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Results demonstrate that the outer membrane receptor of pyoverdine in Pseudomonas aeruginosa is capable of interacting with the pyoverdine. Data indicate that the enzyme-pyoverdin complex adopts atleast two different conformations and exhibit diverse characteristics under in vivo and in vitro conditions.

The solution structure of the N-terminal domain of the calcium sensor calcium vector protein reveals that the domain is largely monomeric and exhibits isotropic Brownian rotational diffusion. The amide backbone groups of the alpha-helices, beta-strands, and the amide groups in calcium-binding loops show restricted but rapid movements.

The dynamics of the ATP binding site of cytidine monophosphate kinase from Escherichaia coli indicate a conformational change of the LID region consequent upon binding by a phosphate donor. Data indicate that this region may wrap around ATP rendering the active site inaccessible to the solvent enabling kinases avoid hydrolysis

The conformation of annexin 2 in both monomeric and heterotetrameric forms changes when calcium binds to the protein. This causes an increase in the major excited-state lifetime and a decrease in flexibility of the W212 tryptophan residue. The flexibility increases when annexin 2 binds to membranes but no further conformational change takes place beyond that induced by calcium.

Research was conducted to examine the effect of pH on the steady-state fluorescence emission spectrum of its single tryptophan residue (Trp187). Circular dichroism calculations provide evidence for a preservation of the native folding of the protein in mild acidic conditions. At high pH of ~11, a fluorescence red-shift of smaller amplitude was also noted. Results indicate that electrostatic interactions may be more essential for the stabilization of domain III folding without the presence of calcium.

Experiments were conducted to measure sodium ion (Na+) concentrations using the fluorescent indicator SBFO. Using global compartmental analysis, the values of four rate constants in excited-state reactions were measured. The global biexponential analyses failed to provide a precise value for fluorescence decay traces of SBFO in the Na+ concentrations. However, estimated rate constant values ranged from 0.465 to 0.127. It was also observed that the excited-state reaction had negligible impact on the fluorimetric estimation of Kd.

Steady-state and time-resolved fluorescence, circular dichroism and ultraviolet (UV)-difference spectroscopy studies indicate the tryptophan 187 in the domain III of annexin V is required for calcium ion binding. Trp 187 exhibits an excited-state reaction that causes hydrogen bond formation in the alpha-carbonyl groups of Thr224. Trp 187 is not seen in crystal structures with high level of calcium ions indicating structural changes that enable the incorporation of the protein on phospholipid membranes.

Site-directed mutagenesis is carried out on FpvA to identify the extracellular loops or parts of these loops involved in the various stages of siderophore pyoverdine-iron (Pvd-Fe) uptake in Pseudomonas aeruginosa. Results suggest that W434, S603 and G701 mutations in loops [L.sub.4], [L.sub.7] and [L.sub.9] respectively are involved on the mechanism of Pvd-Fe intake.

The dynamical behavior of a biomimetic molecule, basket-handle porphyrins (BHP), is studied using several techniques. The results show complimentarity between experiments and theoretical simulations.

The fluorescent dynamic stokes shift method has emphasized a time-dependent dipolar relaxation process around the single tryptophan residue (Trp31) in cytidine monophosphate kinase from E.coli. This Trp residue, located close to the protein surface in a hydrophobic pocket, is weakly accessible to acrylamide, a water-soluble quencher.

Right-handed twisting is a fundamental structural feature of beta-plated sheets in globular proteins, which is critical for their geometry and function. Density functional theory using the B3LYP exchange-correlation functional and the split-valence 6-31G basis set is utilized to examine the structure and conformational transitions of single and double-stranded antiparrallel B-sheet models to determine the driving force for the right-handed twisting.

Domain III in annexin V exhibits a high conformational flexibility at membrane/water interfaces which may indicate its insignificance in the interaction of the protein with the membrane. This is evident in the weak interaction between domain III and the membrane surface, and this observation agrees with atomic force microscopy results. The conformational dynamics of domain III bound to negatively charged phospholipid vesicles of 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine and 1-palmitoyl-2-oleoyl-sn-glycerophosphoserine or combined with reverse micelles of water/sodium bis(2-ethylhexyl) sulfosuccinate in isooctane.

