The plasma membrane, as a highly complex cell organelle, serves as a crucial platform for a multitude of cellular processes. Its collective biophysical properties are largely determined by the structural diversity of the different lipid species it accommodates. Therefore, a detailed investigation of biophysical properties of the plasma membrane is of utmost importance for a comprehensive understanding of biological processes occurring therein. During the past two decades, several environment-sensitive probes have been developed and become popular tools to investigate membrane properties. Although these probes are assumed to report on membrane order in similar ways, their individual mechanisms remain to be elucidated. In this study, using model membrane systems, we characterized the probes Pro12A, NR12S and NR12A in depth and examined their sensitivity to parameters with potential biological implications, such as the degree of lipid saturation, double bond position and configuration (cis versus trans), phospholipid headgroup and cholesterol content. Applying spectral imaging together with atomistic molecular dynamics simulations and time-dependent fluorescent shift analyses, we unravelled individual sensitivities of these probes to different biophysical properties, their distinct localizations and specific relaxation processes in membranes. Overall, Pro12A, NR12S and NR12A serve together as a toolbox with a wide range of applications allowing to select the most appropriate probe for each specific research question.

• Keywords
• MD simulation, environment-sensitive probes, lipid saturation, model membranes, spectral imaging, time-resolved emission shift,
• MeSH
• Cell Membrane chemistry   MeSH
• Cholesterol MeSH
• Fluorescent Dyes * analysis   chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cholesterol MeSH
• Fluorescent Dyes * MeSH

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

Fluorescence of 2-(N,N-dimethylamino)-6-propionylnaphthalene dyes Badan and Prodan is quenched by tryptophan in Brij 58 micelles as well as in two cytochrome P450 proteins (CYP102, CYP119) with Badan covalently attached to a cysteine residue. Formation of nonemissive complexes between a dye molecule and tryptophan accounts for about 76% of the fluorescence intensity quenching in micelles, the rest is due to diffusive encounters. In the absence of tryptophan, fluorescence of Badan-labeled cytochromes decays with triexponential kinetics characterized by lifetimes of about 100 ps, 700-800 ps, and 3 ns. Site mutation of a histidine residue in the vicinity of the Badan label by tryptophan results in shortening of all three decay lifetimes. The relative amplitude of the fastest component increases at the expense of the two slower ones. The average quenching rate constants are 4.5 × 10(8) s(-1) (CYP102) and 3.7 × 10(8) s(-1) (CYP119), at 288 K. Cyclic voltammetry of Prodan in MeCN shows a reversible reduction peak at -1.85 V vs NHE that becomes chemically irreversible and shifts positively upon addition of water. A quasireversible reduction at -0.88 V was observed in an aqueous buffer (pH 7.3). The excited-state reduction potential of Prodan (and Badan) is estimated to vary from about +0.6 V (vs NHE) in polar aprotic media (MeCN) to approximately +1.6 V in water. Tryptophan quenching of Badan/Prodan fluorescence in CYPs and Brij 58 micelles is exergonic by ≤0.5 V and involves tryptophan oxidation by excited Badan/Prodan, coupled with a fast reaction between the reduced dye and water. Photoreduction is a new quenching mechanism for 2-(N,N-dimethylamino)-6-propionylnaphthalene dyes that are often used as solvatochromic polarity probes, FRET donors and acceptors, as well as reporters of solvation dynamics.

