Douglas S. English

Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly.

In biological systems, organic molecules exert a remarkable level of control over the nucleation and mineral phase of inorganic materials such as calcium carbonate and silica, and over the assembly of crystallites and other nanoscale building blocks into complex structures required for biological function. This ability to direct the assembly of nanoscale components into controlled and sophisticated structures has motivated intense efforts to develop assembly methods that mimic or exploit the recognition capabilities and interactions found in biological systems. Of particular value would be methods that could be applied to materials with interesting electronic or optical properties, but natural evolution has not selected for interactions between biomolecules and such materials. However, peptides with limited selectivity for binding to metal surfaces and metal oxide surfaces have been successfully selected. Here we extend this approach and show that combinatorial phage-display libraries can be used to evolve peptides that bind to a range of semiconductor surfaces with high specificity, depending on the crystallographic orientation and composition of the structurally similar materials we have used. As electronic devices contain structurally related materials in close proximity, such peptides may find use for the controlled placement and assembly of a variety of practically important materials, thus broadening the scope for ‘bottom-up’ fabrication approaches.

Direct observation of structural changes in organic light emitting devices during degradation

A method for studying the degradation of organic light emitting devices (OLEDs) in real time is described. Transparent OLEDs allow for the spatial correlation of cathode topographic images with optical images (transmission, photoluminescence, and electroluminescence) of the devices throughout the degradation process. In this study we focused on the evolution of nonemissive, “dark” spots during device operation. We conclude that the electroluminescent dark spots originate as nonconductive regions at the cathode/organic interface and expand or grow as a result of exposure to atmosphere. We propose a mechanism of dark spot growth involving aerobic oxidation of the cathode/organic interfacial region, leading to a highly resistive, carrier blocking interface at the dark spot locations. No initial defects on the cathode surface, which might be responsible for the formation of dark spots, were detected by atomic force microscopy. Structural changes, such as degradation of organic materials and the cathode surfac...

The role of charge in the surfactant-assisted stabilization of the natural product curcumin.

Colloidal solutions of surfactants that form micelles or vesicles are useful for solubilizing and stabilizing hydrophobic molecules that are otherwise sparingly soluble in aqueous solutions. In this paper we investigate the use of micelles and vesicles prepared from ionic surfactants for solubilizing and stabilizing curcumin, a medicinal natural product that undergoes alkaline hydrolysis in water. We identify spectroscopic signatures to evaluate curcumin partitioning and deprotonation in surfactant mixtures containing micelles or vesicles. These spectroscopic signatures allow us to monitor the interaction of curcumin with charged surfactants over a wide range of pH values. Titration data are presented to show the pH dependence of curcumin interactions with negatively and positively charged micelles and vesicles. In solutions of cationic micelles or positively charged vesicles, strong interaction between the Cur(-1) phenoxide ion and the positively charged surfactants results in a change in the acidity of the phenolic hydrogen and a lowering of the apparent lowest pK(a) value for curcumin. In the microenvironments formed by anionic micelles or negatively charged bilayers, our data indicates that curcumin partitions as the Cur(0) species, which is stabilized by interactions with the respective surfactant aggregates, and this leads to an increase in the apparent pK(a) values. Our results may explain some of the discrepancies within the literature with respect to reported pK(a) values and the acidity of the enolic versus phenolic protons. Hydrolysis rates, quantum yields, and molar absorption coefficients are reported for curcumin in a variety of solutions.

A photoprotection strategy for microsecond-resolution single-molecule fluorescence spectroscopy

Time resolution of current single-molecule fluorescence techniques is limited to milliseconds because of dye blinking and bleaching. Here we introduce a photoprotection strategy that affords microsecond resolution by combining efficient triplet quenching by oxygen and Trolox with minimized bleaching via the oxygen radical scavenger cysteamine. Using this approach we resolved the single-molecule microsecond conformational fluctuations of two proteins: the two-state folder α-spectrin SH3 domain and the ultrafast downhill folder BBL.

Single-molecule spectroscopy in oxygen-depleted polymer films

Abstract Single-molecule spectroscopy of the prototypical dye, DiI, has been investigated in rigorously de-oxygenated polymer films. The extraordinarily high signal-to-noise data demonstrate that the intrinsic T 1 → S0 intersystem crossing kinetics of DiI are simple-first-order with little temporal or molecule-to-molecule fluctuations. The results verify that O2 collisions are the major factor responsible for fluctuations of the intersystem crossing rate in non-degassed and partially degassed polymer films. In addition to the intrinsic DiI intersystem kinetics, rare fluctuations are observed in the intersystem crossing rate, which are ascribed to residual impurities and/or to low quantum yield photochemical processes of DiI.

Photoluminescence quenching of silicon nanoparticles in phospholipid vesicle bilayers

Due to its unique optical properties, nanostructured silicon is a promising material for optoelectronic applications, solar energy conversion and chemical sensors. This paper demonstrates the incorporation of organic-capped silicon nanoparticles into the hydrophobic interior of phospholipid vesicle bilayers, and uses photoluminescence quenching to determine the accessibility of membrane-embedded nanoparticles to aqueous and lipid-bound quenchers. Experiments with water-soluble quenchers indicate the existence of two populations of membrane-bound nanoparticles with varying accessibilities to the aqueous phase. Results from membrane-bound quenchers reveal that the major population localizes deep inside the bilayer. The ability of the nanoparticles to fully embed in the bilayer allows studies of their interactions with both aqueous and lipophilic molecules in a biomimetic environment. These composites may prove useful in the development of aqueous-based sensors and provide a model system for studying nanoparticle/cell interactions.