Both steady-state and time-resolved tryptophan fluorescence spectroscopy were used in investigating the interaction between the immunophilin domain of FKBP59 (FKBP59-I) and a number of immunosuppressant drugs. The highly homologous protein FKBP12's weak fluorescence is shown to due to an atypical hydrogen bond interaction between the indole nitrogen and Phe129 benzene ring. FKBP59-I, located on the external hydrophilic side of the 50-60 beta-sheet, is determined to be responsible for over 95% of the observed fluorescence.

The water radial distribution in AOT/iso-octane/waterreverse micelles (RM), used to mimic the membrane–water interface,was examined by excited-state lifetime and transient spectral measurementsof the series of n-(9-anthroyloxy) stearic acids(n-AS), with n= 2, 3, 6, 7, 9,10, and 12. A water gradient in the RM extended from the polar headgroup region up to the middle of the surfactant carbon chains. A fastintramolecular excited-state relaxation, involving the rotation ofthe carboxylic group of the ester bond with respect to the anthracenering, gave rise to a nanosecond time-dependent fluorescence Stokesshifts (TDFSS). In water-filled RMs, we only observed a water-inducedTDFSS occurring over subnano- and nanosecond time scales with decreasingamplitudes and rates as a function of depth, according to the decreasingwater gradient and the slowing down of the anthroyloxy moiety rotationalmotion. This water-induced TDFSS is most likely the result of bothH-bond formation and general dipolar relaxation, as indirectly showedby measurements with DMF (a nonprotic polar solvent) instead of waterin RMs. [ABSTRACT FROM AUTHOR]

The human multidrug-resistance-associated protein 1 (hMRP1/ABCC1) belongs to the large ATP-binding cassette transporter superfamily. In normal tissues, hMRP1 is involved in tissue defense, whereas, in cancer cells, it is overproduced and contributes to resistance to chemotherapy. We previously investigated the folding properties of the predicted transmembrane fragments (TM) TM16, and TM17 from membrane-spanning domain 2 (MSD2). These TMs folded only partially as an α-helix and were located in the polar headgroup region of detergent micelles used as membrane mimics (Vincent et al. in Biochim Biophys Acta 1768:538-552, ; de Foresta et al. in Biochim Biophys Acta 1798:401-414, ). We have now extended these studies to TM4 and TM10, from MSD0 and MSD1, respectively. TM10 may be involved in the substrate translocation pathway whereas the role of TM4 is less predictable, because few studies have focused on MSD0, a domain present in some hMRP1 homologs only. Each TM contained a single Trp residue (W142 or W553) acting as an intrinsic fluorescent probe. The location and dynamics of the TMs in dodecylphosphocholine (DPC) or n-dodecyl-β- d-maltoside (DDM) micelles were studied by Trp steady-state and time-resolved fluorescence, including quenching experiments. Overall TM structure was analyzed by far-UV circular dichroism studies in detergent micelles and TFE. TM10 behaved similarly to TM16 and TM17, with an interfacial location in micelles consistent with a possible role in lining the transport pore. By contrast, TM4 behaved like a classical TM fragment with a high α-helical content, and its transmembrane insertion did not require its interaction with other TMs. [ABSTRACT FROM AUTHOR]

