No abstract

Transducin is a heterotrimer formed by a fatty acylated alpha-subunit and a farnesylated betagamma-subunit. The role of these two covalent modifications and of adjacent hydrophobic and charged amino acid residues in reversible anchoring at disk model membranes is investigated at different pH values, salt concentrations, and lipid packing densities using the monolayer expansion technique and CD spectroscopy. The heterotrimer only binds if the acetylated alpha-subunit is transformed into its surface-active form by divalent cations. In the presence of salts the alpha(GDP)-subunit, the betagamma-complex, and the heterotrimer bind to POPC monolayers at 30 mN/m, estimated to mimic the lateral packing density of disk membranes, with apparent binding constants of Kapp = (1.1 +/- 0.3) x 10(6) M-1 (reflecting the penetration of the fatty acyl chain together with approximately three adjacent hydrophobic amino acid residues), Kapp = (3.5 +/- 0.5) x 10(6) M-1 (reflecting the penetration of the farnesyl chain), and Kapp = (1.6 +/- 0.3) x 10(6) M-1 (reflecting a major contribution of the alpha(GDP)-subunit with only a minor contribution from the betagamma-complex). The apparent binding constant of the alpha(GTP)-subunit is distinctly smaller than that of the alpha(GDP)-subunit. Binding to negatively charged POPC/POPG (75/25 mole/mole) monolayers is reinforced by 2-3 cationic residues for the betagamma-complex. The alpha-subunit shows no electrostatic attraction and the heterotrimer shows even a slight electrostatic repulsion which becomes the dominating force in the absence of salts.

The transforming growth factors-␤ (TGF-␤) are important regulatory peptides for cell growth and differentiation with therapeutic potential for wound healing. Among the several TGF-␤ isoforms TGF-␤3 has a particularly low solubility at physiological pH and easily forms aggregates. A spectroscopic structural analysis of TGF-␤3 in solution has thus been difficult. In this study, circular dichroism spectroscopy was used to determine the secondary structural elements of TGF-␤3. In addition, the aggregation of TGF-␤3 was investigated systematically as a function of pH and salt concentration using a rapid screening method. Sedimentation equilibrium and sedimentation velocity analysis revealed that TGF-␤3 exists predominantly in two major forms: (i) monomers in solution at low pH and (ii) large precipitating aggregates at physiological pH. Under acidic conditions (pH < 3.8) the protein was not aggregated. At pH ϳ3.9, a monomer i dimer equilibrium could be detected that transformed into larger aggregates at pH > 4.1. Aggregation was pronounced in the pH range of 4.3 < pH < 9.8 with the aggregation maximum between pH 6.5 and 8.5. The aggregation process was accompanied by a structural change of the protein. The CD spectra were characterized by an isodichroic point at 209.5 nm indicating a two-state equilibrium between TGF-␤3 dissolved in solution and aggregated TGF-␤3. Aggregated TGF-␤3 showed a higher ␤-sheet content and lower ␤-turn and random coil contributions compared with monomeric TGF-␤3. Both the solution structure and the aggregate structure of TGF-␤3 were different from the crystal structure. This was in contrast to TGF-␤2, which showed very similar crystal and solution structures. Under alkaline conditions (pH > 9.8) the turbidity disappeared and a further conformational change was induced. The pH dependence of the TGF-␤3 conformation in solution in the range of 2.3 < pH < 11.0 was reversible. Aggregation of TGF-␤3 was, furthermore, influenced by the presence of salt. For pH > 3.8 the addition of salt greatly enhanced the tendency to aggregate, even in the very basic domain. Under physiological conditions (pH 7.4, c NaCl ‫؍‬ 164 mM) TGF-␤3 has almost the highest tendency to aggregate and will remain in solution only at nanomolar concentrations.

Over the past few years, polyfunctional thiols present as trace components have been found to play a major role in many food flavors, due to their exceptionally low odor thresholds. Unfortunately, their presence in minute concentration (in ng/kg to a few microg/kg) and their high reactivity make it very difficult to extract and identify them. Furthermore, most of them are not yet commercially available. The aim of this work was to characterize the chromatographic and sensorial properties of 10 synthetic mercaptoketones and mercaptoalcohols. Combinatorial chemistry proved to be a very useful way to synthesize them rapidly. Sulfur-selective sulfur chemiluminescence detection chromatograms coupled with mass spectroscopy enabled the target compounds to be identified. Flavor profiles and best estimate gas chromatography lowest amount detected by sniffing (BE-GC-LOADS) values were further determined by GC-olfactometry. As expected, new, exceptionally odorant molecules (BE-GC-LOADS < 0.1 ng) were revealed by this unusual approach.