EL222 is a blue-light sensitive DNA binding
protein from the bacteria Erythrobacter litoralis. The dark-state
crystal structure of the protein reveals a flavin binding light-oxygen-voltage
(LOV) domain and a DNA binding helix-turn-helix (HTH) domain tightly packed
against each other and thereby blocking the recognition of DNA. Upon blue light
excitation by the flavin moiety, EL222 undergoes conformational changes that ultimately
lead to protein dimerization and association with DNA (1). However, our knowledge of the
light-adapted state and the molecular mechanism by which it is formed remains
incomplete. Our goal is to elucidate the structure of light-state EL222 and the
conformational changes that occur when EL222 is photoactivated.
Fourier transform infrared (FTIR) spectroscopy is a well known method to
characterize protein structure and dynamics. However, the spectral congestion
makes it challenging to assign the observed bands to particular bonds. This
problem can be solved by the introduction of IR-sensitive probe groups in
target residues (2). Here we make use of the cyano group as a
site-specific infrared reporter to track the conformational dynamics of photoactivated
EL222. EL222 variants containing the non-canonical amino acid
p-cyanophenylalanine (CNF) in different locations across the protein were
prepared by amber suppression technology. Screening of a large set of labelled
positions was done by recording the steady-state difference FTIR spectra between
light and dark states spectra. Some mutants showed a clear shift in the
position of the CN stretching vibration, suggesting a change in the local
environment around the probe upon illumination. Selected mutants will then be
further investigated by time resolved IR spectroscopy to detect the site-specific
propagation of conformational changes in EL222 from a few femtoseconds (photon absorption) to several seconds
(interaction with DNA).