The main experimental tools we employ are multi-dimensional NMR spectroscopy, isothermal titration calorimetry, fluorescence spectroscopy and protein engineering methods. We attempt to reconcile results from structural, dynamical, energetic and kinetic results in order to gain deeper insights into molecular mechanisms and biological function.

One project involves several intracellular lipid-binding proteins from the small intestine. These predominately-cytoplasmic proteins are thought to function in the transcellular transport and targeting of lipids and have distinct specificities for long-chain fatty acids, vitamin A, bile salts and cholesterol. They also serve as superb model systems for investigating molecular recognition at atomic resolution. We are using triple-resonance 3-D and 4-D NMR to characterize the solution structures and dynamic properties of the wild-type proteins with and without their native ligands. In addition, we are engineering mutant and chimeric proteins in order to further define the molecular basis for ligand specificity and affinity. Together, this information is being used to develop functional hypotheses that can be tested in gene-disrupted and transgenic animals. Inhibitors with therapeutic potential in type II diabetes and hypercholesterolemia are being designed in collaboration with Doug Covey's laboratory.

A second project involves a putative arachidonate-binding domain of the NMDA receptor. The NMDA receptor is a glutamate-gated calcium channel in the brain that serves an important role in learning and memory. The arachidonate-binding domain is thought to be topologically similar to members of the lipid-binding protein family. Arachidonate potentiates NMDA receptor activity and may contribute to the excitotoxicity and neurodegeneration associated with severe neurological disorders such as Alzheimer's and Huntington's diseases. Functional hypotheses derived from the structural studies at atomic resolution will be tested in Jim Huettner's laboratory using intact receptors expressed in whole cells.

• Toke, O., Monsey, J.D. and Cistola, D.P. Kinetic mechanism of ligand binding in human ileal bile acid binding protein as determined by stopped-flow fluorescence analysis. Biochemistry (E-pub ahead of print.) (2007).
• Anderson, M.A., Ogbay, B. Arimoto, R., Sha, W., Kisselev, O.G., Cistola, D.P. and Marshall, G.R. Relative strength of cation-pi vs salt-bridge interactions: The G_talpha(340-350) peptide/rhodopsin system. JACS 128:7531-7541 (2006)
• Lu, J., Cistola, D.P. and Li, E. Analysis of ligand binding and protein dynamics of human retinoid X receptor alpha ligand-binding doman by nuclear magnetice resonance. Biochemistry 45:1629-1639 (2006).
• Toke, O., Monsey, J.D., DeKoster, G.T., Tochtrop, G.P., Tang, C., and Cistola, D.P. Determinants of cooperativity and site-selectivity in human ileal bile acid binding protein. Biochemistry 45:727-37 (2006).
• Tochtrop, G.P., Dekoster, G.T., Covey, D.F. and Cistola, D.P. A single hydroxyl group governs ligand site selectivity in human ileal bile acid binding protein. J Am Chem Soc 126:11024-11029 (2004).
• Ogbay, B., Dekoster, G. T. and Cistola, D. P. The NMR structure of a stable and compact all-beta-sheet variant of intestinal fatty acid-binding protein. Protein Science 13:1227-1237 (2004).
• Liu, W. Zheng, Y., Cistola, D.P. and Yang, D. Measurement of methyl (13)C-(1)H cross-correlation in uniformly (13)C-, (15)N-, labeled proteins. Journal of Biomolecular NMR 27:351-364 (2003).

• Tochtrop, G.P., Bruns, J.L., Tang, C., Covey, D.F. and Cistola, D.P. Steroid ring hydroxylation patterns govern cooperativity in human bile acid protein. Biochemistry 42:11561-11567 (2003).

• Lu, J., Cistola, D.P., and Li, E. Two homologous rat cellular retinol-binding proteins differ in local conformational flexibility. J Mol Biol 330:799-812 (2003).

• Michalski, M.L., Monsey, J.D., Cistola, D.P. and Weil, G.J. An embryo-associated fatty acid-binding protein in the filarial nematode Brugia malayi. Mol. Biochem. Parasitology 124:1-10 (2002).

• Tochtrop, G.P., DeKoster, G.T., Cistola, D.P. and Covey, D.F. Synthesis of [3,4-(13)c(2)]-enriched bile salts as NMR probes of protein-ligand interactions. J Organic Chem. 67:6764-6771 (2002).

• Tochtrop, G.P., DeKoster, G.T., Cistola, D.P. and Covey, D.F. A Simple Efficient Synthesis of [23,24]-13C(2)-Labeled Bile Salts as NMR Probes of Protein-Ligand Interactions. Bioorganic & Medicinal Chemistry Letters. 12:433-435 (2002).

