Conformational changes and protein interactions underlie the function of many proteins, but are particularly critical for transmembrane proteins, such as ion channels and G-protein coupled receptors, which transfer signals across membranes. Understanding these changes at a structural level is essential for a rational development of future therapeutics that target these proteins. It is proposed to purchase an instrument capable of measuring such conformational changes and of precisely measuring proximity of proteins. The instrument takes advantage of the unique properties of the luminescent lanthanide elements, including Tb3+ and Eu3+. The long, ms, lifetime decays of the luminescence of these elements can be measured with high precision and therefore permits high-precision measurements of resonance energy transfer to nearby acceptor chromophores (LRET). The lifetimes will be measured from proteins expressed in Xenopus oocytes with lanthanide-chelates specifically attached to them. The oocyte expression system provides flexibility in the proteins that can be examined and in the placement of the labels on accessible, cysteine-substituted amino acids. The instrument is commercially available as two components. The first component comprises the optics for excitation of the lanthanides, a microscope for focusing the excitation on the oocyte, and emission optics, which collect time-dependent luminescence decays. The decays will be analyzed using fitting software provided with the instrument. The second component provides electrophysiological voltage clamp of the oocyte and permits measurement of currents through ion channels. Thereby, we obtain a direct correlation of functional changes with the LRET measurements of distance changes. A detailed correlation of structural and functional changes will provide a substantially higher level of understanding of membrane protein function. Many of the proteins of interest in this application are therapeutic targets. This work will improve our ability to target drugs to particular protein conformations and thereby deliver highly specific drug action.
