Are Time-Dependent Fluorescence Shifts at the Tunnel Mouth of Haloalkane Dehalogenase Enzymes Dependent on the Choice of the Chromophore?
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- 作者：Mariana Amaro ; Jan Brezovsk媒 ; Silvia Kov谩膷ov谩 ; Luk谩拧 Maier ; Radka Chaloupkov谩 ; Jan S媒kora ; Kamil Paruch ; Ji艡铆 Damborsk媒 ; Martin Hof
- 刊名：The Journal of Physical Chemistry B
- 出版年：2013
- 出版时间：July 3, 2013
- 年：2013
- 卷：117
- 期：26
- 页码：7898-7906
- 全文大小：348K
- 年卷期：v.117,no.26(July 3, 2013)
- ISSN：1520-5207
文摘
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.