Pawel Kaszycki

Fluorescence-quenching-resolved spectra of melittin in lipid bilayers.

The interaction of bee venom melittin with dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles has been studied by means of fluorescence quenching of the single tryptophan residue of the protein, at lipid-to-peptide ratio, Ri = 50 and at high ionic strength (2 M NaCl). The method of fluorescence-quenching-resolved spectra (FQRS), applied in this study with potassium iodide as a quencher, enabled us to decompose the tryptophan emission spectrum of liposome-bound melittin into components, at temperatures above as well as below the main phase transition temperature (Tt) of DMPC. One of the two resolved spectra exhibits maximum at 342 and 338 nm for experiments above and below Tt, respectively, and is similar to the maximum of tryptophan emission found for tetrameric melittin in solution (340 nm). This spectrum is characterized by the Stern-Volmer quenching constant, Ksv, of about 4 M-1 and it represents the fraction of melittin molecules whose tryptophan residues are exposed to the solvent to a degree comparable with tetrameric species in solution. The other spectrum component, corresponding to the quencher-inaccessible fraction of tryptophan molecules (Ksv = 0 M-1) has its maximum blue-shifted up to 15 nm, indicating a decrease in polarity of the environment. For experiments above Tt, the blue spectrum component revealed the excitation-wavelength dependence, originating probably from the relaxation processes between the excited tryptophan molecules and lipid polar head groups. We conclude that melittin bound to DMPC liposomes exists in two lipid-associated forms; one, with tryptophan residues exposed to the solvent and the other, penetrating the membrane interior, with tryptophan residues located in close proximity to the phospholipid polar head groups of the outer vesicle lipid layer. We also discuss our data with current models of melittin-bilayer interactions.

Tet repressor-tetracycline interaction

Previous studies [Wasylewskiet al. (1996),J. Protein Chem.15, 45–58] have shown that the W43 residue localized within the helix-turn-helix structure domain of Tet repressor can exist in the ground state in two conformational states. In this paper we investigate the fluorescence properties of W43 of TetR upon binding of tetracycline inducer and its chemical analogs such as anhydro- and epitetracycline. Binding of the drug inducer to the protein indicates that the W43 residue still exists in two conformational states; however, its environment changes drastically, as can be judged by the changes in fluorescence parameters. The FQRS (fluorescence-quenching-resolved spectra) method was used to decompose the total emission spectrum. The resolved spectra exhibit maxima of fluorescence at 346 and 332 nm and the component quenchable by KI (346 nm) is shifted 9 nm toward the blue side of the spectrum upon inducer binding. The observed shift does not result from the changes in the exposure of W43, since the bimolecular quenching rate constant remains the same and is equal to about 2.7×109M−1sec−1. The binding of tetracycline leads to drastic decrease of the W43 fluorescence intensity and increase of the tetracycline intensity as well as the decrease of fluorescence lifetime, especially of the W43 component characterized by the emission at 332 nm. The observed energy transfer from W43 to tetracycline is more efficient for the state characterized by the fluorescence emission at 332 nm (88%) than for the component quenchable by iodide (53%) Tetracycline and several of its derivatives were also used to observe how chemical modifications of the hydrophilic groups in tetracycline influence the mechanism of binding of the antibiotic to Tet repressor. By use of pulsed-laser photoacoustic spectroscopy it is shown that the binding of tetracyclines to Tet repressor leads to significant increase of tetracycline fluorescence quantum yields. Steady-state fluorescence quenching of tetracycline analogs in complexes with Tet repressor using potassium iodide as a quencher allowed us to determine the dependence of the exposure of bound antibiotic on the modifications of hydrophilic substituents of tetracycline. Circular dichroism studies of the TetR-[Mg · tc]+ complex do not indicate dramatic changes in the secondary structure of the protein; however, the observed small decrease in the TetR helicity may occur due to partial unfolding of the DNA recognition helix of the protein. The observed changes may play an important role in the process of induction in which tetracycline binding results in the loss of specific DNA binding.

Fluorescence-detected polymerization kinetics of human alpha 1-antitrypsin.

The time dependence of the humanα1-antitrypsin polymerization process was studied by means of the intrinsic fluorescence stopped-flow technique as well as the fluorescence-quenching-resolved spectra (FQRS) method and native PAGE. The polymerization was induced by mild denaturing conditions (1 M GuHCl) and temperature. The data show that the dimer formation reaction under mild conditions was followed by an increase of fluorescence intensity. This phenomenon is highly temperature sensitive. The structure ofα1-antitrypsin dimer resembles the conformation of antithrombin III dimer. In the presence of the denaturant the polymerization process is mainly limited to the dimer state. Theα1-antitrypsin activity measurements confirm monomer-to-dimer transition under these conditions. These results are in contrast to the polymerization process induced by temperature, where the dimer state is an intermediate step leading to long-chain polymers. On the basis of stopped-flow and electrophoretic data it is suggested that both C-sheet as well as A-sheet mechanisms contribute to the polymerization process under mild conditions.