Annexin 3 (ANX A3) represents ∼1% of the total protein of human neutrophils and promotes tight contact between membranes of isolated specific granules in vitro leading to their aggregation. Like for other annexins, the primary molecular events of the action of this protein is likely its binding to negatively charged phospholipid membranes in a Ca2+-dependent manner, via Ca2+-binding sites located on the convex side of the highly conserved core of the molecule. The conformation and dynamics of domain III can be affected by this process, as it was shown for other members of the family. The 20 amino-acid, N-terminal segment of the protein also could be affected and also might play a role in the modulation of its binding to the membranes. The structure and dynamics of these two regions were investigated by fluorescence of the two tryptophan residues of the protein (respectively, W190 in domain III and W5 in the N-terminal segment) in the wild type and in single-tryptophan mutants. By contrast to ANX A5, which shows a closed conformation and a buried W187 residue in the absence of Ca2+, domain III of ANX A3 exhibits an open conformation and a widely solvent-accessible W190 residue in the same conditions. This is in agreement with the three-dimensional structure of the ANX A3-E231A mutant lacking the bidentate Ca2+ ligand in domain III. Ca2+ in the millimolar concentration range provokes nevertheless a large mobility increase of the W190 residue, while interaction with the membranes reduces it slightly. In the N-terminal region, the W5 residue, inserted in the central pore of the protein, is weakly accessible to the solvent and less mobile than W190. Its amplitude of rotation increases upon binding of Ca2+ and returns to its original value when interacting with membranes. Ca2+ concentration for half binding of the W5A mutant to negatively charged membranes is ∼0.5 mM while it increases to ∼1 mM for the ANX A3 wild type and to ∼3 mM for the W190 ANX A3 mutant. In addition to the expected perturbation of the W190 environment at the contact surface between the protein and the membrane bilayer, binding of the protein to Ca2+ and to membranes modulates the flexibility of the ANX A3 hinge region at the opposite of this interface and might affect its membrane permeabilizing properties. [ABSTRACT FROM AUTHOR]

Examines conformation and dynamic properties of adenosine diphosphate (ADP) binding site of cytidine monophosphate kinase (CMPK) from Escherichia coli. Molecular modelling of CMPK complex with Ant-dADP; Spectroscopic characteristics of CMPK-bound Ant-dADP; Rotational dynamics of free and protein-bound Ant-dADP.

A time-resolved fluorescence spectroscopic study of the recombinant human epidermal growth factor (hEGF), a bis(tryptophan)-containing protein (Trp49-Trp50), and of the two single-tryptophan- containing engineered mutants with Trp49 or Trp50 replaced by Phe ([W49F]hEGF, [W50F]hEGF), was undertaken in order to gain insight into the conformational dynamics of the C-terminal region. Quite different position-dependent microenvironments for the two Trp residues are shown by comparing the fluorescence intensity decay of both mutants. Trp50 in the single-tryptophan mutant [W49F]EGF probably undergoes a dominant interaction with the solvent. A more heterogeneous environment of Trp49 in the [W50F]hEGF mutant is found. Moreover, the fluorescence decay of the native hEGF is not simply the additive result of the decays of both mutants: the Trp2 sequence confers a conformation of the C-terminal sequence which is more in contact with the rest of the protein molecule. By contrast, the fluorescence anisotropy decay of the native protein is quite similar to that of the single-tryptophan mutants. A high degree of rotational freedom in the C-terminal region of the protein is demonstrated. The resonance energy transfer, which could contribute to the anisotropy decay, appears therefore not to be highly efficient with respect to the depolarization motions. In addition to these local conformational and dynamic aspects of the hEGF C-terminal sequence, the fluorescence anisotropy decay data demonstrate the existence of a dimerization process of the native protein which is dependent on pH and protein concentration. This phenomenon influences the excited-state lifetime profiles and, therefore, the local conformational equilibrium of the C-terminal region. [ABSTRACT FROM AUTHOR]

