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|Title:||Studies on the Structure and Photochemistry of Bacteriorhodopsin (Proton Pumping, Cation Binding, Synthetic Pigments)|
|Department / Program:||Physiology and Biophysics|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Cation Binding By Bacteriorhodopsin (bR). bR normally has a purple color ((lamda)(,max) = 558 nm), hence the name, purple membrane. Atomic absorption spectrophotometry shows that purple membrane binds approximately 4 Ca('++) and Mg('++) per bR at pH = 6.0. Ca('++) and Mg('++) can be removed by various methods from their binding sites. After removal bR turns blue ((lamda)(,max) = 603 nm, the blue membrane).
Blue membrane has its own photocycle, having a blue-shifted intermediate with a maximum at 500-510 nm in the millisecond time scale. When Ca('++) and Mg('++) are replaced by La('+++), the kinetics of the M intermediate and proton pumping are significantly changed, drastically so at high pH.
Ca('++) and Mg('++) also play an important role in pigment regeneration. In the absence of Ca('++) and Mg('++) the rate of pigment regeneration becomes slower and 430/460 nm regeneration intermediate is absent; moreover, isomeric specificity is altered so that in addition to all-trans and 13-cis isomers, 11-cis retinal could also form a pigment.
Divalent cations may bind to the membrane through surface potential or via a specific conformation of coordination ligands like those found in the binding of Ca('++) to calmodulin. Temperature experiments of purple membrane and elemental analysis of bleached membrane show that in addition to the surface potential, a specific conformation of the protein is required for the divalent cation binding.
Synthetic Pigments. Acyclic retinal analogues and analogues varying in polyene chain length are used to study the mechanism of pigment formation and the role of the cyclohexyl ring and polyene chain in the function of bR. The results indicate that (1) unlike rhodopsin, there is no cyclohexyl ring binding site in bacterioopsin. (2) in order to form a pigment, the chromophore must contain at least three ethylene bonds in the polyene chain. (3) if polyene chain contains four or five ethylenic bonds, a pigment is formed which photocycles and pumps protons. (4) the cyclohexyl ring has no role important in the physiological functions of bR.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1985.
|Date Available in IDEALS:||2014-12-16|
This item appears in the following Collection(s)
Dissertations - Biophysics and Computational Biology
Graduate Dissertations and Theses at Illinois
Graduate Theses and Dissertations at Illinois