A fluorescence study of Tn10-encoded Tet repressor

Steady-state fluorescence quenching and time-resolved measurements have been performed to resolve the fluorescence contributions of the two tryptophan residues, W43 and W75, in the subunit of the homodimer of the Tet repressor fromEscherichia coli. The W43 residue is localized within the helix-turn-helix structural domain, which is responsible for sequence-specific binding of the Tet repressor to thetet operator. The W75 residue is in the protein matrix near the tetracycline-binding site. The assignment of the two residues has been confirmed by use of single-tryptophan mutants carrying either W43 or W75. The FQRS (fluorescence-quenching-resolved-spectra) method has been used to decompose the total emission spectrum of the wild-type protein into spectral components. The resolved spectra have maxima of fluorescence at 349 and 324 nm for the W43 and W75 residues, respectively. The maxima of the resolved spectra are in excellent agreement with those found using single-tryptophan-containing mutants. The fluorescence decay properties of the wild type as well as of both mutants of Tet repressor have been characterized by carrying out a multitemperature study. The decays of the wild-type Tet repressor and W43-containing mutant can be described as being of double-exponential type. The W75 mutant decay can be described by a Gaussian continuous distribution centered at 5.0 nsec with a bandwidth equal to 1.34 nsec. The quenching experiments have shown the presence of two classes of W43 emission. One of the components, exposed to solvent, has a maximum of fluorescence emission at 355 nm, with the second one at about 334 nm. The red-emitting component can be characterized by bimolecular-quenching rate constant,kq equal to 2.6×109, 2.8×109, and 2.0×109 M−1 sec−1 for acrylamide, iodide, and succinimide, respectively. The bluer component is unquenchable by any of the quenchers used. The W75 residue of the Tet repressor has quenching rate constant equal to 0.85×109 and 0.28 × 109 M−1 sec−1 for acrylamide and succinimide, respectively. These values indicate that the W75 is not deeply buried within the protein matrix. Our results indicate that the Tet repressor can exist in its ground state in two distinct conformational states which differ in the microenvironment of the W43 residue.

A fluorescence quenching study on protoporphyrin IX in a model membrane system.

The interaction of protoporphyrin IX (3,7,12,15-tetramethyl-8, 13-divinyl-2,18-porphyrine-dipropionic acid) (PPIX) with unilamellar dimyristoyl-L-alpha-phosphatidylcholine (DMPC) phospholipid vesicles has been studied by means of steady-state fluorescence quenching spectroscopy. The method of fluorescence-quenching-resolved spectroscopy has been applied in order to resolve the complex emission spectrum of a membrane-bound PPIX into two component spectra, attributed to distinct fluorophore species with different accessibilities to the iodide quencher. It is shown that PPIX associated with liposomes exists in two different microenvironments. One part of the fluorophore is embedded inside the lipid bilayer and is inaccessible to iodide. Its fluorescence spectrum exhibits the maximum characteristic of protoporphyrin found in the apolar medium. The other fraction of PPIX is located near the membrane surface, close to the polar phospholipid heads. Its emission is blue-shifted, resembling that of PPIX in a polar environment. It is quenched by iodide, although it reveals significant shielding from the quencher as compared to a buffer PPIX solution. Fluorescence quenching using 1-oxyl-4-oxo-2,2,6,6-tetramethyl-piperidine (TEMPONE) does not discriminate between the two protoporphyrin species. However, the accessibility of protoporphyrin IX to this quencher is much lower in a liposome system than in water.

Fluorescence and Phosphorescence Study of Tet Repressor–Operator Interaction

Fluorescence and phosphorescence measurements have been carried out on single-p tryptophan (Trp 43 or Trp 75)-containing mutants of Tet repressor (Tet R). Tet R containing Trp 43, the residue localized in the DNA recognition helix of the repressor, has been used to observe the binding of Tet R to two 20-bp DNA sequences of tet O1 and tet O2 operators. Binding of Tet R to tet O1 operator leads to a 78% decrease of the repressor fluorescence intensity, with an accompanying 20-nm blue shift of its fluorescence emission maximum to 330 nm. Upon binding of Tet R to tet O2 operator, the Trp 43 fluorescence intensity is quenched by 60%, and a 10-nm shift of its emission maximum to 340 nm occurs. Solute fluorescence quenching studies, using acrylamide, performed at low ionic strength indicate that in both the complex of Tet R with the O1 and that with the O2 operator, Trp 43 is moderately buried, as indicated by a bimolecular rate quenching constant of about 1.8 × 109 M−1 sec−1. In contrast to the Tet R–tet O2 complex, the Stern–Volmer acrylamide quenching constant Ksv of the complex with tet O1 operator changes from 7.5 M−1 at 5 mM NaCl to 22 M−1 at 200 mM NaCl, indicating different exposures of Trp 43 in the two complexes in solutions of higher ionic strength. Phosphorescence studies showed a 0–0 vibronic transition at 408 and 403 nm for Trp 43 and Trp 75, respectively. Upon binding of Tet R to the tet operators, we observed red shifts of 0–0 vibronic bands of Trp 43 to 413 and 412 nm for tet O1 and tet O2 operator, respectively, and the phosphorescence triplet lifetime of Trp 43 at 75 K was quenched from 6.0–5.5 to 3.5–3.3 sec. The thermal phosphorescence quenching profile ranged from −200°C to −20°C, and differed drastically for the two complexes, suggesting different dynamics of the microenvironment of the Trp 43 residue. The luminescence data for Trp 43 of Tet R suggest that the recognition helix of Tet R interacts in different fashions with the tet O1 and tet O2 operators.

