The organization of biomolecules and bioassemblies is highly governed by the nature and extent of their interactions with water. These interactions are of high intricacy and a broad range of methods based on various principles have been introduced to characterize them. As these methods view the hydration phenomena differently (e.g., in terms of time and length scales), a detailed insight in each particular technique is to promote the overall understanding of the stunning "hydration world." In this prospective mini-review we therefore critically examine time-dependent fluorescence shift (TDFS)-an experimental method with a high potential for studying the hydration in the biological systems. We demonstrate that TDFS is very useful especially for phospholipid bilayers for mapping the interfacial region formed by the hydrated lipid headgroups. TDFS, when properly applied, reports on the degree of hydration and mobility of the hydrated phospholipid segments in the close vicinity of the fluorophore embedded in the bilayer. Here, the interpretation of the recorded TDFS parameters are thoroughly discussed, also in the context of the findings obtained by other experimental techniques addressing the hydration phenomena (e.g., molecular dynamics simulations, NMR spectroscopy, scattering techniques, etc.). The differences in the interpretations of TDFS outputs between phospholipid biomembranes and proteins are also addressed. Additionally, prerequisites for the successful TDFS application are presented (i.e., the proper choice of fluorescence dye for TDFS studies, and TDFS instrumentation). Finally, the effects of ions and oxidized phospholipids on the bilayer organization and headgroup packing viewed from TDFS perspective are presented as application examples.

• Keywords
• biomembranes, calcium, cholesterol, hydration, lipid headgroups, membrane dynamics, oxidized phosholipids, time-dependent fluorescence shift,
• Publication type
• Journal Article MeSH
• Review MeSH

Haloalkane dehalogenases are enzymes with a broad application potential in biocatalysis, bioremediation, biosensing and cell imaging. The new haloalkane dehalogenase DmxA originating from the psychrophilic bacterium Marinobacter sp. ELB17 surprisingly possesses the highest thermal stability (apparent melting temperature Tm,app = 65.9 °C) of all biochemically characterized wild type haloalkane dehalogenases belonging to subfamily II. The enzyme was successfully expressed and its crystal structure was solved at 1.45 Å resolution. DmxA structure contains several features distinct from known members of haloalkane dehalogenase family: (i) a unique composition of catalytic residues; (ii) a dimeric state mediated by a disulfide bridge; and (iii) narrow tunnels connecting the enzyme active site with the surrounding solvent. The importance of narrow tunnels in such paradoxically high stability of DmxA enzyme was confirmed by computational protein design and mutagenesis experiments.

• Keywords
• access tunnel, catalytic pentad, dimer, enantiselectivity, haloalkane dehalogenase, psychrophile, thermostability,
• Publication type
• Journal Article MeSH

The haloalkane dehalogenase enzyme DmmA was identified by marine metagenomic screening. Determination of its crystal structure revealed an unusually large active site compared to those of previously characterized haloalkane dehalogenases. Here we present a biochemical characterization of this interesting enzyme with emphasis on its structure-function relationships. DmmA exhibited an exceptionally broad substrate specificity and degraded several halogenated environmental pollutants that are resistant to other members of this enzyme family. In addition to having this unique substrate specificity, the enzyme was highly tolerant to organic cosolvents such as dimethyl sulfoxide, methanol, and acetone. Its broad substrate specificity, high overexpression yield (200 mg of protein per liter of cultivation medium; 50% of total protein), good tolerance to organic cosolvents, and a broad pH range make DmmA an attractive biocatalyst for various biotechnological applications.IMPORTANCE We present a thorough biochemical characterization of the haloalkane dehalogenase DmmA from a marine metagenome. This enzyme with an unusually large active site shows remarkably broad substrate specificity, high overexpression, significant tolerance to organic cosolvents, and activity under a broad range of pH conditions. DmmA is an attractive catalyst for sustainable biotechnology applications, e.g., biocatalysis, biosensing, and biodegradation of halogenated pollutants. We also report its ability to convert multiple halogenated compounds to corresponding polyalcohols.

• Keywords
• biotechnology, cosolvents, enzyme, haloalkane dehalogenase, marine, microbial, stability, substrate specificity,
• MeSH
• Bacteria enzymology   genetics   metabolism   MeSH
• Biocatalysis MeSH
• Biotechnology MeSH
• Hydrolases chemistry   genetics   isolation & purification   metabolism   MeSH
• Catalytic Domain MeSH
• Catalysis MeSH
• Kinetics MeSH
• Hydrogen-Ion Concentration MeSH
• Crystallization MeSH
• Metagenome MeSH
• Microbial Consortia genetics   physiology   MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• haloalkane dehalogenase MeSH Browser
• Hydrolases MeSH

BACKGROUND: Enzyme active sites can be connected to the exterior environment by one or more channels passing through the protein. Despite our current knowledge of enzyme structure and function, surprisingly little is known about how often channels are present or about any structural features such channels may have in common. RESULTS: Here, we analyze the long channels (i.e. >15 Å) leading to the active sites of 4,306 enzyme structures. We find that over 64% of enzymes contain two or more long channels, their typical length being 28 Å. We show that amino acid compositions of the channel significantly differ both to the composition of the active site, surface and interior of the protein. CONCLUSIONS: The majority of enzymes have buried active sites accessible via a network of access channels. This indicates that enzymes tend to have buried active sites, with channels controlling access to, and egress from, them, and that suggests channels may play a key role in helping determine enzyme substrate.

