The fluorescent dye FM1-43 labels nerve terminals in an activity-dependent fashion and has been found increasingly useful in exploring the exo- and endocytosis of synaptic vesicles and other cells by fluorescence methods. The dye distributes between the aqueous phase and the lipid membrane but the physical-chemical parameters characterizing the adsorption/partition equilibrium have not yet been determined. Fluorescence spectroscopy alone is not sufficient for a detailed elucidation of the adsorption mechanism since the method can be applied only in a rather narrow low-concentration window. In addition to fluorescence spectroscopy, we have therefore employed high sensitivity isothermal titration calorimetry (ITC) and deuterium magnetic resonance (2H-NMR). ITC allows the measurement of the adsorption isotherm up to 100 microM dye concentration whereas 2H-NMR provides information on the location of the dye with respect to the plane of the membrane. Dye adsorption/partition isotherms were measured for neutral and negatively-charged phospholipid vesicles. A non-linear dependence between the extent of adsorption and the free dye concentration was observed. Though the adsorption was mainly driven by the insertion of the non-polar part of the dye into the hydrophobic membrane interior, the adsorption equilibrium was further modulated by an electrostatic attraction/repulsion interaction of the cationic dye (z=+2) with the membrane surface. The Gouy-Chapman theory was employed to separate electrostatic and hydrophobic effects. After correcting for electrostatic effects, the dye-membrane interaction could be described by a simple partition equilibrium (Xb=Kcdye) with a partition constant of 103-104 M-1, a partition enthalpy of DeltaH=-2.0 kcal/mol and a free energy of binding of DeltaG=-7.8 kcal/mol. The insertion of FM1-43 into lipid membranes at room temperature is thus an entropy-driven reaction following the classical hydrophobic effect. Deuterium nuclear magnetic resonance provided insight into the structural changes of the lipid bilayer induced by the insertion of FM1-43. The dye disturbed the packing of the fatty acyl chains and decreased the fatty acyl chain order. FM1-43 also induced a conformational change in the phosphocholine headgroup. The -P-N+ dipole was parallel to the membrane surface in the absence of dye and was rotated with its positive end towards the water phase upon dye insertion. The extent of rotation was, however, much smaller than that induced by other cationic molecules of similar charge, suggesting an alignment of FM1-43 such that the POPC phosphate group is sandwiched by the two quaternary FM1-43 ammonium groups. In such an arrangement the two cationic charges counteract each other in a rotation of the -P-N+ dipole.

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.

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