Integral membrane receptor proteins often play a key role in signal transduction across cell membranes in both bacteria and higher organisms. Detailed knowledge of the three-dimensional structures of these receptor proteins would undoubtedly contribute greatly to a general understanding of the molecular mechanisms of signal transduction, but formidable technical challenges prohibit the routine determination of high resolution structures for most membrane proteins at present. Therefore, computer modeling studies are proposed to generate detailed three-dimensional models for a bacterial membrane chemoreceptor, the Trg receptor from Escherichia coli, and a number of mammalian seven helix G protein-coupled receptors, the adrenergic neurotransmitter receptors. Several specific issues of adrenergic receptor structure/function will be addressed utilizing primarily interactive molecular graphics model building techniques and existing experimental data, including: (1) What detailed packing arrangement of the seven helix bundles in adrenergic receptors is most consistent with available ligand binding and structural information for these receptors, while also yielding sensible physicochemical property profiles? (2) Can a single, general three-dimensional adrenergic receptor model that fulfills the conditions imposed on question 1 above explain the overlap, and differences, of ligand binding selectivities for various receptor subtypes? The models generated in this project will be used to design affinity labeling ligands, in collaboration with Dr. Peter Jeffs at Glaxo, that will covalently bind alpha- and beta-adrenergic receptors. These covalent adducts will be characterized using mass spectrometric techniques to provide new information about the structure and composition of the ligand binding site. Finally, a refined three-dimensional model for the beta2 adrenergic receptor will be used to screen the Fine Chemicals data base for novel (i.e. non-adrenergic) compounds that may possess reasonable binding affinities. Three-dimensional data base search methods will be employed, and promising target compounds will be tested in a beta2 receptor ligand binding assay in collaboration with Prof. Raymond Stevens at UC-Berkeley. A second set of modeling studies will be undertaken in collaboration with Prof. Gerald Hazelbauer at Washington State University to generate detailed three-dimensional models for the bacterial Trg chemoreceptor. Utilizing data from sulfhydryl accessibility, crosslinking, and spin labeling studies performed by Prof. Hazelbauer's group for 52 single cysteine mutants of the Trg receptor, structures for the transmembrane and periplasmic domains of the receptor will be constructed, and models for the conformational changes associated with signal transduction will be explored. The model structures will be explored. The model structures will be continually evaluated and refined using new experimental data from Prof. Hazelbauer's laboratory. The combination of detailed model building and close collaboration with experimental groups should yield useful new information about structure and signal transduction mechanisms for two distinct classes of membrane receptor proteins. Information obtained for the adrenergic receptors may also be of use in design of pharmacologic agents targeted to these receptors.
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