Integral membrane proteins (IMP) are an important class of biological targets for drug development because of the key roles they play in active transport, signal transduction, protein expression, energy generation/maintenance, and receptor-mediated responses to extracellular nutrients, drugs, and pathogens.  Integral membrane protein function is typically determined using whole cell or liposome assays, which are often slow, imprecise, cumbersome, indirect, and lack sensitivity.  Development of rapid and sensitive methods for assaying membrane protein function in planar sensor architectures is one of the major goals of this project, having broad practical and fundamental impact in the areas of drug discovery, mechanisms of membrane protein activity & hard-soft material interfaces in medical implants.

In collaboration with Professor Christine Hrycyna in our department, we are currently focused on the membrane-associated enzyme isoprenylcysteine carboxylmethyltransferase (ICMT) as a target IMP for sensor development.  Fabrication of stable, asymmetric supported membranes containing oriented ICMT are in progress such that they can detect a-carboxylmethylation events.  We can detect ICMT activity using either a fluorescence-based assay or a self-assembled interferometric detection method.  The fluorescence approach uses a coupled enzymatic reaction with ICMT and S-adenosylhomocysteine (SAH) hydrolase to generate the thiol homocysteine (Hcy).  Hcy then dequenches a disulfide-linked fluorophore-quencher reporter molecule, F-Q, to generate a fluorescent signal.  In the presence of the substrate N-acetyl-farnesyl cysteine (AFC) and the co-factor S-adenosylmethionine (SAM), methyl transfer activity catalyzed by ICMT should be lowered when exposed to membrane-permeable inhibitor leads, leading to low thiol production and consequently low fluorescence emission.  The most potent inhibitors can then be tested further using cell and membrane-based assays.  We envision a similar method for screening ICMT inhibitor leads via interferometry, except that this approach uses a sandwich assay architecture to bind the initial product of the ICMT reaction, SAH instead of detecting the secondary ICMT product, Hcy, with coupled enzyme reactions.

      References

D. H. Thompson, K. Wong, R. Humphry-Baker, J. Wheeler, J. M. Kim & S. B. Rananavare, "Tetraether Bolaform Amphiphiles as Models of Archaebacterial Membrane Lipids: Raman Spectroscopy, ³¹P-NMR, X-Ray Scattering and Electron Microscopy" Journal of the American Chemical Society 1992 114, 9035-9042.

A. Patwardhan & D. H. Thompson, “Efficient Synthesis of 40- and 48-Membered Tetraether Macrocyclic Bisphosphocholines” Organic Letters 1999 1, 241-244.

J.-M. Kim, A. Patwardhan, A. Bott & D. H. Thompson, “Preparation and Electrochemical Behavior of Gramicidin Bipolar Lipid Monolayer Membranes Supported on Gold Electrodes”, Biochimica et Biophysica Acta 2003 1617, 10-21.

C. Wang, S. Leffler, D. H. Thompson & C. A. Hrycyna, “A General Assay for S-adenosyl Methionine–dependent Enzymes Using a Thiol-activated Fluorescent Molecular Beacon”, Biochemical and Biophysical Research Communications 2005 331, 351-356.

H. B. Hodges, M. Zhou, J. L. Anderson, D. H. Thompson & C. A. Hrycyna, “Inhibition of Membrane-Spanning Metalloproteins by Hydrophobic Metal Chelators”, Bioconjugate Chemistry 2005 16, 490-493.

W. Febo-Ayala, S. Morera-Felix, C. A. Hrycyna & D. H. Thompson, “Functional Reconstitution of the Integral Membrane Enzyme, Isoprenylcysteine Carboxyl Methyltransferase, in Synthetic Bolalipid Membrane Vesicles”, Biochemistry 2006 45, 14683-14694.

W. Febo-Ayala, D. P. Holland, S. A. Bradley, D. H. Thompson, “Lateral Diffusion Coefficients of an Eicosanyl-Based Bisglycerophosphocholine Determined by PFG-NMR and FRAP”, Langmuir 2007 23, 6276-6280.

E. Kang, J.-w. Park, S. McClellan, J.-M. Kim, D. P. Holland, G. U. Lee, E. Franses, K. Park, D. H. Thompson, “Specific Adsorption of Histidine-Tagged Proteins on Silica Surfaces Modified with Ni²⁺:NTA-Derivatized Poly(ethylene glycol)”, Langmuir 2007 23, 6281-6288.