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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]

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]