Functional reconstitution of the human serotonin receptor 5-HT(6) using synthetic transmembrane peptides.

Seven transmembrane (7TM) synthetic peptides mimicking the alpha-helical TM domains of the human serotonin receptor subtype-6 (5-HT(6)) were autonomously reconstituted in detergent micelle and liposome environments. The degree of assembly of the 7TM peptides was characterized by monitoring the fluorescence resonance energy transfer (FRET) between donor and acceptor probes labeled at the amino termini of the second and fourth TM-peptides, respectively. The FRET efficiency of these peptides significantly increased when the 7TM peptides were reconstituted in liposome compare to detergent micelles. Furthermore, the 7TM peptides reconstituted in liposomes selectively bound to free serotonin and serotonin-conjugated magnetic beads, yielding a dissociation constant of 0.84 microM. These results show that the seven individual TM domains of 5-HT(6) can spontaneously assemble into liposomes in a conformation that mimics a native structure, and further demonstrate that specific interactions between TM helices play a critical role in the folding and stabilizing of GPCRs. The autonomous assembly of 7TM-peptides can be applied to the screening of agonists for GPCRs that are difficult to manipulate.

Reversible immobilization of diffusive membrane-associated proteins using a liquid-gel bilayer phase transition: a case study of Annexin V monomers.

We report a novel strategy for reversible immobilization of diffusive membrane-associated proteins in a native orientation using a liquid-gel bilayer phase transition, and its application to the single molecule study of Cy3-labeled Annexin V (A5) monomers on supported lipid bilayers containing phosphatidylserine (PS) in a Ca(2+)-rich environment. Total internal reflection fluorescence single molecule trajectory analysis revealed that, at low membrane occupancy, A5 monomers diffuse randomly on liquid phase bilayers and occasionally collide with other A5 monomers to form short-lived pseudodimers. During the liquid-to-gel bilayer phase transition, diffusive A5 monomers become immobilized mostly as isolated monomers, with some percentage of dimers and trimers. The EDTA-induced unbinding of immobilized Cy3-A5 spots indicates that Ca(2+)-bridges between A5 and PS lipids are preserved in the immobilized A5 monomers, confirming their native orientation on gel phase bilayers. Furthermore, the persistence of Ca(2+)-bridges during the liquid-to-gel phase transition, despite negligible A5 binding affinity to gel phase bilayers, strongly suggests the formation of tightly bound A5(Ca2+)m(PS)n complexes that diffuse and become immobilized as single units during the bilayer phase transition.

A correlated force-optical study on the self-assembly behavior of annexin V on model membranes: effect of dye conjugation.

We have examined the self-assembled membrane-bound aggregates of two annexin V (A5) dye conjugates and compared them to those from native A5. Native A5 and FITC-labeled A5 (A5-FITC) both formed discrete well-defined crystalline monolayer domains of p6 symmetry. However, A5-FITC also showed additional domains with a corrugated appearance not observed in native A5. In contrast, Cy3-labeled A5 (A5-Cy3) showed a mixture of crystalline monolayer and irregular multilayered domains, with the ratio of the two types varying significantly from sample to sample, and also required a much longer incubation time than native A5 and A5-FITC. When A5-FITC and A5-Cy3 were co-incubated on the same bilayer, well-defined crystalline monolayer domains containing both A5-FITC and A5-Cy3 were consistently observed at a much shorter incubation time than that of pure A5-Cy3 alone, indicating that A5-FITC facilitates the inclusion of A5-Cy3. These results suggest that dye labels can affect A5 2D self-assembly and crystal formation on membrane surfaces.

Detection method for ionic species based on the electroacoustic effect.

The development and application of a new ion detection method based on the electroacoustic (EA) effect is described. An EA signal, produced by applying a pulsed-type electric field to an electrolyte solution in an electroacoustic cell, is dependent on the electrical and thermal properties of the electrolyte and can be detected by using a conventional gas microphone system. The EA signals, generated in this fashion, are proportional to the square of the amplitude of the pulsed-type electric field and show an inverse dependence on the modulated frequency, as found in other acoustic detection systems. The results of this study demonstrate that the EA signals observed with the new system display a linear dependence on the concentration of the electrolyte over a 3 order-of-magnitude concentration range (ca. 10(-7)-10(-4) M). The detection limit of this system was shown to be as low as 29.9 ppb for an aqueous solution of HCl. The results also indicate that the EA signal is proportional to the equivalent conductivity of electrolytes in aqueous solution. As a consequence, the new method has the potential of being used as a universal detector for ions in solutions. An important property of this detection system is that it can be applied to in situ ion detection, and as a result, it can be employed in kinetic studies to follow the progress of ionic chemical reactions.

Carbohydrate arrays for functional studies of carbohydrates.

Carbohydrates, as components of glycoproteins, glycolipids and proteoglycans, play an important biological role as recognition markers through carbohydrate-protein interactions. For the most part, biophysical and biochemical methods have been used to analyze these biomolecular interactions. In contrast, less attention has been given to the development of high-throughput procedures to elucidate carbohydrate-protein recognition events. Recently, carbohydrate arrays were developed and employed as a novel high-throughput analytic tool for monitoring carbohydrate-protein interactions. This technique has been used to profile protein binding and enzymatic activity. The results have shown that carbohydrate binding to the corresponding lectins is highly selective and that the relative binding affinities are well correlated with those obtained from solution-based assays. In addition, this effort demonstrated that carbohydrate arrays could be also utilized to identify and characterize novel carbohydrate-binding proteins or carbohydrate-processing enzymes. Finally, the results of this investigation showed that lectin-carbohydrate binding affinities could be quantitatively assessed by determining IC50 values for soluble carbohydrates with the carbohydrate arrays. The results of these studies suggest that carbohydrate arrays have the potential of playing an important role in basic researches, the diagnoses of diseases and drug discovery.

De novo design of non-hydrogen-bonded helical pseudopeptides composed of oxanipecotic acid oligomers.

Ab initio calculation and circular dichroism experiments reveal that Oxa-oligomers adopted pronounced non-hydrogen-bonded helical structures.

Novel consecutive beta- and gamma-turn mimetics composed of alpha-aminooxy tripeptides.

[structure: see text] To develop novel consecutive beta- and gamma-turn mimetics, we designed and characterized alpha-aminooxy tripeptides (trimers) consisting of oxanipecotic acid dimer and alpha-aminooxy acid. According to FT-IR and NMR data, as well as ab initio quantum calculations, the trimers adopted unusual folded structures with consecutive beta- and gamma-turnlike conformations.