Gene Fusion Analysis of Positive Charge-Induced Segment Re-orientation in the Tetracycline Resistance Protein
Abstract
The pBR322 tetracycline resistance protein (Tet) was used as a model system for analysis of control of transmembrane segment orientation in membrane proteins. Two types of Tet fusions were used to test the effects of positively-charged aminoacids and tertiary interactions within the protein on segment orientation. The influence of tertiary interactions was examined using fusions where maltose binding protein (MBP) was linked to the N-terminus of the full-length Tet protein. Local charge control was examined using truncated Tet-alkaline phosphatase (PhoA) fusions where C-terminal segments were deleted from Tet. Three lysine residues were introduced into the third periplasmic domain of Tet to reverse the positive charge balance across its fifth transmembrane segment. The introduction of lysine residues into the intact Tet domain of a MBP-Tet fusion abolished tetracycline resistance of the fusion and made the fusion unstable to proteolysis both in vivo and in vitro. While these data suggest the structure of the Tet domain was altered, rearrangement of segments could not be confirmed directly by proteolysis due to the instability of the protein. A comparison of the alkaline phosphatase activities of wild-type and mutant Tet PhoA fusions indicated that positively-charged residues are sufficient to cause segment inversion in the absence of part of the Tet domain.
Document Details
- Document Type
- Technical Report
- Publication Date
- Feb 01, 1993
- Accession Number
- AD1011287
Entities
People
- Farrell D. Mcafee
Organizations
- Uniformed Services University of the Health Sciences