Are Time-Dependent Fluorescence Shifts at the Tunnel Mouth of Haloalkane Dehalogenase Enzymes Dependent on the Choice of the Chromophore?

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

Diastereoselective Flexible Synthesis of Carbocyclic C‑Nucleosides

Carbocyclic C-nucleosides are quite rare. Our route enables flexible preparation of three classes of these nucleoside analogs from common precursors–properly substituted cyclopentanones, which can be prepared racemic (in six steps) or optically pure (in ten steps) from inexpensive norbornadiene. The methodology allows flexible manipulation of individual positions around the cyclopentane ring, namely highly diastereoselective installation of carbo- and heterocyclic substituents at position 1′, orthogonal functionalization of position 5′, and efficient inversion of stereochemistry at position 2′. Newly prepared carbocyclic C-analog of tubercidine, profiled in MCF7 (breast cancer) and HFF1 (human foreskin fibroblasts) cell cultures, is less potent than tubercidine itself, but more selectively toxic toward the tumorigenic cells

Site-Specific Analysis of Protein Hydration Based on Unnatural Amino Acid Fluorescence

Hydration of proteins profoundly affects their functions. We describe a simple and general method for site-specific analysis of protein hydration based on the in vivo incorporation of fluorescent unnatural amino acids and their analysis by steady-state fluorescence spectroscopy. Using this method, we investigate the hydration of functionally important regions of dehalogenases. The experimental results are compared to findings from molecular dynamics simulations