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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.

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.

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]

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]

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]

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]

“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]