• MeSH
• 2-Naphthylamine analogs & derivatives   chemistry   MeSH
• Archaeal Proteins chemistry   MeSH
• Bacterial Proteins chemistry   MeSH
• Fluorescence MeSH
• Fluorescent Dyes chemistry   MeSH
• Spectrometry, Fluorescence MeSH
• Kinetics MeSH
• Micelles MeSH
• Models, Molecular MeSH
• NADPH-Ferrihemoprotein Reductase chemistry   MeSH
• Cytochrome P-450 Enzyme System chemistry   MeSH
• Tryptophan chemistry   MeSH
• Water MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• 2-Naphthylamine MeSH
• 6-bromoacetyl-2-dimethylaminonaphthalene MeSH Browser
• Archaeal Proteins MeSH
• Bacterial Proteins MeSH
• CYP119 protein, Sulfolobus solfataricus MeSH Browser
• flavocytochrome P450 BM3 monoxygenases MeSH Browser
• Fluorescent Dyes MeSH
• Micelles MeSH
• NADPH-Ferrihemoprotein Reductase MeSH
• prodan MeSH Browser
• Cytochrome P-450 Enzyme System MeSH
• Tryptophan MeSH
• Water MeSH

We emphasize the importance of dynamics and hydration for enzymatic catalysis and protein design by transplanting the active site from a haloalkane dehalogenase with high enantioselectivity to nonselective dehalogenase. Protein crystallography confirms that the active site geometry of the redesigned dehalogenase matches that of the target, but its enantioselectivity remains low. Time-dependent fluorescence shifts and computer simulations revealed that dynamics and hydration at the tunnel mouth differ substantially between the redesigned and target dehalogenase.

• MeSH
• Hydrocarbons, Brominated chemistry   MeSH
• Spectrometry, Fluorescence MeSH
• Hydrolases chemistry   genetics   MeSH
• Catalytic Domain MeSH
• Catalysis MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Molecular Sequence Data MeSH
• Mutagenesis, Site-Directed MeSH
• Protein Engineering * MeSH
• Amino Acid Sequence MeSH
• Molecular Dynamics Simulation * MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
• Stereoisomerism MeSH
• Water chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hydrocarbons, Brominated MeSH
• haloalkane dehalogenase MeSH Browser
• Hydrolases MeSH
• Water MeSH

Haloalkane dehalogenases make up an important class of hydrolytic enzymes which catalyse the cleavage of carbon-halogen bonds in halogenated aliphatic compounds. There is growing interest in these enzymes owing to their potential use in environmental and industrial applications. The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Structural analysis of this enzyme complexed with target ligands was conducted in order to obtain detailed information about the structural limitations of its catalytic properties. In this study, the crystallization and preliminary X-ray analysis of complexes of wild-type DhaA with 2-propanol and with TCP and of complexes of the catalytically inactive variant DhaA13 with the dye coumarin and with TCP are described. The crystals of wild-type DhaA were plate-shaped and belonged to the triclinic space group P1, while the variant DhaA13 can form prism-shaped crystals belonging to the orthorhombic space group P2(1)2(1)2(1) as well as plate-shaped crystals belonging to the triclinic space group P1. Diffraction data for crystals of wild-type DhaA grown from crystallization solutions with different concentrations of 2-propanol were collected to 1.70 and 1.26 Å resolution, respectively. A prism-shaped crystal of DhaA13 complexed with TCP and a plate-shaped crystal of the same variant complexed with the dye coumarin diffracted X-rays to 1.60 and 1.33 Å resolution, respectively. A crystal of wild-type DhaA and a plate-shaped crystal of DhaA13, both complexed with TCP, diffracted to atomic resolutions of 1.04 and 0.97 Å, respectively.

• MeSH
• 2-Propanol MeSH
• Bacterial Proteins chemistry   MeSH
• X-Ray Diffraction MeSH
• Hydrolases chemistry   genetics   metabolism   MeSH
• Hydrolysis MeSH
• Isoenzymes chemistry   genetics   MeSH
• Catalysis MeSH
• Crystallization MeSH
• Crystallography, X-Ray methods   MeSH
• Ligands MeSH
• Propane analogs & derivatives   MeSH
• Rhodococcus enzymology   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• 1,2,3-trichloropropane MeSH Browser
• 2-Propanol MeSH
• Bacterial Proteins MeSH
• haloalkane dehalogenase MeSH Browser
• Hydrolases MeSH
• Isoenzymes MeSH
• Ligands MeSH
• Propane MeSH