This proposal seeks to initiate efforts at The Johns Hopkins University to carry out high resolution structural investigations of membrane proteins with the acquisition of a CM 120 Philips electron microscope system. The CM 120 is a high quality transmission electron microscope which is suitable for studies of biological specimens at atomic and near-atomic resolution. Our goal is to use both the electron diffraction and imaging capabilities of the CM 120 microscope to understand the structural basis for the function of a variety of membrane proteins. Membrane proteins play a central role in executing key cellular functions such as energy conversion, signal transduction and ion transport. Yet, the structures of only seven membrane proteins are known at atomic resolution, in contrast to the nearly one thousand structures known for water-soluble proteins. Recent developments in high resolution electron microscopy have the potential to alter this situation dramatically. Of the seven membrane protein structures known at atomic resolution, two have been solved recently by applying new techniques in high resolution microscopy to two-dimensional, rather than three-dimensional protein crystals. We anticipate that our use of the CM 120 microscope for structural studies will occur in two phases. In the first phase, we will initiate work on the following five projects, four of which represent direct extensions of experiments already being carried out in collaborative efforts with external electron microscopy centers: Structural "snapshots" of steps in energy conversion by bacteriorhodopsin, a light-driven proton pump (S. Subramaniam, with Dr. R. Henderson at MRC, Cambridge, England) Structural basis of water transport by aquaporin, the water channel in red blood cells (P. Agre, with Dr. A. Engel at the Biozentrum, Basel, Switzerland) Structural basis of the association of myosin-I with the cell membrane (T. Pollard, with Dr. R. Milliga n at the Scripps Research Institute, La Jolla, CA) Structure of the voltage-gated potassium channel from Drosophila (M. Li, with Dr. N. Unwin at MRC, Cambridge, England) Structure determination of the IP3 receptor, a ligand-gated calcium channel (W. Agnew and S. Subramaniam) From these initial efforts, our goal is to lay the foundation for an institutional resource to carry out high resolution electron microscopy. A number of biologically important membrane receptors including a variety of sensory transducers and ion channels are currently being studied in laboratories at Johns Hopkins University. We fully expect that in the second phase of research facilitated by the the microscope, the structures of these receptors, as well as those of a variety of other cellular components can be successfully analyzed.