• Tochtrop, G. P., Richter, K, Tang, C., Toner, J. J., Covey, D. F. & Cistola, D. P. Energetics by NMR: Site-specific binding in a positively cooperative system. Proc. Natl. Acad Sci. USA. 99:1847-1852 (2002).

• Wu, F., Corsico, B., Flach, C. R., Cistola, D. P., Storch, J. and Mendelsohn, R. Deletion of the helical motif in the intestinal fatty acid binding protein reduces its interactions with membrane monolayers: Brewster angle microscopy, IR reflection-absorption spectroscopy, and surface pressure studies. Biochemistry 40:1976-1983 (2001).

• Lu, J., Lin, C.-L., Tang, C., Ponder, J. W., Kao, J. L. F., Cistola, D. P. and Li, E. Binding of retinal induces changes in rat cellular retinal-binding protein II conformation and backbone dynamics. J. Mol. Biol. 300:619-632 (2000).

• Wen, J., Lin, L., Tang, C., Ponder, J. W., Kao, J. L. and Cistola, D. P. The NMR Structure of Cellular Retinol Binding Protein II: Implications for the Mechanism of Entry. Gastroenterology, 116, A585 (1999).

• Lu, J., Lin, C.-L., Tang, C., Ponder, J. W., Kao, J. L. F., Cistola, D.P., and Li, E. The structure and dynamics of rat apo-cellular retinol-binding protein II in solution: Comparision with the X-ray crystal structure. J. Mol. Biol. 286:1179-1195, (1999).

• Steele, R.A., Emmert, D.A., Kao, J., Hodsdon, M.E., Frieden, C. and Cistola, D.P. The three-dimensional structure of a helix-less variant of intestinal fatty acid-binding protein. Protein Science 7, 1332-1339 (1998).

• Cistola, D.P. Fat sites found! News and Views article. Nature Structural Biology 5, 751-753 (1998).

• Corsico, B., Cistola, D.P., Frieden, C., and Storch, J. The helical domain of intestinal fatty acid binding protein is critical for collisional transfer of fatty acids to phospholipid membranes. Proc. Natl. Acad. Sci. USA 95, 12174-12178 (1998).

• Hodsdon, M. E., and Cistola, D. P. Discrete backbone disorder in the NMR structure of apo intestinal fatty acid-binding protein. Implications for the mechanism of ligand entry. Biochemistry 36, 1450-146(1997).

• Hodsdon, M. E., and Cistola, D. P. Ligand binding alters the backbone mobility of intestinal fatty acid-binding protein as monitored by N-15 relaxation and H-1 exchange. Biochemistry 36, 2278-2290 (1997).

• Hodsdon, M. E., Ponder, J. W., and Cistola, D. P. The NMR solution structure of intestinal fatty acid-binding protein complexed with palmitate. Application of a novel distance geometry algorithm. J. Mol. Biol., 264, 585-602 (1996).

• Cistola, D. P., Kim, K., Rogl, H., and Frieden, C. Fatty acid interactions with a helix-less variant of intestinal fatty acid-binding protein. Biochemistry 35, 7559-7565 (1996).

• Kim, K., Cistola, D. P., and Frieden, C. Intestinal fatty acid-binding protein: the structure and stability of a helix-less variant. Biochemistry 35, 7553-7558 (1996).

• Hodsdon, M.E., Toner, J. J. and Cistola, D. P. ¹H, ¹³C and ¹⁵N assignments and chemical shift-derived secondary structure of intestinal fatty acid-binding protein. J. Biomol. NMR 6, 198-210 (1995).

• Jakoby, M. G., Covey, D. F. and Cistola, D. P. (1995) Localization of tolbutamide binding sites on human serum albumin using titration calorimetry and heteronuclear 2-D NMR. Biochemistry 34, 8780-8787.

• Cistola, D. P., and Hall, K. B. Probing internal water molecules in proteins using two-dimensional ¹⁹F-¹H NMR. J. Biomol. NMR (Rapid Communication) 5, 415-419 (1995).

• Frieden, C., Jiang, N., and Cistola, D. P. Intestinal Fatty Acid Binding Protein: Folding of Fluorescein-Modified Proteins. Biochemistry 34, 2724-2730 (1995).

• Jakoby, M. G., Miller, K. R., Toner, J. J., Bauman, A., Cheng, L., Li, E., and Cistola, D. P. Ligand-protein electrostatic interactions govern the specificity of retinol- and fatty acid-binding proteins. Biochemistry 32, 872-878 (1993).