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Title:Cation effects on the purple membrane of Halobacterium halobium
Author(s):Jonas, Roy Edward
Doctoral Committee Chair(s):Ebrey, Thomas G.
Department / Program:Biophysics and Computational Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biophysics, General
Abstract:This thesis is a report of cation effects on the purple membrane of Halobacterium halobium. Included are proton and divalent cation studies using purple, blue, and pink membranes and the apoprotein. The blue species of bacteriorhodopsin is produced from purple membrane at low pH or by removal of divalent cations. Studies contrasting purple and blue membranes have been quite useful for investigating the environment of retinal and the proton-pumping mechanism. I have also obtained interesting results from pink membrane, a stable photoproduct of blue membrane in red light.
Experiments concerning changes involved in the light-induced formation of blue and purple membranes from pink and Ca$\sp{2+}$-pink membranes, respectively, are discussed. Fourier transform infrared spectroscopy and pH studies demonstrate in part that structural and/or protonation changes taking place during these transformations may involve four specific carboxylic amino acids which seem to be important in the bacteriorhodopsin photocycle. Pink membrane is also found to contain exclusively 9-cis retinal, an isomer not normally found in purple membrane. These results suggest a significant modification of the chromophore environment and bacteriorhodopsin active site is able to take place in deionized membrane.
Initial evidence from atomic absorption, chemical modifications, pH titrations, and theoretical calculations showed that 5-6 divalent cations are physically bound to purple membrane by carboxylic amino acids. Cooperativity between binding of retinal and a portion of the divalent cations was also suggested by results such as reduced cation binding to the apomembrane and irreversible inhibition of chromophore regeneration using deionized-bleached membrane.
My more recent research shows that the binding of one divalent cation is directly related to the blue-to-purple transition of bacteriorhodopsin; whereas, four other divalent cation binding sites involving carboxyl groups on the bacteriorhodopsin surface do not directly affect the color change. The intrinsic pK$\sb{\rm a}$ of the binding of the special divalent cation and of the color transition is at about pH 2. The events involve the exchange of one divalent cation for two protons; I suggest that the two groups which release a proton upon binding of the divalent cation are aspartates-85 and -212 in the bacteriorhodopsin active site.
Issue Date:1991
Rights Information:Copyright 1991 Jonas, Roy Edward
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9210852
OCLC Identifier:(UMI)AAI9210852

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