Photoinduced dynamics of EL222 monitored by multi-site-specific infrared spectroscopy

Aditya S. Chaudhari, Prokopis C. Andrikopoulos, Yingliang Liu, Bohdan Schneider, Gustavo Fuertes

Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Průmyslová 595, CZ-252 50 Vestec, Czechia

 adityasuresh.chaudhari@ibt.cas.cz

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).

1. Nash AI, McNulty R, Shillito ME, Swartz TE, Bogomolni RA, Luecke H, et al. Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein. Proceedings of the National Academy of Sciences. 2011;108(23):9449-54.

2. Ramos S, Thielges MC. Site-Specific 1D and 2D IR Spectroscopy to Characterize the Conformations and Dynamics of Protein Molecular Recognition. The Journal of Physical Chemistry B. 2019;123(17):3551-66.