Comparison of the Structural Dynamics of Bacterial Luciferases Using Time-Resolved Luminescence Techniques

The structural dynamics of two proteins—luciferases from bacteria Vibrio harveyi and Photobacterium leiognathi, was studied using time-resolved fluorescence and phosphorescence of the tryptophan residues at 5–35°C. Earlier, the temperature effects on functional properties of these luciferases were found to be different. Revealing the structural and dynamic basis of the difference will contribute to understanding the principles of molecular adaptation of cells for living in unfavorable conditions. Nanosecond-resolved shifts of the fluorescence spectra were analyzed and dipolar relaxation times of the tryptophans microenvironment were obtained: τR1 = 0.22–0.49 ns and τR2 = 8.04–9.37 ns for P. leiognathi luciferase and τR1 = 0.18–0.27 ns and τR2 = 6.3–9.0 ns for V. harveyi luciferase. Temperature dependences of τR1 indicate a more rigid structure of the latter protein. Microsecond-resolved phosphorescence decays were measured and phosphorescence lifetimes τp1 and τp2 were determined: 0.15–0.30 and 0.91–2.77 ms for P. leiognathi luciferase and 0.16–0.33 and 2.27–5.45 ms for V. harveyi luciferase, respectively. The increased values of τp2 for V. harveyi luciferase also indicate more rigid microenvironment of the tryptophans within this protein. The experimental data were compared with the molecular modeling results on the mobility of tryptophans and adjacent residues in bacterial luciferase structures under temperature variations.