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Biomedical subjects

Matthew A Holden

Publications and source records attributed to Matthew A Holden.

10 recordsLinked to original sources
PubMed2006

Direct transfer of membrane proteins from bacteria to planar bilayers for rapid screening by single-channel recording.

Although the examination of membrane proteins in planar bilayers is a powerful methodology for evaluating their pharmacology and physiological roles, introducing membrane proteins into bilayers is often a difficult process. Here, we use a mechanical probe to transfer membrane proteins directly from Escherichia coli expression colonies to artificial lipid bilayers. In this way, single-channel electrical recordings can be made from both of the major classes of membrane proteins, alpha-helix bundles and beta barrels, which are represented respectively by a K(+) channel and a bacterial pore-forming toxin. Further, we examined the bicomponent toxin leukocidin (Luk), which is composed of LukF and LukS subunits. We mixed separate LukF- and LukS-expressing colonies and transferred the mixture to a planar bilayer, which generated functional Luk pores. By this means, we rapidly screened binary combinations of mutant Luk subunits for a specific function: the ability to bind a molecular adaptor. We suggest that direct transfer from cells to bilayers will be useful in several aspects of membrane proteomics and in the construction of sensor arrays.

Bacterial Proteins↗
PubMed2005

Direct introduction of single protein channels and pores into lipid bilayers.

We have developed a mechanical method for inserting single pores and channels into lipid bilayers. A hand-operated hydrogel probe, coated with a layer of proteins, is mechanically engaged with the lipid bilayer. The two major classes of membrane proteins (beta barrels and alpha-helix bundles) that can be inserted, thereby demonstrating the wide applicability of the approach. Recordings from the proteins show that they retain electrical properties that are the same as those of proteins inserted from solution. Protein-loaded probes can be used repeatedly, allowing individual pores to be rapidly screened one at a time. The method has implications for fundamental studies of cell membranes, array fabrication, and chemical screening.

Bacterial Proteins↗
PubMed2005

Microfluidic tools for studying the specific binding, adsorption, and displacement of proteins at interfaces.

A combination of temperature and concentration gradient microfluidic devices were employed to study the mechanistic details of biomacromolecule interactions at oxide interfaces. These lab-on-a-chip techniques allowed high-throughput, multiplexed data collection using only nanoliters of analyte. The three examples presented demonstrate rapid data acquisition relative to standard methods. First, we show ligand-receptor binding data for multivalent binding between membrane-bound ligands and incoming aqueous proteins with several binding pockets. A model is described for obtaining both the first and second dissociation constant for the reaction. The second example employs temperature gradient microfluidics to study the thermoresponsive properties of polymers and proteins. Both the folding mechanism and subsequent formation of an aqueous two-phase system were followed. Finally, these microfluidic techniques were combined with fluorescence microscopy and nonlinear optical spectroscopy to elucidate the mechanism of fibrinogen displacement from silica surfaces. This combination of methods enabled both direct and indirect observation of protein conformational changes.

Adsorption↗
PubMed2004

Creating fluid and air-stable solid supported lipid bilayers.

Solid supported lipid bilayers are rapidly delaminated when drawn through the air/water interface. We have discovered that a close packed monolayer of specifically bound protein prevents this process. The protection mechanism worked in two ways. First, when protein-protected bilayers were drawn through the air/water interface, a thin bulk water layer was visible over the entire bilayer region, thereby preventing air from contacting the surface. Second, a stream of nitrogen was used to remove all bulk water from a protected bilayer, which remained fully intact as determined by fluorescence microscopy. The condition of this dried bilayer was further probed by fluorescence recovery after photobleaching. It was found that lipids were not two-dimensionally mobile in dry air. However, when the bilayer was placed in a humid environment, 91% of the bleached fluorescence signal was recovered, indicating long-range two-dimensional mobility. The diffusion coefficient of lipids under humid conditions was an order of magnitude slower than the same bilayer under water. Protected bilayers could be rehydrated after drying, and their characteristic diffusion coefficient was reestablished. Insights into the mechanism of bilayer preservation were suggested.

Air↗
PubMed2004

Patterning enzymes inside microfluidic channels via photoattachment chemistry.

