Format

Send to

Choose Destination
Biochemistry. 2013 Sep 24;52(38):6653-61. doi: 10.1021/bi401040m. Epub 2013 Sep 12.

Blue light-induced dimerization of a bacterial LOV-HTH DNA-binding protein.

Author information

1
Departments of Biophysics and Biochemistry, University of Texas Southwestern Medical Center , Dallas, Texas 75390-8816, United States.

Abstract

With their utilization of light-driven allostery to control biochemical activities, photosensory proteins are of great interest as model systems and novel reagents for use by the basic science and engineering communities. One such protein, the light-activated EL222 transcription factor, from the marine bacterium Erythrobacter litoralis HTCC2594, is appealing for such studies, as it harnesses blue light to drive the reorientation of light-oxygen-voltage (LOV) sensory and helix-turn-helix (HTH) effector domains to allow photoactivation of gene transcription in natural and artificial systems. The protein conformational changes required for this process are not well understood, in part because of the relatively short lifetime of the EL222 photoexcited state (τ ∼ 29 s), which complicates its characterization via certain biophysical methods. Here we report how we have circumvented this limitation by creating an EL222 variant harboring V41I, L52I, A79Q, and V121I point mutations (AQTrip) that stabilizes the photoactivated state. Using the wild-type and AQTrip EL222 proteins, we have probed EL222 activation using a combination of solution scattering, nuclear magnetic resonance (NMR), and electromobility shift assays. Size-exclusion chromatography and light scattering indicate that AQTrip oligomerizes in the absence of DNA and selects for an EL222 dimer-DNA complex in the presence of DNA substrates. These results are confirmed in wild-type EL222 with a high-affinity DNA-binding site that stabilizes the complex. NMR analyses of the EL222-DNA complex confirm a 2:1 stoichiometry in the presence of a previously characterized DNA substrate. Combined, these novel approaches have validated a key mechanistic step, whereby blue light induces EL222 dimerization through LOV and HTH interfaces.

PMID:
23992349
PMCID:
PMC3813961
DOI:
10.1021/bi401040m
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for American Chemical Society Icon for PubMed Central
Loading ...
Support Center