Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO

My research interests as a postdoctoral scholar in Michael Stowell's lab focused on the area of membrane protein structural biology. It is estimated that 20-30% of the human genome encodes for membrane proteins, with many of these proteins being pharmaceutical drug targets.  Structural information, produced by methods such as X-ray crystallography, will be important for understanding how these proteins function and for the future development of potential therapeutics that target these proteins. My research in this area involved both methodology development (i.e., a thermal shift assay testing membrane protein thermostability) as well as on a particular biological system (P2X receptors), both of which are described in more detail below.

Membrane Protein Thermostability Assay

Membrane protein structural information has been relatively slow in coming due to challenges in the production, purification, and crystallization of these proteins. One aspect of my research focused on the development of a thermal shift assay which may aid in the X-ray crystallographic structure determination of membrane proteins. Finding the optimal conditions under which a given detergent-solubilized membrane protein is most thermodynamically stable may help increase the chances that a particular membrane protein will not denature during the course of a crystallization trial, which is a key step in the structure determination process.

The assay that we developed involved the use of commercially available fluorophores (e.g., SYPRO Orange and NanoOrange) and real-time PCR instrumentation. Initial testing was done using hen egg-white lysozyme (HEWL) and served as proof of concept by yielding thermal denaturation temperatures (Tm  of ~75oC) very close to those determined by well-established methods (Figure 1).

Figure 1. (a) Melting curves of HEWL at two different concentrations (1 mg/mL in orange and 2 mg/mL in blue) in 100 mM Bis-Tris propane buffer, pH 8, monitored using the MJ Research real-time PCR instrument and 1:2,500 diluted SYPRO Orange as reporter dye. Melting curves are of duplicate samples at each concentration, showing the reproducibility of the assay. (b) First derivative from the data in Figure 1a showing the reproducible determination of the Tm. The total protein used was 25 and 50 micrograms, respectively. 

Since our ultimate goal is for this assay to be used in aiding membrane protein structure determination, we subsequently tested the assay with several membrane proteins, including the acetylcholine receptor (AChR) from the electric ray Torpedo marmorata, the ABC transporter BtuCD from E. coli, and the mechanosensitive channels of both small (MscS) and large (MscL) conductance from E. coli. Testing yielded melting curves for three of the four membrane proteins (AchR, BtuCD, and MscS), showing the feasibility of this assay for membrane proteins (Figure 2). The journal article fully detailing our findings can be found at Yeh et al., Acta Cryst. D, 2006.

Figure 2. (a) Melting curves for the membrane protein AChR solubilized with Brij-35 detergent measured for three different concentrations using 1:50 diluted Nano Orange as the reporter dye. (b) Melting curves for BtuCD solubilized with LDAO detergent measured for three different concentrations using 1:2,500 diluted SYPRO Orange as reporter dye. (c) Melting curves for 0.2 mg/mL MscS solubilized with FOS-CHOLINE-14 detergent measured for four different pH values using 1:50 diluted NanoOrange as the reporter dye. These measurements demonstrate the sensitivity and feasibility of using this method for screening optimal conditions for the crystallization of a membrane protein.

P2X Receptors

Another area of my research focused on the membrane protein P2X, an ATP-gated receptor. Besides its prevalent role in bioenergetics, ATP has been shown in recent years to act also as an extracellular signaling molecule, being released into the extracellular environment via either transmembrane transport, exocytotic release from synaptic vesicles, or cell lysis due to inflammation and cell death. The effects of extracellular ATP are mediated in part by a family of ATP-gated cation channels known as the P2X receptors. P2X receptors, of which seven subtypes have been cloned from mammalian species (P2X1-P2X7), are widely distributed in many cell types ranging from excitable cells (e.g., neurons, smooth and skeletal muscle) to immune cells (e.g., macrophages, lymphocytes, and dendritic cells. While their precise physiological functions and mechanism of action are still unclear, they have thus far been found to be involved in a diverse range of biological processes including muscle contraction, pain perception, immunomodulation, inflammation, and apoptotic cell death. Their involvement in such processes has raised the possibility of their being potential therapeutic drug targets for a variety of pathologies including cancer and inflammation. In order to further our understanding of the functioning of these receptors, I worked on the cloning, expression, and purification of the receptors for structural analysis.

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