Research Interests

 

1. Structure, function and regulation of the mitochondrial ATP synthase.

 

2. Folding of CFTR and Other Multidomain Membrane Proteins

Current Funding: NIH R01GM066223, R21HL094951 (06/2009), and Cystic Fibrosis Foundation

Selected Publications

The Mitochondrial ATP Synthase

The research interest of this laboratory encompasses two distinct topics: protein structure and function as related to the mitochondrial ATP synthase and understanding the biochemical basis of Batten disease. Yeast Saccharomyces cerevisiae is used as a model organism because the enzyme is highly conserved from yeast to mammals and because of the powerful tools afforded by yeast.

The ATP synthase is a multimeric enzyme that is responsible for the synthesis of ATP via oxidative phosphorylation. The catalytic site is in a water-soluble portion of the enzyme, the F1, which is bound to the membrane by a membrane bound portion, the Fo. The crystal structure of the water-soluble bovine F1 portion was determined in the laboratory of Dr. John Walker at the Laboratory of Molecular Biology, Cambridge, U.K., making it one of the largest nonsymmetrical protein structures solved to date. In collaboration with Drs. John Walker and Andrew Leslie in the MRC in Cambridge, U.K., we now have the 2.8A map of the yeast F1-ATPase. This is a major advance as it resnow allows us to investigate the structure/function relationship of the ATP synthase by a combination of genetic, biochemical, and x-ray crystallographic methods. Some of the aims that are being pursued are:

1. How does the gamma subunit cause the energy transduction step?

2. How does the binding of nucleotides affect the conformation of the active site?

3. What is the coupling mechanism?

4. What is the crystal structure of the F1F0 ATP synthase?

These questions are being addressed using a combination of genetic, biochemical and x-ray crystallographic methods. The yeast system is currently the only system available that can use all of these techniques to answer these questions. We are also involved in a collaborative project with Dr. Richard Berry from Oxford. Dr. Berry is studying the ATPase by single molecular methods thereby measuring the torque of the rotary mechanism. Dr. Berry, in part, will be using the yeast ATPase to address specific questions relevant to the mechanism of ATP synthesis.

CFTR

 

This is a new project looking at the folding and structure of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR). CFTR is a prime example of a critical protein whose structural analysis has been restricted by the inability to express large amounts of the protein. The high-resolution crystal structure of CFTR could be invaluable in the development of new drugs and therapy.

Rational drug design against novel and known proteins is just one application of knowledge obtained from their high-resolution crystal structure. Frequently, the inability to obtain a large amount of the corrected folded protein precludes the ability to solve the high-resolution structure. This project tests a novel hypothesis, which if correct, will provide a rational approach for successful expression of both membrane and water soluble proteins. As a test case, human CFTR will be expressed in a human cell line. This project has the potential to have a very high impact on biomedical research.