Cafiso, David S.
- AB, Biophysics, University of California, Berkeley, CA
- PhD, Biophysics, University of California, Berkeley, CA
- Postdoc, University of California, Berkeley, CA
- Postdoc, Stanford University
Molecular Mechanisms for Membrane Transport and Cell Signaling
Membranes and membrane proteins participate in some of the most important and interesting cellular processes. Energy transduction, cell signaling, membrane excitability, secretion and immune recognition are examples of a few of the processes mediated by membrane proteins. However, the molecular mechanisms by which lipids and membrane proteins accomplish these tasks are largely unknown. Approximately 30 to 40 percent of the proteins in a genome are membrane proteins and 70 percent or more of current pharmaceuticals appear to act on membrane proteins. For these reasons, membrane proteins represent one of the most important challenges in the field of structural biology.
Work in our laboratory is directed at studying membranes and membrane proteins, and there are currently two general areas of research ongoing in our laboratory. One area of investigation involves studies on the mechanisms by which proteins become attached to membrane surfaces.This attachment is critical for cell-signaling because it controls protein-protein interactions and the access of enzymes to lipid substrates. For example the oncogenic form of the src tyrosine kinase is not active and fails to transform cells until it becomes attached to membranes. We are currently studying the structure and electrostatic interactions made by MARCKS (the myristoylated alanine rich C-kinase substrate), which has a highly positively charged domain that attaches to the membrane interface. This protein functions to regulate the levels of highly phosphorylated inositol lipids, such as PIP2, in the cell. We are also studying the membrane binding of protein domains such as C2 and PH (pleckstrin homology) domains, which are found in a wide range of proteins involved in cell-signaling. C2 domains function attach to membranes in a Ca++ dependent fashion, and PH domains translocate to the membrane interface and bind to PIP2.
A second area of investigation involves membrane transport. We are currently investigating the molecular mechanisms that function to transport solutes across lipid bilayers. For example, in gram negative bacteria, such as E. coli, we are examining the molecular mechanisms by which vitamin B-12 and iron are transported across the outer membrane. The outer membrane transport proteins in E. coli are beta-barrel structures, which function to drive the accumulation of iron and vitamin B-12. They represent one of only a few classes of transport proteins for which accurate high-resolution structural models have been obtained.
The primary tools that we use in our work include EPR spectroscopy, high-resolution NMR and solid-state NMR. The application of EPR spectroscopy to membrane protein structure is relatively recent. Using EPR spectroscopy in combination with site-directed mutagenesis and spin-labeling, we are probing the structures and conformational transitions in large membrane proteins that would normally be inaccessible using other structural methods.