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Membrane assembly of the outer membrane protein OmpA of Escherichia coli
M Klose1, A Störiko, Y D Stierhof
1Max-Planck-Institut für Biologie, Tübingen, Federal Republic of Germany.
Abstract:
The membrane part (residues 1 to approximately 170) of the 325-residue Escherichia coli outer membrane protein OmpA is thought to exist in the membrane as an 8-stranded beta-barrel, subdividing this part into four segments. The influence of proline residues on membrane assembly of the protein has been studied. These were introduced, using site-directed mutagenesis, into each of seven of the antiparallel beta-strands. One important parameter for allowing or not allowing membrane assembly was the potential H beta (i) which is the potential to form an amphiphilic beta-strand. When H beta (i) remained unaltered, 2 prolines were tolerated. Lowering H beta (i) in most cases caused failure of assembly when 2 such residues were present. An insert of 10 residues, including 3 prolines, did not alter H beta(i) and was tolerated, but caused "looping out" of the strand to the outer face of the membrane; displacement to its inner side would not have allowed for an amphiphilic beta-strand. Thus, a beta-structured protein is as adaptable as it has been shown for an alpha-helix. The wild type segment order 1-2-3-4 has been changed to 1-3-3-4 and 1-4-3-4. Since the proteins were found associated with the outer membrane but could not be incorporated into it, it appears that sorting is less sensitive to alterations than assembly. A regulatory circuit was affected (missense mutants of outer membrane proteins can cause inhibition of synthesis of other such proteins); expression of the two rearranged genes effected a strong inhibition of synthesis of the unrelated porins OmpC and F as well as that of the maltoporin LamB and wild type OmpA. Hence, outer membrane proteins are designed not only for efficient membrane assembly but also for proper regulation of their synthesis.
Insights
Proline residues influence Escherichia coli outer membrane protein A (OmpA) assembly and regulation. Altering beta-strand amphipathicity or segment order affects membrane integration and synthesis of other outer membrane proteins.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- The Escherichia coli outer membrane protein A (OmpA) forms an 8-stranded beta-barrel structure within the bacterial outer membrane.
- Understanding the assembly and regulation of outer membrane proteins is crucial for bacterial physiology.
Purpose of the Study:
- To investigate the impact of proline residues on the membrane assembly of OmpA.
- To explore how alterations in beta-strand amphipathicity and segment order affect OmpA's integration into the outer membrane.
- To examine the regulatory consequences of modified OmpA assembly on the synthesis of other outer membrane proteins.
Main Methods:
- Site-directed mutagenesis was employed to introduce proline residues into specific beta-strands of OmpA.
- Analysis of beta-strand amphipathic potential (H beta (i)) was performed.
- OmpA variants with altered segment orders (1-3-3-4 and 1-4-3-4) were constructed.
- Protein association with the outer membrane and synthesis of other outer membrane proteins (OmpC, OmpF, LamB, wild type OmpA) were assessed.
Main Results:
- The amphipathic potential of beta-strands significantly influences OmpA membrane assembly; alterations can lead to assembly failure.
- Introduction of proline residues can be tolerated if amphipathicity is maintained, but may cause strand "looping out".
- Rearranged OmpA gene segments (1-3-3-4, 1-4-3-4) associated with the outer membrane but failed to integrate, indicating sorting is less sensitive than assembly.
- Expression of rearranged OmpA variants strongly inhibited the synthesis of unrelated porins (OmpC, F) and maltoporin (LamB).
Conclusions:
- OmpA's beta-barrel structure exhibits adaptability, similar to alpha-helical proteins, in response to proline incorporation.
- Membrane assembly is more sensitive to structural perturbations than protein sorting into the outer membrane.
- Outer membrane proteins possess inherent regulatory mechanisms controlling their synthesis, ensuring proper cellular function.