The ELIBIO (Structural dynamics of biomolecular systems) project explores new frontiers in light and optics to create breakthrough science in biology, chemistry and physics. The research team of ELIBIO is formed at the interface between two complementary research centres, ELI Beamlines oriented to photon science, and the Institute of Biotechnology (IBT) oriented to biomedical, protein engineering and structural biology research. The milestones include the creation of biologically oriented research facilities (Biolab), and the strengthening of research infrastructure at the IBT. We have developed new instrumentation and methods, in particular, genetic code expansion technology, and a dual transient visible absorption and femtosecond-stimulated Raman spectroscopy (FSRS) set-up. The unique combination of genetically encoded non-canonical amino acids carrying vibrational probes, and ultrafast time-resolved optical spectroscopy (UV/Visible, infrared and Raman) down to the femtosecond time scale has allowed us to gain new insights in signal propagation pathways. We have applied these novel methodologies to study photosensory receptors (EL222, CarH, and KR2) and non-photoactive proteins (myoglobin). In addition, quantum chemistry calculations have permitted comprehensive assignments of the experimentally-determined spectral bands, and have made testable predictions. We have also complemented our results with other techniques, like nuclear magnetic resonance spectroscopy, X-ray crystallography, and small-angle scattering. We conclude that the joint use of unnatural amino acids and time-resolved methods can reveal non-equilibrium protein structural dynamics residue-by-residue.
The work was supported by the project ELIBIO: Structural dynamics of biomolecular systems (CZ.02.1.01/0.0/0.0/15_003/0000447) from the European Regional Development Fund and the Ministry of Education, Youth and Sports (MEYS) of the Czech Republic. The Institute of Biotechnology of the Czech Academy of Sciences acknowledges the institutional grant RVO 86652036.