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Cristol 300A |
Cristol 300C |
e-mail: Falke@Colorado.EDU
Lab page: http://spot.colorado.edu/~falke/
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Chemistry and Biochemistry
Professor (Biochemistry and Biophysics). Director of Interdepartmental Biophysics Program. Ph.D., California Institute of Technology, 1985; National Institutes of Health Postdoctoral Fellow, University of California, Berkeley, 1985-87.
T
he goal of the Falke group is a molecular understanding of biological sensory and signaling pathways. Toward this end, the group is using a structure-based approach to probe the switching of receptors and signaling proteins between their 'on' and 'off' signaling states. The approach involves the development of novel techniques to analyze the structures of these switch proteins, as well as the structural changes and docking events triggered by their activation. Current studies focus on (1) cell-surface chemosensory receptors, and (2) cytoplasmic Ca2+-signaling motifs. These two separate-but-equal research programs of the group are funded by distinct NIH grants.Cell-surface receptors. Transmembrane chemosensory receptors serve to gather chemical or hormonal information at the cell surface, then trigger the appropriate intracellular pathways. Because of their critical role in the initiation of cellular signals and their accessibility to extracellular hormones and drugs, chemoreceptors are rapidly becoming a prominent research target in the fields of signaling biology and medicine. The Falke group is developing an array of approaches that uses elements of molecular biology, protein engineering, pathway reconstitution, site-directed cysteine chemistry and spectroscopy to probe the structure and mechanism of prototypical chemoreceptors. A combination of these approaches has recently been successfully applied to elucidate the transmembrane conformational signal of the E. coli aspartate chemoreceptor, providing the first molecular picture of the membrane-spanning conformational signal in a receptor protein. Current research is beginning to reveal the mechanism by which the transmembrane and adaptation signals regulate kinase activity. A new direction of research focuses on the ubiquitous family of eukaryotic G protein-coupled receptors (GPCR). The same methods proven successful in the aspartate receptor are being applied to representative GPCR systems that will serve as models for the large superfamily. Receptors under study include the yeast pheromone receptor, the mammalian somatostatin receptor, and
a2-adrenergic receptor, all of which can be overexpressed in yeast to yield large quantities of receptor protein for structural and mechanistic studies. Goals include the determination of receptor structure, the mechanism of transmembrane signaling, and the molecular basis of G protein regulation.Ca2+-signaling motifs. Most eukaryotic signaling events directly or indirectly involve essential intracellular Ca2+ signals, which transmit information between different regions within the cell. Such signals regulate a broad array of cytoplasmic and nuclear proteins. Typically, these proteins contain a conserved Ca2+ binding motif that recognizes the messenger Ca2+ ion and activates the appropriate molecular responses. The two most widespread types of motif are the EF-hand and C2 domain, which serve as trigger elements in the majority of Ca2+-regulated pathways. Recent work in the Falke group has elucidated the Ca2+-binding stoichiometry, cooperativity, and activation-inactivation cycle of a representative C2 motif. Current work is targeting the mechanism by which Ca2+ binding drives membrane binding, and the molecular nature of the protein-membrane interaction. Expression clones have been obtained for C2 domains from cytosolic phospholipase A2, protein kinase C, synaptotagmin I and VII, rabphilin and Nedd4. Comparison of the Ca2+- and membrane-binding parameters of these domains will shed light on the mechanisms by which the activation thresholds and lipid specificities of different C2 domains are optimized for different cellular pathways. Recent studies of the EF-hand motif have probed the structural and electrostatic principles used by evolution to optimize this motif for different signaling pathways. A model has been developed to explain the tuning of Ca2+-activation kinetics by the size and charge of a specific side chain, termed the gateway residue, that controls access to the site. Sequence comparisons confirm that the gateway residue defines whether an EF-hand protein responds rapidly or slowly to Ca2+ signals. The EF-hand proteins currently under study include cardiac troponin C and calmodulin.
Methods utilized. The Falke group uses an array of methods spanning biophysics, biochemistry, protein engineering, molecular biology, cell biology, proteomics and genomics. Specific approaches include fluorescence, EPR and NMR spectroscopies, mass spectrometry, stopped flow kinetics, X-ray crystallography, molecular graphics and modelling, site-directed mutagenesis, site-directed cysteine chemistry and spectroscopy, DNA sequencing, gene cloning, protein purification, in vivo and in vivo analysis of mutant protein activities, homology searches to identify related proteins, and sequence alignment to detect structure-function patterns.
Selected Publications
Bass, R.B. and J.J. Falke (1999) The aspartate receptor cytoplasmic domain: In situ chemical analysis of structure, mechanism and dynamics. Structure 7, 829-40.
Bass, R.B., Coleman M.D. and J.J. Falke (1999) Signaling domain of the aspartate receptor is a helical hairpin with a localized kinase docking surface: Cysteine and disulfide scanning studies. Biochemistry 38, 9317-27.
Trammell, M.A. and J.J. Falke (1999) Identification of a site critical for kinase regulation on the central processing unit (CPU) helix of the aspartate receptor. Biochemistry 38, 329-36.
Nalefski, E.A. and J.J. Falke (1998) Location of the membrane-docking face on the Ca2+-activated C2 domain of cytosolic phospholipase A2. Biochemistry 37, 17642-50 (Accelerated Publication).
Hazard, A.L., S.C. Kohout, N.L. Stricker, J.A. Putkey, and J.J. Falke (1998) The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements. Protein Science 7, 2451-9.
Bass, R.B. and J.J. Falke (1998) Detection of a conserved a-helix in the kinase docking region of the aspartate receptor by cysteine and disulfide scanning. J. Biol. Chem. 273, 25006-25014.
