Doo Wan Boo

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.

Photochromism of diarylethene derivatives in rigid polymer matrix: structural dependence, matrix effect, and kinetics

Abstract Photochromic reactions of three diarylethene derivatives embedded in three types of polymer matrices were studied using a variety of spectroscopic techniques and quantum ab initio computations. It was found that the photochromic efficiencies of diarylethene molecules depend on the structural and electronic characteristics of Franck–Condon region in the excited states, and also depend strongly on the sizes of free inner spaces of the matrices. The steady-state and time-resolved fluorescence studies revealed the detailed kinetics of the planar and twisted conformers in the excited states. The observed fluorescence lifetimes of the planar and twisted conformers were ∼2.6 ns and 8∼30 ns.

Photoluminescence characteristics of coupled CdSe∕ZnS quantum dots on self-assembled silica nanospheres

The photoluminescence (PL) characteristics of the self-assembled silica nanospheres containing coupled CdSe∕ZnS core-shell quantum dots (QDs) on the surface of the sphere were studied. The degree of QD-coupling and the distribution of the coupled QDs were controlled by generating spatially separated QD-binding sites on the surface of the spheres and varying the QD concentration during the attachment process. When the sizes of QD spots on the surface probed by atomic force microscopy gradually increased, the band gap energy shifted to lower energy via strong Forster energy transfer between nearby QDs, and when UV-exposure time increased, the band gap energy shifted to slightly higher energy, accompanied by a large enhancement in the emission intensity. Moreover, the Bragg diffraction of the self-assembled crystal structure of the spheres affected the PL characteristics of QDs by shifting the entire emission spectra toward longer wavelength as the incident angles were increased.

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.

Functional reconstitution of the human serotonin receptor 5-HT6 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.

Di-oxanipecotic acids as more stable turn motifs than di-nipecotic acids

The folded structures of peptidomimetics containing dimers of oxanipecotic acid (Oxa) in loop segments were characterized and compared with those of the corresponding nipecotic acid (Nip)-based ones. According to structural studies using FT-IR and NMR spectroscopies, di-oxanipecotic acid adopted more stable turn conformations than di-nipecotic acid, and for tetramers, L,(S)-Oxa,(S)-Oxa,L and L,(S)-Oxa,(R)-Oxa,L formed hairpin-like structures but only L,(R)-Nip,(S)-Nip,L promoted the stable folded conformations.

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.

A novel scheme for probing mode-specific proton transfer in proton-bound homodimers: vibrational predissociation and ab initio study on CH5+·CH4

Abstract A novel experimental scheme for probing mode-specific proton transfer in proton-bound homodimers based on the vibrational predissociation spectroscopy combined with the techniques of mass-selected detection and isotope substitution is described. Its application to CH 5 + ·CH 4 suggests that rapid, non-mode specific proton transfer occurs when three C–H stretch modes are excited. To explain the experimental result, the transition state and the minimum energy path (MEP) for proton transfer in CH 5 + ·CH 4 are further characterized by ab initio method.

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.