Protein Hydration and Dynamics Seen by Fluorescence: Studies on Haloalkane-Dehalogenases
29 April 2021
Prof. Martin Hof, Dr. DSc.
J. Heyrovsky Institute of Physical Chemistry, Prague, Czech Republic
"Our research group is interested in the application of fluorescence spectroscopy, microscopy and single-molecule techniques for studying biological systems.
Specifically, we aim at:
- Monitoring the structure, functionality and dynamics of biomembranes
- Elucidating the role of gangliosides in neurodegenerative diseases
- Studying aggregation phenomena of peptides and proteins and their interaction with biomembranes
- Development and application of new fluorescence methods.
- Investigating the protein dynamics and hydration"
See more information at Prof. Hof´s research group website.
About the lecture
The hydration and mobility of proteins are believed to profoundly affect their function1. However, only a few approaches for monitoring these characteristics within the relevant protein regions are available. Here, we describe two fluorescence methods for site-specific analysis of the extent of hydration and degree of the mobility in enzyme Haloalkane Dehalogenase.
The first approach is based on recording „time dependent fluorescence shift“ (TDFS)2 placing the dye in the tunnel mouth of this enzyme3,4. In a second approach, environment sensitive coumarin dye is inserted in the selected region employing the technology of the “unnatural aminoacid”5. By means of the steady state spectroscopy the degree of hydration can be determined including the presence of ‘structured water’6. Finally, the „gating“ dynamics of the enzymes can be traced by following the photoinduced electron transfer (PET) between the selected tryprophan and properly positioned fluorescence dye7. Both the hydration and dynamics monitored within the biologically relevant regions of the dehalogenase enzymes is then compared with their enzyme kinetics of various mutants, which can bring the deeper insight into the functioning of these enzymes.
(1) Levy, Y.; Onuchic, J. N. Annu. Rev. Biophys. Biomolec. Struct. 2006, 35, 389.
(2) Jesenská, A., J. Sýkora, A. Olżyńska, J. Brezovský, Z. Zdráhal, J. Damborský, M. Hof. J. Am. Chem. Soc., 2009. 131(2): p. 494-501
(3) Amaro, M.; Brezovsky, J.; Kovacova, S.; Maier, L.; Chaoupkova, R.; Sykora, J.; Paruch, K.; Damborsky, J.; Hof, M. J. Phys. Chem. B 2013, 117, 7898.
(4) Sykora, J.; Brezovsky, J.; Koudelakova, T.; Lahoda, M.; Fortova, A.; Chernovets, T.; Chaloupkova, R.; Stepankova, V.; Prokop, Z.; Kuta Smatanova, I.; Hof, M.; Damborsky, J. Nat. Chem. Biol. 2014, 10, 428.
(5) Summerer, D.; Chen, S.; Wu, N.; Deiters, A.; Chin, J. W.; Schultz, P. G. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 9785.
(6) Amaro, M.; Brezovsky, J.; Kovacova, S.; Sykora, J.; Bednar, D.; Nemec, V.; Liskova, V.; Kurumbang, N. P.; Beerens, K.; Chaloupkova, R.; Paruch, K.; Hof, M.; Damborsky, J. J. Am. Chem. Soc. 2015, 137, 4988.
(7) Kokkonen P, Sykora J, Prokop Z, Ghose A, Bednar D, Amaro M, Beerens K, Bidmanova S, Slanska M, Brezovsky J, Damborsky J, Hof M. J. Am. Chem. Soc., 2018 140 51 17999-18008