Mutagenesis and Functional Reconstitution of Chlamydial Major Outer Membrane Proteins: VS4 Domains Are Not Required for Pore Formation but Modify Channel Function
| Autor: | Richard H. Ashley, K. M. Shaw, E. S. Hughes |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Lipid Bilayers
Molecular Sequence Data Immunology Porins Chlamydiae Chlamydia trachomatis urologic and male genital diseases Microbiology Ion Channels Epitope Amino Acid Sequence Cloning Molecular Ion channel biology Electric Conductivity biology.organism_classification Molecular Pathogenesis Fusion protein female genital diseases and pregnancy complications Recombinant Proteins General bacterial porin family Protein Structure Tertiary Cell biology Infectious Diseases Biochemistry Membrane protein Mutagenesis Porin bacteria Parasitology Bacterial outer membrane Bacterial Outer Membrane Proteins |
| Zdroj: | Infection and Immunity. 69:1671-1678 |
| ISSN: | 1098-5522 0019-9567 |
| DOI: | 10.1128/iai.69.3.1671-1678.2001 |
| Popis: | Chlamidial organisms are obligate intracellular pathogens containing highly antigenic porin-like major outer membrane proteins (MOMPs). MOMP epitopes are of substantial medical interest, and they cluster within four relatively short variable (VS) domains. If MOMPs adopt a β-barrel fold, like bacterial porins, the VS domains may form extramembranous loops and the conserved regions of the protein may correspond to predicted membrane-located β-strands. However, molecular studies on native MOMPs have been hampered by the need to culture chlamydiae in eukaryotic host cells and purification and reconstitution remain problematic. In addition, the organisms are difficult to manipulate genetically, and it has also been difficult to functionally reconstitute recombinant MOMPs. To help overcome these problems and improve our understanding of MOMP structure and function, we cloned and expressedC. trachomatisandC. psittaciMOMPs and functionally reconstituted them at the single-channel level. We measured significant functional differences between the two proteins, and by removing and exchanging VS4, we tested the hypothesis that the largest variable domain forms an extramembranous loop that contributes to these differences. Proteins in which VS4 was deleted continued to form functional ion channels, consistent with the idea that the domain forms an extramembranous protein loop and incompatible with models in which it contributes to predicted membrane-located β-strands. Additionally, the properties of the chimeric proteins strongly suggested that the VS4 domain interacts closely with other regions of the protein to form the channel entrance or vestibule. Our approach can be used to probe structure-function relationships in chlamydial MOMPs and may have implications for the generation of effective antichlamydial vaccines. |
| Databáze: | OpenAIRE |
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