Thomas received his Ph.D. from the Program in Molecular Biophysics, Johns Hopkins School of Medicine, in December of 2014. During his Ph.D., he performed structural and biochemical studies on extracellular components of the Hedgehog signaling pathway, as well as biochemical studies of the EGFR/HER/ErbB family of receptors. In these studies, the Hedgehog receptor Patched, a twelve-pass alpha-helical integral membrane protein, was expressed recombinantly in insect cells, purified, and its interactions with other extracellular pathway components were studied (Cleveland et al., 2014). In addition, Thomas determined the x-ray crystal structure of an extracellular fragment of the Neogenin receptor. Finally, he performed cell-based biochemical studies on the human EGFR and HER4/ErbB4 receptors, which demonstrated the activation of receptor dimers by a single ligand (Liu, Cleveland, et al., 2012).
In his work at the NIST Center for Neutron Research, Thomas is using Small-Angle Neutron Scattering (SANS) to study the process of membrane protein incorporation and crystallization from the lipidic cubic phase (LCP). In the LCP crystallization technique, membrane proteins are purified and then mixed with certain lipids (most commonly monoolein) to form the cubic phase. This LCP is a system-spanning porous bilayer with a “sponge-like” structure that allows diffusion of the protein in three dimensions. Unlike detergent micelles, the cubic phase allows for protein contacts involving the transmembrane portions of the protein, which are incorporated in the same contiguous bilayer. Crystals can be grown directly from LCP-embedded membrane protein by simply overlaying precipitants. This technique has been used to solve several significant membrane protein structures, particularly of GPCRs.
SANS is ideally suited for the study of membrane proteins in LCP, because it enables the use of contrast matching to eliminate scattering from the lipid matrix. This allows scattering from the membrane protein to be measured directly.
In addition to his studies on the cubic phase, Thomas has also performed studies on nanolipoprotein particles (NLPs), also known as “nanodiscs.” NLPs allow membrane proteins to be maintained in solution for biochemical or structural studies, and may provide a more stable environment than detergent micelles. In this technique, a small “patch” of membrane is isolated and stabilized in solution by a protein “belt,” which covers the hydrophobic core of the bilayer at the rim. It is possible to “package” integral membrane proteins within NLPs by mixing all the detergent-solubilized components at an optimal ratio (lipids, belt protein, and membrane protein), followed by controlled detergent removal. However, for new proteins, this traditional preparation process requires case-by-case optimization that can be time-consuming and expensive.
An alternative NLP preparation process is to use cell-free expression of the belt and membrane proteins in the presence of small lipid vesicles, which results in insertion and formation of NLPs without the need for detergent solubilization or removal. While this procedure is convenient, and is more amenable to high-throughput studies, it has not been as well-studied as the traditional procedures. Therefore, Thomas is using SANS to compare the structure of NLPs produced by both methods to verify their equivalence.
Cleveland, T.E. IV, Kelman, Z. 2015. Isotopic Labeling of Proteins in Halobacterium salinarum. Methods in Enzymology 565:147–165.. doi:10.1016/bs.mie.2015.06.002
Cleveland, T.E. IV, McCabe, J.M., Leahy, D.J. 2014. Detergent-solubilized Patched purified from Sf9 cells fails to interact strongly with cognate Hedgehog or Ihog homologs. Protein Expression and Purification 104:92–102. doi:10.1016/j.pep.2014.09.012
Liu, P., Cleveland, T.E. IV, Bouyain, S., Byrne, P.O., Longo, P.A., Leahy, D.J. 2012. A Single Ligand is Sufficient to Activate EGFR Dimers. PNAS 109(27):10861–6. doi:10.1073/pnas.1201114109
Cleveland, T.E. IV, Hussey, D.S., Chen, Z.Y., Jacobson, D.L., Brown, R.L., Carter-Wientjes, C., Cleveland, T.E. III, Arif, M. 2008. The Use of Neutron Tomography for the Structural Analysis of Corn Kernels. Journal of Cereal Science 48(2):517–525.