BACTERIAL POPULATION DYNAMICS IN WASTE OILY EMULSIONS FROM THE METAL-PROCESSING INDUSTRY

Oil-containing wastewaters are regarded as main industrial pollutants of soil and water environments. They can occur as free-floating oil, unstable or stable oil-in-water (O/W) emulsions, and in the case of extreme organic load, as water-in-oil (W/O) emulsions. In this study two types of oily effluents, a typical O/W emulsion marked as E1 and a W/O emulsion E2, both discharged by local metal processing plants were examined to test their toxicity to microbial communities and the ability to serve as nutrient sources for bacterial growth. The organic contaminant load of the samples was evaluated on the basis of chemical oxygen demand (COD) parameter values and was equal to 48 200 mg O 2 ·dm -3 and >300 000 mg O 2 ·dm -3 for E1 and E2, respectively. Both emulsions proved to be non toxic to bacterial communities and were shown to contain biodiverse autochthonous microflora consisting of several bacterial strains adapted to the presence of xenobiotics (the total of 1.36 · 10 6 CFU·cm -3 and 1.72 · 10 5 CFU·cm -3 was determined for E1 and E2, respectively). These indigenous bacteria as well as exogenously inoculated specialized allochthonous microorganisms were biostimulated so as to proliferate within the wastewater environment whose organic content served as the only source of carbon. The most favorable cultivation conditions were determined as fully aerobic growth at the temperature of 25 oC. In 9 to 18 day-tests, autochthonous as well as bioaugmented allochthonous bacterial population dynamics were monitored. For both emulsions tested there was a dramatic increase (up to three orders of magnitude) in bacterial frequency, as compared to the respective initial values. The resultant high biomass densities suggest that the effluents are sus ceptible to bioremediation. A preliminary xenobiotic biodegradation test confirmed that mixed auto- and allochthonous bacterial consortia obtained upon inoculation of the samples with microbiocenoses preselected for efficient hydrocarbon biodegrada tion led to a decrease in the organic pollution level.

Bioremediation Of A Spent Metalworking Fluid With Auto- And Allochthonous Bacterial Consortia

Abstract Spent mineral oil-based metalworking fluids are waste products of the machining processes and contribute substantially to the global industrial pollution with petroleum oil products. Wastewaters containing oily emulsions are ecologically hazardous and thus a variety of methods have been implemented to prevent these effluents from affecting the natural environment. Most of these methods rely upon physical-chemical treatment and phase separation; however, none of them proved to be effective enough to meet tightening environmental regulations. Therefore, novel technologies need to be elaborated and there is growing interest in implementing biological treatment methods based on microbial bioremediation. In this study an oil/water emulsion obtained from a waste stream of the metal-processing industry was tested for biodegradability of its organic constituents. This liquid waste was found non-toxic to bacterial consortia and was colonized with indigenous microorganisms (approx. 107 cfu · cm−3). The total load of organic content was determined as a chemical oxygen demand (COD) value of 48 200 mg O2 · dm−3. Emulsion treatment was carried out using a threefold wastewater dilution and employing two variants of biostimulated aerobic bacterial communities: (1) uninoculated emulsion, where bioremediation was carried out by the autochthonous bacteria alone, and (2) wastewater samples inoculated with a ZB-01 microbial consortium which served as a source of specialized bacteria for process bioaugmentation. Biodegradation efficiency achieved in a 14-day test was monitored by measuring both the COD parameter and the concentration of high-boiling organic compounds. Both approaches yielded satisfactory results showing significant reduction of the emulsion organic fraction; however, the resultant decrease of wastewater load tended to be more efficient for the case where the process was bioaugmented with the inoculated consortium. Gas chromatography analyses coupled with mass spectrometric detection (GC-MS) confirmed high degradation yields obtained for both cases studied (58 and 71%, respectively) in a 28-day test. It is concluded that oil-based metalworking emulsions can undergo efficient biological treatment under conditions enabling aerobic bacterial proliferation and that xenobiotic biodegradation kinetics can be accelerated by bioaugmenting the process with allochthonous microbial consortia.