Butler, S.B. and J.J. Falke (1998) Cysteine and disulfide scanning reveals two amphiphillic helices in the linker region of the aspartate chemoreceptor. Biochemistry 37, 10746-10756.
Nalefski, N.A., T. McDonagh, W. Somers, J. Seehra, J.J. Falke, and J.D. Clark (1998) Independent folding and ligand specificity of the C2 domain of cPLA2. J. Biol. Chem. 273, 1365-1372.
Danielson, M.A., R.B. Bass and J.J. Falke (1997) Cysteine and disulfide scanning reveals a regulatory a-helix in the cytoplasmic domain of the aspartate receptor. J. Biol. Chem. 272, 32878-32888.
Nalefski, E.A., M.M. Slazas and J.J. Falke (1997) The calcium signaling cycle of a membrane-docking C2 domain. Biochemistry 36, 12011-12018 (Accelerated Publication).
Falke, J.J., R.B. Bass, S.L. Butler, S.A. Chervitz and M.A. Danielson (1997) Bacterial chemotaxis: A molecular view of signal transduction by receptors, kinases and signaling enzymes. Ann. Rev. Cell. Devel. Biol. 13: 457-512.
Drake, S.K., M.A. Zimmer, C. Miller and J.J. Falke (1997) Optimizing the metal binding parameters of an EF-hand-like calcium chelation loop: Coordinating side chains play a more important tuning role than chelation loop flexibility. Biochemistry 36: 9917-9926.
Drake, S.K., M.A. Zimmer, C. Kundrot and J.J. Falke (1997) Molecular tuning of an EF-hand-like calcium binding loop: Contributions of loop position 3. J. Gen. Physiol. 110, 173-184.
Peersen, O.B., T.S. Madsen, and J.J. Falke (1997) Intramolecular tuning of calmodulin by target peptides and proteins: Differential effects on calcium binding and implications for kinase regulation. Prot. Science 6:794-807.
Nalefski, E.A. and J.J. Falke (1996) The C2 domain calcium binding motif: Structural and functional diversity. Protein Sci. 5:2375-2390.
Chervitz, S.A. and J.J. Falke (1996) Molecular mechanism of transmembrane signaling by the aspartate receptor: A model. PNAS 93:2545-2550.
Danielson, M.A. and J.J. Falke (1996) Use of 19F NMR to probe protein structure and conformational changes. Ann. Rev. Biophys. Biomol. Struct. 25:163-195.
Butler, S.L. and J.J. Falke (1996) Effects of protein stabilizing agents on thermal backbone motions: A disulfide trapping study. Biochemistry 35:10595-10600. (Accelerated Publication).
Drake, S.K. and J.J. Falke (1996) Kinetic tuning of the EF-hand calcium binding motif: The gateway residue independently adjusts (i) barrier height and (ii) equilibrium. Biochemistry 35:1753-1760.
Drake, S.K. and J.J. Falke (1996) Tuning the equilibrium ion affinity and selectivity of the EF-hand calcium binding motif: Substitutions at the gateway position. Biochemistry 35:6697-6705.
Chervitz, S.A and J.J. Falke (1995) Lock on/off disulfides identify the transmembrane signaling helix of the aspartate receptor. J. Biol. Chem. 270, 24043-24053.
Chervitz,S.A., C. Lin, and J.J. Falke (1995) Transmembrane signaling by the aspartate receptor: Engineered disulfides reveal static regions of the subunit interface. Biochemistry 34, 9722-9733.
Careaga, C.L., J. Sutherland, J. Sabeti, and J.J. Falke. (1995) Thermal hinge-twisting motions of protein domains in the D-galactose chemosensory receptor: detection by disulfide trapping. Biochemistry 34, 3048-3055.
Falke, J.J., D.F. Blair, T.J. Silhavy, and R. Schmitt (1995) BLAST 1995: International conference on bacterial locomotion and signal transduction. Molecular Microbiology 16, 1037-1050.
Falke, J.J., S.K. Drake, A.L. Hazard, and O.B. Peersen (1994) Molecular tuning of ion binding to calcium signaling proteins. Quart. Rev. Biophys. 27, 219-290.
Danielson, M.A., H.P. Biemann, D.E. Koshland and J.J. Falke (1994) Attractant- and disulfide-induced conformational changes in the ligand binding domain of the chemotaxis aspartate receptor: A 19F NMR study. Biochemistry 33, 6100-6109.
Needham, J.V., T.Y. Chen and J.J. Falke (1993) Novel ion specificity of a carboxylate cluster magnesium binding site in CheY: Strong charge selectivity and weak size selectivity. Biochemistry 32, 3363-3367.
Drake, S.K., R.B. Bourret, L.A. Luck, M.I. Simon, and J.J. Falke (1993) Activation of the phosphosignaling protein CheY. I. Analysis of the phosphorylated conformation by 19F NMR and protein engineering. J. Biol.. Chem. 268, 13081-13088.
Bourret, R.B., S.K. Drake, S.A. Chervitz, M.I. Simon, and J.J. Falke (1993) Activation of the phosphosignaling protein CheY. II. Analysis of activated mutants by 19F NMR and protein engineering. R.B. J. Biol. Chem. 268, 13089-13096.
Renner, M., M.A. Danielson, and J.J. Falke (1993) Kinetic control of calcium signaling: Tuning the ion dissociation rates of EF-hand calcium binding sites. PNAS 90, 6493-6497.
Careaga, C.L. and J.J. Falke (1992) Thermal motions of surface a-helices in the D-galactose chemosensory receptor: Detection by disulfide trapping. J. Mol. Biol. 226, 1219-1235.
Falke, J.J. and D.E. Koshland (1987) Global flexibility in a sensory receptor: A site-directed disulfide bond study. Science 237, 1596-1600.