeric, V. frigida aureochrome1 fragment containing a basic leucine zipper (bZIP) domain and a light–oxygen–voltage (LOV)-sensing domain. PZ dimerizes upon photoexcitation and consequently increases its affinity for the target sequence. In the present study, to understand the equilibria among DNA complexes of PZ, DNA binding by PZ and mutational variants was quantitatively investigated by electrophoretic-mobility-shift assay and fluorescence-correlation spectroscopy in the dark and light states. DNA binding by PZ was sequence-specific and light-dependent. The half-maximal effective concentration of PZ for binding to the target DNA sequence was ~40 nM in the light, which was >10-fold less than the value in the dark. By contrast, the dimeric PZ-S2C variant (with intermolecular disulfide bonds) had higher affinity for the target sequence, with dissociation constants of ~4 nM, irrespective of the light conditions. Substitutions of Glu159 and Lys164 in the leucine zipper region decreased the affinity of PZ for the target sequence, especially in the light, suggesting that these residues form inter-helical salt bridges between leucine zipper regions, stabilizing the dimer–DNA complex. Our quantitative analyses of the equilibria in PZ–DNA-complex formation suggest that the blue-light-induced dimerization of LOV domains and coiled-coil formation by leucine zipper regions are the primary determinants of the affinity of PZ for the target sequence.

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© 2018 THE BIOPHYSICAL SOCIETY OF JAPAN
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