Structural Analysis of Calcium Regulation in Muscle

The overall goal of the proposed research is to determine the structural basis for the Ca-ATPase (calcium pump) regulation mechanisms in both skeletal and cardiac sarcoplasmic reticulum (SR). Calcium is the principal intracellular messenger for activation of many cells, and is the carrier of contractile stimuli in muscle. Its homeostatic balance is maintained by Ca-ATPase, a 110 kDa integral membrane protein. Phospholamban (PLN), expressed in cardiac muscle, and sarcolipin (SLN), expressed in skeletal muscle, inhibit Ca-ATPase activity and this inhibition is relieved by phosphorolation via protein kinase A (PKA).

Our immediate goals are

1) solve the structures of the two Ca-ATPases modulators in membrane environments, and

2) determine their interactions with Ca-ATPase.

Structure and Molecular Mechanisms of Selective Neurotoxicants

The overall goal of the proposed research is to determine the structural and molecular bases for the selective neurotoxicity of organotins via their interactions with the protein stannin. Organotins (compounds with Sn-C bonds) are ubiquitous in the environment (Gadd 2000). In 1985, their annual worldwide production was approximately 35,000 tons (Fent 1996). Organotin compounds are used in the materials sciences as antifouling agents, in agriculture as fungicides (Fent 1996), and in biomedicine as anticancer drugs (Gielen and others 1990). Incidental exposure to organotins in humans and laboratory testing on rats both have resulted in severe behavioral changes, ranging from disorientation, seizures, aggressiveness to death (Boyer 1989; Feldman and others 1995; Kreyberg and others 1992). Unlike other heavy metals and organometallic compounds, organotins display highly selective activity, damaging specific areas of the brain (Philbert and others 2000).

To better understand the selective toxicity of these compounds, we plan to:

1) Determine the high-resolution structures of free and TMT-bound stannin in detergent micelles.

2) Characterize the dynamics of free and TMT-bound stannin in detergent micelles.

3) Elucidate of stannin's topology in lipid bilayers using solid-state NMR spectroscopy.

Molecular modeling of NMR data: Using Dipolar Pattern for Rapid Folding of Protein Structures

The goal of the project is to use minimal information from NMR spectroscopy to determine the three-dimensional structure of proteins. We have made significant steps for the interpretation of the residual dipolar couplings in terms of the relative orientations of the different protein secondary structure domains. We have found a new, general equation to interpret the spatial orientation of any secondary structure domain, including α-helices, β-sheets, π-helices, and 310-helices (Mascioni and Veglia 2003).