Lewis Kay Professor

Ph.D., Yale University, 1988
Laboratory of Chemical Physics, NIH 1988-92
McMurrich Building, Room 1233
416-978-0741
kay@pound.med.utoronto.ca

Development and Application of NMR Methods for The Study of Protein Structure and Dynamics
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Research Synopsis
 
The research in my laboratory focuses on the development of NMR techniques for studying macromolecular structure and dynamics and the application of NMR techniques to problems of biological and clinical importance. In particular the research is divided into the following three areas:

We are presently studying the solution structures of several fragments of the cystic fibrosis gene product. Cystic fibrosis (CF) is an inherited disease affecting one in every 2000 Caucasians and often leads to death by the age of 30. Recently, the gene responsible for CF was identified. It codes for a large protein called the CF transmembrane conductance regulator (CFTR). Studies have shown that CFTR functions as a channel through cell membranes, allowing chloride ions to flow. Sequence analysis of the protein suggests that it consists of five domains organized into two repeated units, each containing a membrane-spanning segment and a cytoplasmic nucleotide-binding domain, and separated by regulatory domain. The major mutation in patients with CF is a deletion mutation whereby phenylalanine 508 is deleted in the amino-terminal nucleotide binding domain, NBD 1, of CFTR.

We are currently determining the high resolution solution structures of small (70 amino acid) fragments of NBD1 both with and without the deletion mutation in an effort to understand at the molecular level how this mutation results in a dysfunctional protein. We are also studying the solution structure of fragments of the regulatory domain (R domain) of CFTR. Recently, it has been shown that the R domain functions as a molecular plug by controlling the flow of Cl- ions through the CFTR channel. The opening and closing of the channel is regulated by phosphorylation of the R domain by cAMP-dependent protein kinases. Solution structures of the R domain with and without phosphorylation will provide an understanding of how phosphorylation regulates this plug.

A second area of research involves the structural aspects of cell signaling. In particular, we are studying the solution structures of SH2 and SH3 domains from cytoplasmic tyrosine kinases. These domains bind tyrosine phosphorylated membrane bound tyrosine kinases, implicated in normal signaling and cellular transformation. Tyrosine phosphorylation of the membrane bound kinases acts as a molecular switch to induce the binding of SH2 domains forming protein complexes at the cell membrane. The formation of these complexes controls signal pathways involving tyrosine kinases. Solution structures of SH2 domains with and without tyrosine phosphorylated peptides will provide a framework for understanding cell signaling at the molecular level. Once these structures have been determined, a detailed study of the sidechain and backbone dynamics will be initiated in order to understand how binding of the phosphorylated peptide affects protein dynamics. A modulation of protein motion may be an important way of transferring information.

The laboratory is also very active in developing novel multi-dimensional NMR experiments for studying structural and dynamic properties of proteins. Examples of such experiments include the three- and four-dimensional triple resonance NMR experiments for protein backbone assignment as well as four-dimensional NOE spectroscopy.
 


Selected Publications
Four-Dimensional Heteronuclear Triple-Resonance NMR Spectroscopy of Interleukin-1b in Solution. Kay, L. E., Clore, G. M. , Bax, A. and Gronenborn, A. M. (1990) Science 249, 411-414.

Three-Dimensional Triple-Resonance Spectroscopy of Isotopically Enriched Proteins. Kay, L.E., Ikura, M., Tschudin, R. and Bax, A. (1990) J. Magn. Reson. 89, 496-514.

Solution Structure of a Polypeptide Dimer Comprising the Fourth Ca2+-Binding Site of Troponin C by Nuclear Magnetic Resonance Spectroscopy. Kay, L.E., Forman-Kay, J. D., McCubbin, W. D. and Kay, C. M. (1991) Biochemistry 30, 4323-4333.

Backbone Dynamics of Proteins As Studied by 15N Inverse Detected Heteronuclear NMR Spectroscopy: Application to Staphylococcal Nuclease. Kay, L. E., Torchia, D. A. and Bax, A. (1989) Biochemistry 28, 8972-8979.
  Recent publications on PubMed.

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