Department of Chemistry and Biochemistry
Cristol Chemistry 354
University of Colorado Boulder
Boulder, CO 80309-0345
Our research combines in vitro spectroscopic and biophysical techniques, protein design and engineering for the development of novel probes, and cellular imaging studies to elucidate the mechanism of cellular signaling pathways. We are focused on understanding pathways involved in metal/ion homeostasis and apoptosis as they contribute to disease (Alzheimer’s disease and cancer) or pathogenic processes (neurodegeneration).
Our lab uses a variety of techniques (molecular evolution, phage display, rational protein design, and peptide synthesis) to develop sensors and reporters based on fluorescence resonance energy transfer (FRET). The sensors make use of a wide array of fluorescent proteins that undergo energy transfer when in close proximity and are designed such that binding of the target causes a change in FRET that can be monitored with a fluorometer (in vitro) or with a fluorescent microscope (in cells). Because the sensors can be genetically encoded, they can be targeted to specific subcellular locations, enabling us to examine the spatial variability and compartmentalization of different signaling processes within the context of live cells. The advantage of such sensors is that they permit the study of reactions while preserving the complex network of interactions that occurs inside a cell and preserve the temporal control/dynamics of different processes. In addition to visualizing cellular processes, we are developing peptide-based probes that permit acute perturbation of reaction pathways to enhance our understanding of critical signaling reactions.
A powerful complement to these cellular studies are spectroscopic and biophysical methods (absorption, circular dichroism, electron paramagnetic resonance, fluorescence, fluorescence anisotropy, stopped-flow, and surface plasmon resonance) that provide detailed information on the bonding nature, kinetics, and thermodynamics of protein-metal, protein-small molecule, and protein-protein interactions.
Specific projects in the lab include 1) the development of genetically encoded zinc sensors and examination of the role of zinc in neuronal signal transduction, 2) the design of peptide-based tools to perturb protein-protein interactions, and 3) development of probes to study the connection between amyloid-beta and calcium dysregulation.
Palmer, A. E., Jin, C, Reed, J. C., Tsien, R. Y., Bcl-2 mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor, Proc. Natl.
Acad. Sci., 2004, 101:50, 17404-17409 * This work was highlighted in the following journals: BioTechniques, 2005, 38:1, p17; Nature Reviews: Molecular Cell Biology, 2005, 6, p92
Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N.G., Palmer, A.E., Tsien, R.Y. Improved monomeric red, orange, and yellow fluorescent proteins derived from Discoma red fluorescent protein, Nature Biotechnology, 2004, 22, 1567-1572
Palmer, A.E., Szilagyi, R.K., Cherry, J.R., Jones, A., Xu, F., Solomon, E.I. Spectroscopic characterization of the Leu513His variant of fungal laccase: effect of increased axial ligand interaction on the geometric and electronic structure of the Type 1 Cu site Inorg. Chem., 2003, 42, 4006-4017
Campbell, R.E., Tour, O., Palmer, A.E., Steinbach, P.A., Baird, G. S., Zacharias, D.A., Tsien, R.Y. A Monomeric Red Fluorescent Protein Proc. Natl. Acad. Sci., 2002, 99, 7877-7882
Palmer, A.E., Quintanar, L., Severance, S., Wang, T.-P., Kosman, D. J., and Solomon, E.I. Spectroscopic characterization and O2 reactivity of the trinuclear Cu cluster of mutants of the multicopper oxidase Fet3p, Biochemistry, 2002, 41, 6438-6448