• MeSH
• Amino Acids chemistry   genetics   MeSH
• Enzymes chemistry   genetics   MeSH
• Ion Channels physiology   MeSH
• Catalytic Domain MeSH
• Protein Conformation MeSH
• Humans MeSH
• Models, Molecular MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amino Acids MeSH
• Enzymes MeSH
• Ion Channels MeSH

WrbA is a novel multimeric flavodoxin-like protein of unknown function. A recent high-resolution X-ray crystal structure of E. coli WrbA holoprotein revealed a methionine sulfoxide residue with full occupancy in the FMN-binding site, a finding that was confirmed by mass spectrometry. In an effort to evaluate whether methionine sulfoxide may have a role in WrbA function, the present analyses were undertaken using molecular dynamics simulations in combination with further mass spectrometry of the protein. Methionine sulfoxide formation upon reconstitution of purified apoWrbA with oxidized FMN is fast as judged by kinetic mass spectrometry, being complete in ∼5 h and resulting in complete conversion at the active-site methionine with minor extents of conversion at heterogeneous second sites. Analysis of methionine oxidation states during purification of holoWrbA from bacterial cells reveals that methionine is not oxidized prior to reconstitution, indicating that methionine sulfoxide is unlikely to be relevant to the function of WrbA in vivo. Although the simulation results, the first reported for WrbA, led to no hypotheses about the role of methionine sulfoxide that could be tested experimentally, they elucidated the origins of the two major differences between apo- and holoWrbA crystal structures, an alteration of inter-subunit distance and a rotational shift within the tetrameric assembly.

• MeSH
• Apoproteins chemistry   isolation & purification   metabolism   MeSH
• Flavin Mononucleotide chemistry   metabolism   MeSH
• Spectrometry, Mass, Electrospray Ionization MeSH
• Kinetics MeSH
• Protein Conformation MeSH
• Methionine analogs & derivatives   chemistry   metabolism   MeSH
• Oxidation-Reduction MeSH
• Escherichia coli Proteins chemistry   isolation & purification   metabolism   MeSH
• Repressor Proteins chemistry   isolation & purification   metabolism   MeSH
• Molecular Dynamics Simulation * MeSH
• Protein Stability MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Apoproteins MeSH
• Flavin Mononucleotide MeSH
• Methionine MeSH
• methionine sulfoxide MeSH Browser
• Escherichia coli Proteins MeSH
• Repressor Proteins MeSH
• WrbA protein, E coli MeSH Browser

The effect of non-denaturing concentrations of three different organic solvents, formamide, acetone and isopropanol, on the structure of haloalkane dehalogenases DhaA, LinB, and DbjA at the protein-solvent interface was studied using molecular dynamics simulations. Analysis of B-factors revealed that the presence of a given organic solvent mainly affects the dynamical behavior of the specificity-determining cap domain, with the exception of DbjA in acetone. Orientation of organic solvent molecules on the protein surface during the simulations was clearly dependent on their interaction with hydrophobic or hydrophilic surface patches, and the simulations suggest that the behavior of studied organic solvents in the vicinity of hyrophobic patches on the surface is similar to the air/water interface. DbjA was the only dimeric enzyme among studied haloalkane dehalogenases and provided an opportunity to explore effects of organic solvents on the quaternary structure. Penetration and trapping of organic solvents in the network of interactions between both monomers depends on the physico-chemical properties of the organic solvents. Consequently, both monomers of this enzyme oscillate differently in different organic solvents. With the exception of LinB in acetone, the structures of studied enzymes were stabilized in water-miscible organic solvents.

• MeSH
• 2-Propanol chemistry   pharmacology   MeSH
• Acetone chemistry   pharmacology   MeSH
• Formamides chemistry   pharmacology   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Hydrolases chemistry   MeSH
• Crystallography, X-Ray MeSH
• Protein Structure, Quaternary drug effects   MeSH
• Models, Molecular MeSH
• Solvents chemistry   MeSH
• Molecular Dynamics Simulation MeSH
• Protein Structure, Tertiary drug effects   MeSH
• Water chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 2-Propanol MeSH
• Acetone MeSH
• formamide MeSH Browser
• Formamides MeSH
• haloalkane dehalogenase MeSH Browser
• Hydrolases MeSH
• Solvents MeSH
• Water MeSH