The influence of corticosteroids on the lipid polymorphism of dielaidoylglycerophosphoethanolamine was studied by31P NMR spectroscopy and differential scanning calorimetry. Both techniques evidenced two transitions in the pure lipid samples. The first one corresponded to the gel→liquid crystalline phase transition. It occurred at a temperature of 38.9 °C, as measured by differential scanning calorimetry and at 35– 40 &degC as detected by 31 P NMR. The second transition corresponded to the bilayer→hexagonal H11 phase transition. It occurred at 64.3 °C a measured by differential scanning calorimetry and at 60 C as detected by NMR. Addition of corticosteroids led to different specific effects on the bilayer→hexagonal II,, phase transition, according to their chemical structure. These effects appear to be the result of low amounts of incorporated steroids, according to binding studies (partition coefficient values range between 5 and 54). The presence of a conjugated 3-keto group in the steroid molecule (progesterone) promoted a downward shift in the bilayer→hexagonal H11 phase transition temperature by about 6–7 °C as compared to the 3β-OH-bearing compound (pregnenolone), which did not exhibit any appreciable effect. No change in the ΔHof transition could be measured. The presence of the 21-OH group (like in deoxycorticosterone) induced the formation of a structure, characterized by an isotropic lineshape of the 31P NMR spectrum at temperatures where the ‘hexagonal‘ type of lineshape is present, without steroid. The transition from the bilayer to this other structure occurred at a slightly higher temperature than the bilayer→hexagonal HII, phase transition. It corresponded to a peak in differential scanning calorimetry scans with a ΔH of 2.1 kJ·mol-1. The presence of the I7βaOH group as present in 17β-OH-progesterone and 11-deoxycortisol suppressed the two former effects. These compounds had no influence on the bilayer→hexagonal HII phase transition. The additional presence of the 11β-OH group like in corticosterone and cortisol, evoked a stabilization of the bilayer organization as the bilayer→hexagonal HII phase transition temperature is shifted upward by about 10°C. This was accompanied by a decrease of the Δto 0.8 kJ· mol-1. Besides this, the corticosteroids did not affect to a large extent the ge→liquid crystalline phase transition: a general slight downward shift of the transition temperature and a small broadening of the transition were observed without significant change in the &DeltaH. The effects of corticosieroids on the bilayer→hexagonal HII phase transition are qualitatively correlated with their influence on biological membranes. [ABSTRACT FROM AUTHOR]

The sequence situated around Trp242 in Bordetella pertussis adenylate cyclase, a bifunctional protein of 1706 amino acid residues, forms the core of the calmodulin-binding site. Peptides varying in size and in affinity for calmodulin, and preserving the same sequence around Trp242 were analyzed by time-resolved fluorescence spectroscopy. Their dynamic properties were compared to those of the catalytic domain of B. pertussis adenylate cyclase corresponding to the first 400 amino acid residues of the protein and in which the Trp69 residue was replaced by Phe. The heterogeneity of the fluorescence intensity decays of Trp242 is likely due to the existence of conformers in equilibrium as is suggested by the effect of trifluoroethanol both the secondary structure content and the lifetime distributions. Binding to calmodulin leads to striking effects on the lifetime distribution profiles by selecting a major excited state population and therefore one major conformer. Trp242 still presents some degree of rotational freedom in the complexes. The reduction of rotational freedom is more important for the shorter peptides than for the longest one. A similar selection of one major conformer with the same lifetime was also observed for the Trp242 in the mutant protein when bound to calmodulin, as in the complexes with the peptides. We conclude that the site of interaction of B. pertussis adenylate cyclase with calmodulin has similar conformational flexibility as that evidenced in the isolated peptides. This property of the molecule allows a better adjustment of the enzyme upon interaction with calmodulin. [ABSTRACT FROM AUTHOR]

The binding effect of enantiomeric substrate analogs under micellar from on the local conformation and dynamics of the N-terminal region of porcine pancreas phospholipase A2 was examined by time-resolved fluorescence measurements of its single tryptophan residue (Trp3). The complexity of the fluorescence intensity decay of the unliganded protein (four excited-state lifetime populations) suggests a conformational heterogeneity of the N-terminal region of the protein. A considerable simplification of the excited-state lifetime profile was specifically observed in the complex with one of the stereoisomers [(R)-2-tetradecanoylamino)-hexanol-phosphocholine] at low inhibitor/protein molar ration of ≈ 9. This indicated the existence of a definite conformation of the N-terminal region of the protein in the complex. No effect was detected for the S-enantiomer. In parallel, the rotational mobility of the Trp residue in the complex with the R-enantiomer was reduced. At a higher inhibitor/protein molar ratio of ≈ 130, the stereospecificity of the interaction was lost and complexes were formed with both stereoisomers. These complexes were, however, not similar to complexes were formed with both stereoisomers. These complexes were, however, not similar to the specific one either in terms of the local Trp3 environment or of the volume of the rotating unit. The local effects of low amounts of monomeric inhibitors added to a preformed protein/micelle complex of a phospholipase A2 double mutant in which a Trp residue was genetically inserted near the active site at position 31 while the natural Trp3 was replaced by Phe [Kuipers, O., Vincent, M., Brochon, J. C., Verheij, H. M., de Haas, G. H. & Gallay, J. (1991) Biochemistry 30, 877–8785], were also monitored by time-resolved fluorescence of this single Trp residue. A stereospecific dependence of the local perturbations was again observed. These results support the idea that the active conformation of the protein is reached in solution only after formation of a ternary complex: protein-interface-inhibitor. [ABSTRACT FROM AUTHOR]

