Nanosecond time-dependent Stokes shift at the tunnel mouth of haloalkane dehalogenases
Language English Country United States Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
19113888
DOI
10.1021/ja804020q
PII: 10.1021/ja804020q
Knihovny.cz E-resources
- MeSH
- Acrylamide chemistry metabolism MeSH
- Anisotropy MeSH
- Fluorescent Dyes chemistry metabolism MeSH
- Spectrometry, Fluorescence MeSH
- Hydrolases chemistry genetics metabolism MeSH
- Catalytic Domain MeSH
- Kinetics MeSH
- Coumarins chemistry metabolism MeSH
- Quantum Theory MeSH
- Models, Molecular MeSH
- Mutagenesis, Site-Directed MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
- Stochastic Processes MeSH
- Structure-Activity Relationship MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Acrylamide MeSH
- Fluorescent Dyes MeSH
- haloalkane dehalogenase MeSH Browser
- Hydrolases MeSH
- Coumarins MeSH
The tunnel mouths are evolutionally the most variable regions in the structures of haloalkane dehalogenases originating from different bacterial species, suggesting their importance for adaptation of enzymes to various substrates. We decided to monitor the dynamics of this particular region by means of time-resolved fluorescence spectroscopy and molecular dynamic simulations. To label the enzyme specifically, we adapted a novel procedure that utilizes a coumarin dye containing a halide-hydrocarbon linker, which serves as a substrate for enzymatic reaction. The procedure leads to a coumarin dye covalently attached and specifically located in the tunnel mouth of the enzyme. In this manner, we stained two haloalkane dehalogenase mutants, DbjA-H280F and DhaA-H272F. The measurements of time-resolved fluorescence anisotropy, acrylamide quenching, and time-resolved emission spectra reveal differences in the polarity, accessibility and mobility of the dye and its microenvironment for both of the mutants. The obtained experimental data are consistent with the results obtained by molecular dynamics calculations and correlate with the anatomy of the tunnel mouths, which were proposed to have a strong impact on the catalytic activity and specificity of the examined mutants. Interestingly, the kinetics of the recorded time-dependent Stokes shift is unusual slow; it occurs on the nanosecond time-scale, suggesting that the protein dynamics is extremely slowed down at the region involved in the exchange of ligands between the active-site cavity and bulk solvent.
References provided by Crossref.org
What Does Time-Dependent Fluorescence Shift (TDFS) in Biomembranes (and Proteins) Report on?
Dynamics and hydration explain failed functional transformation in dehalogenase design
