STRUCTURE AND FUNCTION OF BACTERIORHODOPSIN

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The structure of bacteriorhodopsin, a 27,000 dalton membrane protein from purple membrane of Halobacterium halobium is to be defined by high resolution electron diffraction of membranes, and of membranes with heavy metal groups covalently attached. Building on our previous studies, we can now determine the site of attachment of such labels in sequence and structure. Thus a primary objective is to determine the order and orientation in which the seven transmembrane sequences occurs in the structure. We have previously determined the phases of electron diffraction amplitudes to 3.7 Angstroms experimentally (i.e., from images), and recorded diffraction to 2.65 Angstroms at -120 C and this is crucial to difference mapping. We have defined an extended resolution 7x10 Angstrom 3-D structure that shows additional features helpful in building a model of the protein structure: Having defined the retinal attachment site in sequence, we will identify side chains involved in the mechanism of proton pumping using site-specific mutagenesized proteins, and using mutants under study in a closely complementary proposal of H. Boyer at UCSF. The site-specific changes will be based on structure, and both structural change and functional variance will be assessed to define a mechanism of light-energy conversion, insertion into the membrane, membrane protein stability, the role of prolines, the role of peptides between helices in the aqueous phase, and with the end goal of understanding the mechanisms in an archetype of transmembrane protein-mediated phenomena. Increasing similarities to rhodopsin in the eye is emerging. An overall goal is to understand membrane mediated phenomena, and this protein is focussed on as an archetype likely to yield much fundamental insight sooner than others.