The Smith Group of analytical and bioanalytical chemists develops imaging instrumentation and methods, and they apply these techniques to study enzymatic catalysis, polymer films and the cell membrane. 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 organization and dynamics, and the development of instrumentation for analysis of enzymatic catalysis and polymer films, which has applications in developing improved biofuels and energy storage and capture devices. 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, Basic Energy Sciences through Ames Laboratory.
Measurements of Cell Membrane Organization and Dynamics
Fluorescence Analysis Techniques used: Fluorescence Resonance Energy Transfer (FRET); Fluorescence Recovery After Photobleaching (FRAP); Single Particle Tracking; Sub-diffraction Stimulated Emission Depletion Imaging.
Overview: 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 clustering 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 reporter peptides, 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 or membrane proteins in altering the clustering and diffusion of integrin cell membrane receptors. The method involves the combination of FRET or FRAP and methods to selectively reduce the expression of a target protein. We have identified a number of cytoplasmic and membrane proteins that alter integrin diffusion and clustering.
Another research effort has been measuring the role of cholesterol, and cholesterol-enriched membrane nanodomains in integrin clustering and diffusion (Figure 2). Our methodology was to remove cholesterol from the cell membrane by extraction, and then measure changes in integrin properties with reduced cholesterol levels in the membrane. We have shown that integrin mutants with altered 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 and The Ames Laboratory, we are measuring cellular structures and organization that are smaller in size than the diffraction limit of light using sub-diffraction imaging techniques.
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
Overview: We developed an instrument that can provide chemical specific depth-profiling data with sub-diffraction spatial resolution, and are applying the instrument to study heterogeneous catalytic systems and polymer films.
Recent Findings: We have set up 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 tens of nanometer spatial resolution have been achieved. This is a roughly one order of magnitude improvement relative to confocal Raman microscopy.
We have measured the Raman scatter from an interface containing a thin gold film to obtain surface plasmon resonance and enhanced Raman scattering measurements simultaneously. We showed that the Raman signal as a function of incident angle is reproducible and can be modeled by the calculated mean square electric field at the interface.
We are currently using the instrument to study heterogeneous enzymatic catalytic systems and thin polymer films that have uses in energy storage and capture devices. This project is in collaboration with Professors Jacob Petrich and Ning Fang, Department of Chemistry at Iowa State University and The Ames Laboratory.
Analytical Methods for Biofuels Research
Techniques used: Raman spectroscopy; enzyme immobilization
Goals: We are developing analysis methods that enable glucose and ethanol product yields to be measured in the conversion of biomass to biofuels. We also develop methods for improved product yield, and to characterize the composition of 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.
We demonstrated that near IR excitation Raman spectroscopy can be used in combination with chemometrics to measure the lignin monomer composition of herbaceous, angiosperm and gymnosperm biomass. The technique is facile, rapid and suitable for in situ measurements.
Immobilization of enzymes onto solid supports can increase product yield in enzymatic reactions. We have shown that silica-immobilized cellulase increases ethanol yield in the simultaneous saccharification and fermentation of cellulose compared to reactions performed with enzyme in solution. This work lays the foundation for cheaper ethanol from renewable biomass.