We present a simple method to characterize vesicles and determine, at the same time, the membrane permeability to specific molecules. The method is based on encapsulating highly hydrophilic 3,3',3' '-phosphinidynetris-benzenesulfonic acid (PH) into vesicles and subsequently monitoring its reaction with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB). We tested the method by measuring the membrane permeability of vesicles formed from a series of poly(ethylene oxide)-co-polybutylene oxide (EB) copolymers and egg yolk phosphatidylcholine. We found that the experimental data are in good agreement with calculations based on Fick's first law. We therefore quantified the DTNB permeability across vesicle membranes, finding that polymeric EB membranes have a more selective permeability toward polar molecules compared to phospholipids membranes.

Efforts to develop a quantitative understanding of molecular recognition rely on the additivity of individual intermolecular interactions, and cooperativity represents one of the major potential stumbling blocks. A chemical double-mutant cycle has been used to experimentally measure cooperativity between functional group interactions within a complex framework. The interaction between two aromatic groups varies by 0.2 +/- 0.4 kJ mol(-1) in synthetic H-bonded complexes that differ by 8-13 kJ mol(-1) in overall stability. In these systems, the free energies associated with individual intermolecular interactions can therefore be reliably treated in an additive fashion. The results suggest that alternative explanations should be considered for cooperative phenomena observed in other systems, and a rationale based on the population of partially bound states in flexible molecules is proposed to account for the enthalpic chelate effect and enthalpy-entropy compensation.

Linear oligomeric supramolecular assemblies of defined length have been generated using the Vernier principle. Two molecules, containing a different number (n and m) of mutually complementary binding sites, separated by the same distance, interact with each other to form an assembly of length (n x m). The assembly grows in the same way as simple supramolecular polymers, but at a molecular stop signal, when the binding sites come into register, the assembly terminates giving an oligomer of defined length. This strategy has been realized using tin and zinc porphyrin oligomers as the molecular building blocks. In the presence of isonicotinic acid, a zinc porphyrin trimer and a tin porphyrin dimer form a 3:4 triple stranded Vernier assembly six porphyrins long. The triple strand Vernier architecture introduced here adds an additional level of cooperativity, yielding a stability and selectivity that cannot be achieved via a simple Vernier approach. The assembly properties of the system were characterized using fluorescence titrations and size-exclusion chromatography (SEC). Assembly of the Vernier complex is efficient at micromolar concentrations in nonpolar solvents, and under more competitive conditions, a variety of fragmentation assemblies can be detected, allowing determination of the stability constants for this system and detailed speciation profiles to be constructed.

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Thanks largely to a cooperative chelate effect, clustered membrane-embedded proteins favourably bind to multivalent ligands in solution and, conversely, a multivalent receptor can induce the clustering of membrane-embedded proteins. Here, we use a chemical model to show that the binding of a monovalent ligand and the clustering of a membrane-embedded receptor are closely related processes that modulate each other without the contribution of any apparent multivalence effect. Clearly, the confinement of the receptor within the surface reveals cooperative effects between clustering and binding that are too weak to detect in bulk-solution systems. This work shows that for membrane-embedded receptors that undergo some degree of spontaneous clustering, analyses based on multivalence-mediated cooperativity are insufficient to describe fully the molecular recognition events induced by ligands in solution. Instead, a binding-clustering thermodynamic cycle is proposed for the analysis of the interaction of any kind of ligand with membrane-embedded receptors.