Many proteins are located in cellular membranes. To function in such an environment, proteins must insert and assume their native state in the lipid bilayer. The insertion and folding processes are aided by molecular machinery in vivo. In vitro, however, some membrane proteins do not require folding factors; the molecular determinants of folding and assembly are encoded by their amino acid sequences. How a sequence encodes a structure that spontaneously folds and assembles in a membrane is not yet understood.
To define the principles that govern membrane protein folding and assembly, we have cloned and purified nine E. coli outer membrane proteins (OMPs): OmpX, OmpA, OmpW, the crcA gene product (PagP), OmpT, outer membrane phospholipase A (OmpLa), the fadl gene product (FadL), the yaet gene product (Omp85) and OmpF. These proteins have no sequence similarity but share a common β-barrel transmembrane motif. To determine how environmental factors influence folding, we measured the yield of folded OMPs in a large variety of conditions. Each experimental condition, however, yielded a different fraction of folded protein for each OMP. Therefore, intrinsic properties specific to each protein must also affect folding.
To determine the causes for the fraction folded variations observed in any single condition, we monitored the folding kinetics and tested the resistance to thermal denaturation of each OMP via SDS-PAGE. We also measured their propensities to aggregate by sedimentation velocity analytical ultracentrifugation and observed the effect of aggregation on folding. In each study, OMPs exhibited a wide variety of complex behaviors.
Apart from the folding of the β-barrel, several OMPs require self-association to become active. To determine the molecular interactions that drive protein self-association during assembly, we used sedimentation equilibrium analytical ultracentrifugation to directly measure the strength of the association between OmpF monomers. We discovered that this association is much stronger than that which was previously measured for OmpLa.
This broad scan of OMP folding revealed that the β-barrel transmembrane motif has a wide variety of folding and association abilities. Therefore, the sequence-structure relationship of this single fold is extraordinarily versatile.
|Advisor:||Fleming, Karen G.|
|School:||The Johns Hopkins University|
|School Location:||United States -- Maryland|
|Source:||DAI-B 70/05, Dissertation Abstracts International|
|Keywords:||Aggregation, Analytical centrifugation, Beta-barrel protein, Membrane protein self-association, Outer membrane proteins, Protein folding|
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