We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/Kr+ laser, the fluorophore moieties were bleached to create highly reactive species. These activated molecules subsequently attached themselves to the adsorbed proteins on the microchannel walls and floor via a singlet oxygen-dependent mechanism. Enzymes linked to streptavidin or avidin could then be immobilized via (strept)avidin/biotin binding. Using this process, we were able to pattern multiple patches of streptavidin-linked alkaline phosphatase inside a straight microfluidic channel without the use of valves under exclusively aqueous conditions. The density of alkaline phosphatase in the patches was calculated to be approximately 5% of the maximum possible density by comparison with known standards. Turnover was observed via fluorogenic substrate conversion and fluorescence microscopy. A more complex two-step enzyme reaction was also designed. In this case, avidin-linked glucose oxidase and streptavidin-linked horseradish peroxidase were sequentially patterned in separate patches inside straight microfluidic channels. Product formed at the glucose oxidase patch became the substrate for horseradish peroxidase, patterned downstream, where fluorogenic substrate turnover was recorded.

Animals↗
PubMed2003

The Vroman effect: a molecular level description of fibrinogen displacement.

The molecular level details of the displacement of surface adsorbed fibrinogen from silica substrates were studied by atomic force microscopy, immunochemical assays, fluorescence microscopy, and vibrational sum frequency spectroscopy. The results showed that human plasma fibrinogen (HPF) can be readily displaced from the interface by other plasma proteins near neutral pH because the positively charged alpha C domains on HPF sit between the rest of the macromolecule and the underlying surface. The alpha C domains make weak electrostatic contact with the substrate, which is manifest by a high degree of alignment of Lys and Arg residues. Upon cycling through acidic pH, however, the alpha C domains are irreversibly removed from this position and the rest of the macromolecule is free to engage in stronger hydrogen bonding, van der Waals, and hydrophobic interactions with the surface. This results in a 170-fold decrease in the rate at which HPF can be displaced from the interface by other proteins in human plasma.

Adsorption↗
PubMed2003

Light activated patterning of dye-labeled molecules on surfaces.

This Communication describes a novel method for patterning proteins, ligands, or other organic/inorganic species onto solid substrates. The process works by linking the moiety of interest to a fluorophore that can be subsequently photobleached and bound to the interface. The technique can be performed in aqueous solution and allows several species to be addressed onto the surface simultaneously by using different frequencies of light. Because the method can be used with fluorophores excited by visible light, shorter and more damaging wavelengths can be avoided.

Chemistry Techniques, Analytical↗
PubMed2003

Investigations of bivalent antibody binding on fluid-supported phospholipid membranes: the effect of hapten density.

Investigations of ligand-receptor binding between bivalent antibodies and membrane-bound ligands are presented. The purpose of these studies was to explore binding as a function of hapten density in a two-dimensionally fluid environment. A novel microfluidic strategy in conjunction with total internal reflection fluorescence microscopy was designed to achieve this. The method allowed binding curves to be acquired with excellent signal-to-noise ratios while using only minute quantities of protein solution. The specific system investigated was the interaction between anti-DNP antibodies and phospholipid membranes containing DNP-conjugated lipids. Binding curves for ligand densities ranging from 0.1 to 5.0 mol % were obtained. Two individual dissociation constants could be extracted from the data corresponding to the two sequential binding events. The first dissociation constant, K(D1), was 2.46 x 10(-)(5) M, while the second was K(D2) = 1.37 x 10(-)(8) mol/m(2). This corresponded to a positively cooperative binding effect with an entropic difference between the two events of 62.3 +/- 2.7 J/(mol.K). Furthermore, the percentage of monovalently and bivalently bound protein was determined at each ligand density.

2,4-Dinitrophenol↗
PubMed2002

Reusable platforms for high-throughput on-chip temperature gradient assays.

This paper describes a reusable platform that can apply a linear temperature gradient to a lab-on-a-chip device. When a planar microfluidic device with a series of microchannels is placed on top of the platform with the channels perpendicular to the gradient, each channel is held at a discrete temperature. This allows temperature-dependent data for chemical or biochemical species flowed into the device to be obtained in a concurrent fashion. As a demonstration, a melting curve for dsDNA is performed by collecting all the data simultaneously. The gradient is stable enough to easily distinguish between 30-mers where the complement strand contains a single C-A mismatch or a single T-G mismatch or is a perfect match. On the other hand, a temperature gradient can be formed parallel to the direction of flow of the microchannels. This allows the temperature in each channel to vary continuously as the liquid flows downstream. If each microchannel in the array contains a distinct pH value, ionic strength, species concentration, or chemical composition, then a high-throughput two-variable experiment can be performed. We demonstrate this mode of data collection by measuring the fluorescence yield of fluorescein dye molecules in aqueous solution simultaneously as a function of concentration and temperature.

Journal Article↗