Drugs of abuse act through binding to membrane receptors, neurotransmitter transporters, and ligand-gated ion channels such as the 5-HT3-receptor. To gain mechanistic understanding and expand therapeutic options, structural information is essential, but membrane proteins have been difficult to obtain in the crystalline state needed for detailed study by x-ray or electron crystallography. We propose a novel approach to identify membrane proteins that readily form two-dimensional (2D) crystals using Xenopus oocytes as expression host. A single Xenopus oocyte can produce many times the amount of protein needed to solve its structure to near atomic resolution by electron crystallography, yet at the same time is remarkably deficient in endogenous membrane proteins. Equally important, most eukaryotic membrane proteins can be expressed in oocytes, and in some cases such expression leads to spontaneous 2D-crystallization in vivo. Proteins that thus show propensity to form 2D-crystals are highly promising targets for structural studies. This feasibility study aims, at developing new approaches to find 2D-crystals that form in vivo or to encourage in situ crystallization in cases where crystallization does not occur spontaneously.
Aim 1 : Develop an assay to detect spontaneous formation of 2D-crystals in vivo. The extent to which spontaneous clustering of membrane proteins correlates with spontaneous formation of 2D-crystals in vivo has never been investigated. Since clustering of fluorescently labeled membrane proteins can easily be detected by fluorescence microscopy, we will determine whether fluorescence labeling and screening for punctuate staining can identify membrane proteins that readily form 2D-crystals. To achieve this goal, we will use fluorescently labeled """"""""positive controls"""""""" (connexin32 and 50) and """"""""unknowns"""""""" such as the 5-HT3-receptor.
Aim 2 : Develop a novel method to induce in situ 2D-crystallization of membrane proteins that do no crystallize spontaneously. The low background of endogenous membrane proteins in oocyte membranes may allow any recombinant membrane protein to be crystallized in situ by removing excess lipid. Traditionally, detergents are used for this purpose. However, the action of detergents is difficult to control it only small amounts of membranes are available. Therefore, we will test whether lipid-binding proteins can replace detergents in delipidation protocols. This approach may make a large number of membrane proteins amenable to 2D-crystallization and structure determination.
| Aller, Stephen G; Eng, Edward T; De Feo, Christopher J et al. (2004) Eukaryotic CTR copper uptake transporters require two faces of the third transmembrane domain for helix packing, oligomerization, and function. J Biol Chem 279:53435-41 |
