Martin Hof

Martin Hof; J. Heyrovský

Institute of Physical Chemistry, Czech Academy of Sciences, Dolejškova 3, 18223 Prague 8, Czech Republic

The hydration and mobility of proteins are believed to profoundly affect their function. 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 class of haloalkane dehalogenases. The first approach is based on recording time dependent fluorescence shift (TDFS) [1] placing the dye in the tunnel mouth of this enzyme [2]. Secondly, the „gating" dynamics of the enzymes can be traced by fluorescence correlation spectroscopy following the photoinduced electron transfer (PET-FCS) between the selected tryptophan and properly positioned fluorescence dye [3]. The hydration and dynamics monitored within the biologically relevant regions of the dehalogenase enzymes is then compared with the enantioselectivity as well as catalytic efficiency of various mutants, which brings fundamental insights into the functioning of such enzymes. Finally, the application of the TDFS approach for integral membrane proteins will be discussed.

1- Jesenská, A., J. Sýkora, A. Olżyńska, J. Brezovský, Z. Zdráhal, J. Damborský, M. Hof.  J. Am. Chem. Soc., 2009. 131(2): p. 494-501. 2- 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. 3- 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