Correlation between surfactant/micelle structure and the stability of bacteriorhodopsin in solution. Manipulation of the photocycle of bacteriorhodopsin via systematic structural modification of site-specific protein-bound organic cations

Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)




Robert R. Birge


bleaching, bolaform, Chemistry, Biochemistry, Biophysics

Subject Categories



The process of solubilization/isothermal bleaching of bacteriorhodopsin (bR) in a series of alkylammonium surfactants is studied. The rate of bleaching increases initially with increasing surfactant concentration but decreases at higher concentrations. The kinetic data indicate penetration of the surfactant into the protein interior. The rate curves are a function of the micellar environment, the length of the surfactant tail, the size of the headgroup and the hydrophobicity of the headgroup. The micelle surface charge is relatively unimportaat. Isothermal bleaching of bR in micelle solution is biphasic where the initial rapid reaction is attributed to protein solubilization. A counterion that binds more strongly at the micelle surface provides a more stable solubilization environment. The stability of solubilized bR in salt solution can be rationalized based on a multi-equilibrium scheme for protein solubilization plus variation in the micelle structure. We conclude that the subsequent fall in the bleaching rate is due to the micelle structure getting more compact at high detergent concentrations. The kinetic data are consistent with a reaction scheme where the free and solubilized protein are in a pH-dependent dynamic equilibrium. We conclude that the hydrophobic portion of the micelle is most important in determining protein stability.

Bacteriorhodopsin is regenerated from the blue membrane with the addition of a series of mono and bis (quaternary ammonium) (bolaform) cations. The bolaform cations bind at specific locations within bacteriorhodopsin. Molecular orbital simulations of the chromophore-adjacent binding site but minimized for the bolaform cations indicate that the bolaform cations bind with greater affinity versus the monovalent ones due to a steric match mechanism which is operative only for the former. The M and O states are more stable for a bigger cation. Our data suggest that in the native protein, the calcium ion enters the proton channel to prevent back transfer of the proton. We conclude that the motion of the cation into a position of electrostatic stabilization of the $\rm ASP\sb{85}$ anion is required for the $\rm O\to bR$ reaction. Our results demonstrate the viability of manipulating the bR photocycle by selection of the appropriate organic cation.


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