Ph.D.: California Institute of Technology, 1985
Postdoctoral Fellow: NIH Postdoctoral Fellow at University of California, Berkeley, 1985-87
Biochemistry and Biophysics of Signaling Proteins
The 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 a 2-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.
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.