Heterogeneity of the Bc1 Complex Subunits and Qo Site Occupants From Rhodobacter Sphaeroides
Luna-Chavez, Cesar
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https://hdl.handle.net/2142/85453
Description
Title
Heterogeneity of the Bc1 Complex Subunits and Qo Site Occupants From Rhodobacter Sphaeroides
Author(s)
Luna-Chavez, Cesar
Issue Date
2006
Doctoral Committee Chair(s)
Gennis, Robert B.
Department of Study
Biophysics and Computational Biology
Discipline
Biophysics and Computational Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biophysics, Medical
Language
eng
Abstract
For decades there has only been anecdotal data regarding how difficult it is to crystallize membrane proteins but these facts are never rigorously evaluated, because virtually no one can report all the reasons why membrane proteins do not crystallize easily. Trials to crystallize bc1 complex from R. sphaeroides, a membrane-bound four-subunit respiratory protein that shares physical and catalytic properties with the vertebrate bc1 complex, have been undertaken and results help understand the problems of heterogeneity and solutions that can remedy the situation. The over-expressed four-subunit enzyme is extracted from the membrane fraction using dodecyl-maltoside in a heterogeneous form. Separation and quantitation of bc1 complex and its subunits was carried out by a combination of chromatography and ultracentrifugation to achieve a nearly homogeneous preparation of the complex and its subunits. bc1 complex is in equimolar stoichiometry of its four subunits and all redox cofactors expected to be associated with the enzyme. A heme-based extinction coefficient by the pyridine hemochrome method has been determined for the reduced-oxidized difference spectrum. Crystal leads of bc1 complex have been grown by vapor diffusion within weeks using sodium citrate buffer, PEG precipitant and additives. A new aerobic system for the oxidoreductase activity of bc1 complex in the presence of bacterial succinate-ubiquinone reductase and appropriate quinones was developed to characterize the best samples for crystallization based on enzymatic activity. The presence of all cofactors in bc1 complex is required for proper function and is required to be of equimolar stoichiometry to ensure homogeneity---a critical requirement for characterization and crystallization. One cofactor not decisively determined to be single or double occupant at the Qo site of bc1 complex is a lipid soluble quinone. To address this issue, reintroduction of ubiquinone analogs into the Qo site was utilized to attempt reloading any absent quinones and ultimately experimental conditions were narrowed down to reach satisfactory homogeneous preparations and find unexpectedly more quinones. When inhibitors were added, there was not as much displacement of quinones in contrast to other studies with bc1 complex from different organisms. EPR spectral changes associated with occupancy at the Qo site suggest double occupancy.
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