title: Are time-dependent fluorescence shifts at the tunnel mouth of haloalkane dehalogenase enzymes dependent on the choice of the chromophore?
abstract: Time-dependent fluorescence shifts (TDFS) of chromophores selectively attached to proteins may give information on the dynamics of the probed protein moieties and their degree of hydration. Previously, we demonstrated that a coumarin dye selectively labeling the tunnel mouth of different haloalkane dehalogenases (HLDs) can distinguish between different widths of tunnel mouth openings. In order to generalize those findings analogous experiments were performed using a different chromophore probing the same region of these enzymes. To this end we synthesized and characterized three new fluorescent probes derived from dimethylaminonaphthalene bearing a linker almost identical to that of the coumarin dye used in our previous study. Labeling efficiencies, acrylamide quenching, fluorescence anisotropies, and TDFS for the examined fluorescent substrates confirm the picture gained from the coumarin studies: the different tunnel mouth opening, predicted by crystal structures, is reflected in the hydration and tunnel mouth dynamics of the investigated HLDs. Comparison of the TDFS reported by the coumarin dye with those obtained with the new dimethylaminonaphthalene dyes shows that the choice of chromophore may strongly influence the recorded TDFS characteristics. The intrinsic design of our labeling strategy and the variation of the linker length ensure that both dyes probe the identical enzyme region; moreover, the covalently fixed position of the chromophore does not allow for a major relocalization within the HLD structures. Our study shows, for the first time, that TDFS may strongly depend on the choice of the chromophore, even though the identical region of a protein is explored.
authors.affiliation: J. Heyrovsky Institute of Physical Chemistry of the ASCR, v. v. i., Academy of Sciences of the Czech Republic, Dolejskova 3, 182 23 Praha, Czech Republic.
authors.forename: Mariana
authors.initials: M
authors.lastname: Amaro
authors.forename: Jan
authors.initials: J
authors.lastname: Brezovský
authors.forename: Silvia
authors.initials: S
authors.lastname: Kováčová
authors.forename: Lukáš
authors.initials: L
authors.lastname: Maier
authors.forename: Radka
authors.initials: R
authors.lastname: Chaloupková
authors.forename: Jan
authors.initials: J
authors.lastname: Sýkora
authors.forename: Kamil
authors.initials: K
authors.lastname: Paruch
authors.forename: Jiří
authors.initials: J
authors.lastname: Damborský
authors.forename: Martin
authors.initials: M
authors.lastname: Hof
mesh_terms: D020369:Bradyrhizobium / Q000201:enzymology*; D003374:Coumarins / Q000737:chemistry*; D005453:Fluorescence; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry*; D006867:Hydrolases / Q000737:chemistry*; D009281:Naphthalenes / Q000737:chemistry*; D012240:Rhodococcus / Q000201:enzymology*
chemical_list: D003374:Coumarins; D005456:Fluorescent Dyes; D009281:Naphthalenes; C030123:coumarin; D006867:Hydrolases; C046137:haloalkane dehalogenase