Human recombinant interferon α2 belongs a to family Of proteins active against a wide range of viruses. It contains two tryptophan residues located at positions 77 and 141 in the peptide sequence. The fluorescence emission spectrum of these tryptophan residues displays a maximum at 335 nm. The fluorescence intensity decay is described by one broad excited-state-lifetime population centered around a value of 1.7 ns (full width at half maximum, 1.5 ns). These observations suggest that in the native protein, both tryptophan residues emit from similar environments, not directly exposed to the surrounding solvent. The anisotropy decay is essentially biexponential. The correlation-time value characterizing the Brownian rotation of the protein varies linearly with the viscosity/temperature ratio. The calculated hydrodynamic volumes are compatible with the existence of a dimer and a tetramer, at pH 5.5 and 9.4, respectively. Addition of urea at pH 5.5 disrupts the dimer and modifies to some extent the excited-state-lifetime distribution which becomes more heterogeneous. Disulfide-bond reduction also dissociates the dimer and leads to a highly heterogeneous fluorescence-intensity decay with four excited-state-lifetime populations. An opening of the local structure in the Trp region of the protein is likely to occur in these conditions. The fast-anisotropy-decay components can be due to either fast rotation or energy transfer between the indoles. Close proximity of the two Trp residues (less than 1 nm) is suggested from steady-state and time-resolved fiuorescence-anisotropy measurements in vitrified medium [95% (by mass) glycerol at -38°C]. This suggestion is in agreement with the recently published three-dimensional structure of the homologous protein murine interferon β [Senda, T., Shimazu, T., Matsuda, S. Kawano, G., Shimizu, H., Nakamura, K. T. & Mitsui, Y. (1992) EMBO J. 11, 3193–3201]. [ABSTRACT FROM AUTHOR]

The interaction of recombinant human epidermal growth factor with small unilamellar phospholipid vesicles was studied by steady-state and time-resolved fluorescence of the bis-tryptophan sequence (Trp-Trp). Steady-state anisotropy measurements demonstrate that strong binding occurred with small unilamellar vesicles made up of acidic phospholipids at acidic pH only (pH≤ 4.7). An apparent stoichiometry for 1,2-dimyristoyl- sn-phosphoglycerol of about 12 phospholipid molecules per molecule of human epidermal growth factor was estimated. The binding appears to be more efficient at temperatures above the gel to liquid-crystalline phase transition. The conformation and the environment of the Trp-Trp sequence are not greatly modified after binding, as judged from the invariance of the excited state lifetime distribution and from that of the fast processes affecting the anisotropy decay. This suggests that the Trp-Trp sequence is not embedded within the bilayer, in contrast to the situation in surfactant micelles (Mayo et al. 1987; Kohda and Inigaki 1992). [ABSTRACT FROM AUTHOR]

“Solubilization” of a beef adrenal microsomal enzyme: 3-oxosteroid δ5–δ4-isomerase has been obtained by high concentrations of divalent cation (0.5–2 M CaCl2). A study of the solubilization yield as a function of ion concentration and pit has shown that optimum yield is obtained with 1 M CaCl2 near pH 7. After elimination of calcium from the “soluble” enzyme, precipitation occurs and enzymatic activity is irreversibly lost in both precipitate and supernatant. The values of the kinetic parameters determined at pH 8.5 and 35 °C are: V = 410 and 920 nmol · min-1 · mg-1 and Km = 36 and 11 μM for 5-androstene-3,17-dione and 5-pregnene-3,20-dione respectively. Our results suggest the existence of only one C19 and C21 3- oxosteroid isomerase in the beef adrenal glands. [ABSTRACT FROM AUTHOR]