Exploring one-state downhill protein folding in single molecules

A one-state downhill protein folding process is barrierless at all conditions, resulting in gradual melting of native structure that permits resolving folding mechanisms step-by-step at atomic resolution. Experimental studies of one-state downhill folding have typically focused on the thermal denaturation of proteins that fold near the speed limit (ca. 106 s-1) at their unfolding temperature, thus being several orders of magnitude too fast for current single-molecule methods, such as single-molecule FRET. An important open question is whether one-state downhill folding kinetics can be slowed down to make them accessible to single-molecule approaches without turning the protein into a conventional activated folder. Here we address this question on the small helical protein BBL, a paradigm of one-state downhill thermal (un)folding. We decreased 200-fold the BBL folding-unfolding rate by combining chemical denaturation and low temperature, and carried out free-diffusion single-molecule FRET experiments with 50-μs resolution and maximal photoprotection using a recently developed Trolox-cysteamine cocktail. These experiments revealed a single conformational ensemble at all denaturing conditions. The chemical unfolding of BBL was then manifested by the gradual change of this unique ensemble, which shifts from high to low FRET efficiency and becomes broader at increasing denaturant. Furthermore, using detailed quantitative analysis, we could rule out the possibility that the BBL single-molecule data are produced by partly overlapping folded and unfolded peaks. Thus, our results demonstrate the one-state downhill folding regime at the single-molecule level and highlight that this folding scenario is not necessarily associated with ultrafast kinetics.

Carbohydrate modified catanionic vesicles: probing multivalent binding at the bilayer interface.

This article reports on the synthesis, characterization, and binding studies of surface-functionalized, negatively charged catanionic vesicles. These studies demonstrate that the distribution of glycoconjugates in the membrane leaflet can be controlled by small alterations of the chemical structure of the conjugate. The ability to control the glycoconjugate concentration in the membrane provides a method to explore the relationship between ligand separation distance and multivalent lectin binding at the bilayer interface. The binding results using the O-linked glucosyl conjugate were consistent with a simple model in which binding kinetics are governed by the density of noninteracting glucose ligands, whereas the N-linked glycoconjugate exhibited binding kinetics consistent with interacting or clustering conjugates. From the noninteracting ligand model, an effective binding site separation of the sugar sites for concanavalin A of 3.6-4.3 nm was determined and a critical ligand density above which binding kinetics are zeroth order with respect to the amount of glycoconjugate present at the bilayer was observed. We also report cryo-transmission electron microscopy (cryo-TEM) images of conjugated vesicles showing morphological changes (multilayering) upon aggregation of unilamellar vesicles with concanavalin A.

Carbohydrate-functionalized catanionic surfactant vesicles: preparation and lectin-binding studies

In this paper we describe the modification of anionic surfactant vesicles with surfactant-based glycoconjugates of glucose, lactose, maltose and maltotriose. Catanionic vesicles were prepared by mixing cetyltrimethylammonium tosylate and sodium dodecylbenzenesulfonate to obtain thermodynamically stable, spontaneously formed vesicles. Carbohydrate moieties were linked to a hydrocarbon chain via an N-glycosyl linkage to produce an amphiphilic glycoconjugate that undergoes hydrophobic insertion into the outer bilayer leaflet of the surfactant vesicle. Once inserted, the bioavailability of the surface-adsorbed carbohydrates was determined using agglutination of the modified vesicles through selective binding of the lectins concanavalin A and peanut agglutinin. In these studies, glycoconjugates were present in the vesicle bilayer at a carbohydrate-to-surfactant ratio of approximately 1 : 100. At this coverage, vesicle formation is uninhibited, the carbohydrate groups are displayed on the vesicle outer surface and bind selectively to lectins in solution. The preparation of stable, carbohydrate-functionalized, anionic surfactant vesicles may prove useful in the targeted delivery of molecular payloads such as dyes or drugs to cells with high selectivity for a wide range of biotechnological applications.

Gradual Disordering of the Native State on a Slow Two-State Folding Protein Monitored by Single-Molecule Fluorescence Spectroscopy and NMR

Theory predicts that folding free energy landscapes are intrinsically malleable and as such are expected to respond to perturbations in topographically complex ways. Structural changes upon perturbation have been observed experimentally for unfolded ensembles, folding transition states, and fast downhill folding proteins. However, the native state of proteins that fold in a two-state fashion is conventionally assumed to be structurally invariant during unfolding. Here we investigate how the native and unfolded states of the chicken α-spectrin SH3 domain (a well characterized slow two-state folder) change in response to chemical denaturants and/or temperature. We can resolve the individual properties of the two end-states across the chemical unfolding transition employing single-molecule fluorescence spectroscopy (SM-FRET) and across the thermal unfolding transition by NMR because SH3 folds-unfolds in the slow chemical exchange regime. Our results demonstrate that α-spectrin SH3 unfolds in a canonical way in the sense that it converts between the native state and an unfolded ensemble that expands in response to chemical denaturants. However, as conditions become increasingly destabilizing, the native state also expands gradually, and a large fraction of its native intramolecular hydrogen bonds break up. This gradual disordering of the native state takes place in times shorter than the 100 μs resolution of our SM-FRET experiments. α-Spectrin SH3 thus showcases the extreme plasticity of folding landscapes, which extends to the native state of slow two-state proteins. Our results point to the idea that folding mechanisms under physiological conditions might be quite different from those obtained by linear extrapolation from denaturing conditions. Furthermore, they highlight a pressing need for re-evaluating the conventional procedures for analyzing and interpreting folding experiments, which may be based on too-simplistic assumptions.