The main focus of our research is to develop nuclear magnetic resonance (NMR) techniques and use them together with other biophysical approaches to elucidate the molecular structure, dynamics and mechanisms of membrane-targeting proteins that regulate phototransduction in vision and other signal transduction processes. We are currently studying retinal recoverin, a calcium-myristoyl switch protein in vision, and the guanylate cyclase activating proteins (GCAPs), implicated in autosomal dominant cone dystrophy. Our studies also include neuronal calcium sensor (NCS) homologs, such as the DREAM protein in the brain that serves as a transcriptional repressor for pain modulation as well as a subclass of EF-hand proteins (CaBPs) that modulate the activity of neuronal Ca2+ channels. This emerging family of proteins is important for signal transduction generally because Ca2+-induced conformational changes in these proteins control their cellular location and capacity to interact with membrane-bound targets and/or DNA elements. The long-term goal of our work is to develop an atomic-level understanding of how calcium-myristoyl switch proteins operate in signal transduction and disease processes. In particular, we want to understand how covalently attached myristoyl groups work in concert with calcium-binding sites and target proteins to guide this family of proteins to specific membrane-bound targets.

1. Calcium-myristoyl Switch Proteins in Vision. Recoverin, a 23 kdal protein and member of the EF-hand superfamily, serves as a calcium sensor in retinal rod cells. The Ca2+-bound form of recoverin prolongs the lifetime of photoexcited rhodopsin by inhibiting rhodoposin kinase. Recoverin contains an amino-terminal myristoyl or related fatty acyl group and four EF-hands. The binding of two Ca2+ to myristoylated, but not unmyristoylated, recoverin leads to its translocation from the cytosol to retinal rod outer segment membranes. We solved the structure of myristoylated recoverin by nuclear magnetic resonance (NMR) spectroscopy. In the Ca2+-free protein, the myristoyl group is sequestered in a deep hydrophobic box, where it is clamped by multiple residues contributed by three of the EF-hands. We have used both solution and solid-state NMR to show that Ca2+ induces the unclamping and extrusion of the myristoyl group that in turn interacts with lipid bilayer membranes. The Ca2+-induced exposure of the myristoyl group is accompanied by a 45� rotation of the amino-terminal domain relative to the carboxy-terminal domain, and many hydrophobic residues are exposed. Ca2+-induced extrusion of the amino-terminal myristoyl group is a key structural determinant for membrane localization by recoverin and may be a general mechanism for controlling the transport of signaling proteins to cellular membrane targets. For more details about our studies on recoverin, see Structure(2010) 18:9, Biochemistry (2009) 48:850, J. Biol. Chem. (2006) 281:37237, Biochemistry (200342:6333, Biochemistry 2002 41:5776 and Nature (1997) 389:198.

2. Evolution of the Ca2+-myristoyl switch. Recoverin and GCAPs belong to a large family of myristoylated calcium sensor proteins that are highly conserved in all eukaryotes from yeast to humans. Mammalian brain and spinal cord contain more than 20 homologs such as hippocalcin, several neurocalcins and frequenins. Why the abundance and diversity of Ca2+-myristoyl switches in the central nervous system? One possibility is that the neuronal homologs may potentiate the mobilization of intracellular calcium by seven-helix receptors. Recoverin homologs in the brain also regulate gene expression (DREAM), interact with presenilins (calsenilin) and regulate voltage-gated K+ channels (KChiPs). Surprisingly, recoverin has a clearly recognizable homolog in yeast that is more than 60% identical to mammalian frequenin and has all the hallmarks of a Ca2+-myristoyl switch protein. What signaling processes in yeast does this ancient homolog bring calcium tidings? The trail begins here and by following it, we may catch glimpses of how the molecular circuitry underlying excitability and memory arose. For more details about myristoyl switch proteins in yeast, see J. Biol. Chem. (2010) 285: 4405, J. Biol. Chem (2007) 282:30949, J. Biol. Chem (2004) 279:12744, J. Biol. Chem. (2003) 278:4862 and Nat. Cell Biol. (1999) 1:234.

3. Calcium Sensor Proteins (CaBPs) that Regulate Neuronal Calcium Channels. CaBPs represent a new sub-class of neuronal calcium sensor proteins that regulate multiple Ca2+ channels in the brain and retina. In particular, CaBP1 has been shown to interact with and modulate the activity of inositol 1,4,5-trisphosphate receptors that serve as calcium release channels on the ER membrane. CaBP1 also interacts with P/Q-type voltage-gated Ca2+ channels, L-type channels and TRPC5. Recently, CaBP4 has been shown to regulate L-type channels only in the retina. My lab is using NMR and other biophysical approaches to elucidate the Ca2+-induced conformational changes of CaBPs at the atomic level to understand how they regulate the opening and closing of Ca2+ channel targets in signal transduction and disease. For more details about our studies on CaBP1, see J. Biol. Chem (2009) 284:2472, J. Biol. Chem. (2005)280:37461.