Molecular contacts in the transmembrane c-subunit oligomer of F-ATPases identified by tryptophan substitution mutagenesis
| Autor: | Lucy R. Forrest, Mark S.P. Sansom, Georg Groth, Claudia Schnick |
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| Rok vydání: | 2000 |
| Předmět: |
Models
Molecular Molecular model Protein subunit Immunoblotting Biophysics Biochemistry Oligomer Fluorescence chemistry.chemical_compound Adenosine Triphosphate F1F0 ATP synthase Escherichia coli Side chain Computer Simulation Binding Sites ATP synthase biology Cell Membrane Tryptophan Subunit c Cell Biology Transmembrane protein Culture Media Oligomer structure Proton-Translocating ATPases Crystallography Transmembrane domain Monomer Amino Acid Substitution chemistry Mutation Mutagenesis Site-Directed biology.protein Protons Plasmids |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1459:49-60 |
| ISSN: | 0005-2728 |
| DOI: | 10.1016/s0005-2728(00)00112-2 |
| Popis: | When isolated in its monomeric form, subunit c of the proton transporting ATP synthase of Escherichia coli was shown to fold in a hairpin-like structure consisting of two hydrophobic membrane spanning helices and a short connecting hydrophilic loop. In the plasma membrane of Escherichia coli , however, about 9–12 c -subunit monomers form an oligomeric complex that functions in transmembrane proton conduction and in energy transduction to the catalytic F 1 domain. The arrangement of the monomers and the molecular architecture of the complex were studied by tryptophan scanning mutagenesis and restrained MD simulations. Residues 12–24 of the N-terminal transmembrane segment of subunit c were individually substituted by the large and moderately hydrophobic tryptophan side chain. Effects on the activity of the mutant proteins were studied in selective growth experiments and various ATP synthase specific activity assays. The results identify potential intersubunit contacts and structurally non-distorted, accessible residues in the c -oligomer and add constraints to the arrangement of monomers in the oligomeric complex. Results from our mutagenesis experiments were interpreted in structural models of the c -oligomer that have been obtained by restrained MD simulations. Different stoichiometries and monomer orientations were applied in these calculations. A cylindrical complex consisting of 10 monomers that are arranged in two concentric rings with the N-terminal helices of the monomers located at the periphery shows the best match with the experimental data. |
| Databáze: | OpenAIRE |
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