Abstract
The topology of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides has been examined by generating gene fusions with alkaline phosphatase. Gene fusions were generated at random locations within the fbcB gene encoding the cytochrome b subunit. These fusion products were expressed in Escherichia coli and were screened for alkaline phosphatase activity on chromogenic plates. 33 in-frame fusions which showed activity were further characterized. The fusion junctions of all those fusions which had a high specific activity were clustered in three regions of the cytochrome b polypeptide, and thus these regions were tentatively assigned as being near the periplasmic surface. The data are consistent with a model containing eight transmembrane helices. In order to explore the validity of the gene fusion approach for a protein not normally expressed in E. coli, the topology of the L-subunit of the photosynthetic reaction center from R. sphaeroides was also explored using phoA gene fusions. A similar protocol was used as with the cytochrome b subunit. The gene fusions with high specific activity were shown to be in regions of the L-subunit polypeptide known to be at or near the periplasmic surface, as defined by the high resolution structure determined by X-ray crystallography. These data demonstrate the utility of this approach for determining membrane protein topology and extend potential applications to include at least some proteins not normally expressed in E. coli.
Highlights
The topology of the cytochrome b subunit of the bcl in-depth understanding of their structure
In order to explore the validity of the gene fusion and Argos [14] pointed out that the fourth putative memapproach for a protein not normally expressed in E. coli, the topology of the L-subunit of the photosynthetic reaction center fromR . sphaeroides was explored using phoA gene fusions
The gene fusions models with only eight membrane-spanning helices in which with high specificactivity were shown to be in regionhselix IV is removedfrom themembrane.Thiseight-span of the L-subunit polypeptide known to be a t or near model has been remarkably successfulin mapping all the the periplasmic surface, as defined by the high resolu- mutationsresulting in resistance at the Q Z site to oneside of the membrane, and those affect
Summary
Materials-All restriction endonucleases andnucleic acid modifying enzymes were obtained from Bethesda Research Laboratories, New England BioLabs, BoehringerMannheim,andInternational Biotechnologies, Inc. Bacteria and Plasmids-Bacterial strains and plasmidussed in this fragment of pUI310. Fusion LP150 was prepared by ligating a PstI/ BstYI (Klenow filled-in) fragment of pUI704 with pUI310 treated with XbaI and Klenow, followed by PstI. Fusion LP260 was constructed by ligating a PstIISalI fragment of pUI705 with PstIISalIcut pUI320. Plasmids were maintained in the presence of ampicillin (50pg/ml)ortetracycline(15pg/ml). DhO A sphaeroides strain Gawas grown aerobically in Sistrom’s minimal medium [33] by vigorous shaking. Strains containing pRK415 derivatives were supplemented with 1pg/ml tetracycline. Screening forAlkaline Phosphatase Actiuity-Colonies with alkaline phosphatase activity were selected on LB (Luria broth) plates pBC I. Recombinant DNA Procedures-Restriction enzyme digestions, ligation, electrophoresis, and plasmid transformationws ere carried out as described by Maniatis et al [34]
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