Date of Award:

5-1-1988

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biology

Department name when degree awarded

Biology

Committee Chair(s)

Jon Y. Takemoto

Committee

Jon Y. Takemoto

Committee

Andy Anderson

Committee

Reed P. Warren

Abstract

The purpose of this work was to determine the membrane topography of the B875 light-harvesting complex of wild-type Rhodobacter sphaeroides and to determine if any precursors to the B875 polypeptides exist. To investigate the membrane topography of the B875 complex, I purified the B875 light-harvesting complex and membrane vesicles (chromatophores, and spheroplast-derived vesicles) from the intracellular photosynthetic membrane (ICM) of wild-type R. sphaeroides and treated each with proteinase K or trypsin. The B875-⍺ and -β polypeptides were analyzed by electrophoretic, and immunochemical methods. Proteinase K digested both polypeptides in the purified complex and completely eliminated the A875 spectral peak, characteristic of the B875 complex. Trypsin digested the α polypeptide and reduced the A875 peak by 50%. Proteinase K cleaved the β polypeptide of the chromatophores and the α polypeptide of spheroplast-derived vesicles. I conclude that the β polypeptide is exposed on the cytoplasmic surface of the ICM. The difference in the digestion patterns between the spheroplast-derived vesicles and chromatophores suggests that the α polypeptide is exposed on the periplasmic surface. De novo synthesis of the B875 complex polypeptides occurs when the oxygen concentration of a culture is lowered. This easily controllable environmental factor makes R. sphaeroides an excellent model system to study the assembly and insertion of integral membrane proteins in developing membranes. The possibility that mature B875 polypeptides could be formed from precursor polypeptides was investigated by shifting R. sphaeroides from respiratory to photosynthetic energy production by lowering the oxygen concentration of growth cultures. Two proteins with molecular weights of 37,000 and 20,000 were present in high-oxygen cells and reacted specifically with antibodies against the B875 polypeptides. These proteins were treated with subtilisin to produce peptide maps. Subtilisin digested the 37,000 dalton polypeptide to form fragments which migrated to the same gel positions as the B875-α and -β polypeptides. Subtilisin did not digest the 20,000 dalton polypeptide, although a hydrolysis fragment of it was formed which migrated to the same gel position as the α polypeptide. The results suggest that the 37,000 and 20,000 dalton polypeptides are precursors to the B875 polypeptides.

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