Research

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The Smith Group of analytical and bioanalytical chemists develops imaging instrumentation and methods, and they apply these techniques to study enzymatic catalysis and processes in cultured cells and tissue samples. The team uses imaging techniques including fluorescence and Raman scattering.

The Smith Group’s research efforts in the laboratory have two main objectives: the development of methods to study cell membrane dynamics, and the development of instrumentation for analysis of enzymatic catalysis, which has applications in developing improved biofuels. The former is funded by the National Science Foundation, Divisions of Chemistry and Molecular Biology, and formerly by the Roy J. Carver Charitable Trust; the latter area of focus is funded by the Department of Energy, Office of Science, and formerly by the Iowa State University Plant Sciences Institute.

Measurements of Cell Membrane Dynamics

Analysis Techniques used: Fluorescence Resonance Energy Transfer (FRET); Fluorescence Recovery After Photobleaching; Single Particle Tracking; Sub-diffraction Imaging

Goals: The cell membrane is a complex organization of lipids, proteins, carbohydrates and small molecules that has a dynamic interface with its environment. We aim to measure the small-scale organization of the cell membrane in live cells, as well as the extracellular and intracellular cues that cause a rearrangement of this organization. We seek to develop the relationship between cell membrane organization and cell signaling across the membrane.

Some Recent Findings: We previously developed a noninvasive FRET assay for measuring the microclustering of wild-type and mutant cell membrane receptors, termed integrins (Figure 1). The assay uses donor and acceptor FRET reporter peptides that cluster with integrins. Energy transfer from donor to acceptor FRET reporters, when the two are in close proximity, is used to measure integrin clustering within the cell membrane of cultured cells.

We have also developed a method to measure the role of cytoplasmic proteins in altering the clustering of integrin cell membrane receptors. The method involves the combination of our novel fluorescence resonance energy transfer assay and methods to selectively reduce the expression of a target cytoplasmic protein. Changes in integrin clustering provide evidence for the role of the targeted protein in altering integrin microclustering.

Another main research effort has been to measure the role of cholesterol, and cholesterol-enriched membrane nanodomains in integrin microclustering. Our methodology was to remove cholesterol from the cell membrane by extraction, and then measure changes in integrin microclustering with reduced cholesterol levels in the membrane. We have shown that integrin mutants with different ligand affinities have differing clustering properties when cholesterol is extracted from the membrane.

In collaboration with Professor Jacob Petrich, Department of Chemistry at Iowa State University, we are measuring small scale membrane organization that is smaller in size than the diffraction limit of light using sub-diffraction imaging techniques (Figure 2).

In collaboration with Professor Javier Vela, Department of Chemistry, Iowa State University, we are pursuing single particle tracking methods to measure the diffusion of integrins in the plasma membrane (Figure 3). These studies will provide information about heterogeneous receptor diffusion, which cannot be elucidated with bulk fluorescence studies.

Total Internal Reflection Raman Microscopy

Goals: We seek to develop an instrument that can provide chemical specific depth-profiling data with sub-diffraction spatial resolution, and then apply it to study heterogeneous catalytic systems.

Recent Findings: We have finalized the setup of a novel instrument for obtaining chemically specific depth-profiling measurements, termed scanning angle total internal reflection Raman microscopy (Figure 4). The instrument platform is an inverted optical microscope with added automated variable-angle optics to control the angle of an incident laser on a prism/sample interface. Axial measurements with 34 nm spatial resolution have been achieved, which has not been demonstrated with other Raman techniques. This is a roughly 30-fold improvement relative to confocal Raman microscopy. We are currently using the instrument to study heterogeneous enzymatic catalytic systems.

Analysis Methods for Biofuels Research

Techniques used: Raman spectroscopy; enzyme immobilization

Goals: We are developing analysis methods that enable glucose and ethanol concentrations to be measured in hydrolysate and fermentation media that are suitable to screen product yields in the conversion of biomass to biofuels. We also develop methods for improved product yield, and to characterize plant materials.

Recent Findings: The conversion of plant materials to a usable portable fuel requires several conversion steps (Figure 5). In order to obtain the highest yield of usable fuel, each step in the process must be optimized. We are working on developing several analysis techniques that will benefit the utilization of biomass as a renewable source of energy and chemicals. We have recently demonstrated that Raman spectroscopy can be used to simultaneously measure glucose and xylose in corn stover hydrolysate with an accuracy comparable to other more laborious analysis methods.