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J Mol Biol. 2012 May 25;419(1-2):89-101. doi: 10.1016/j.jmb.2012.02.043. Epub 2012 Mar 3.

Engineered oligomerization state of OmpF protein through computational design decouples oligomer dissociation from unfolding.

Author information

1
Department of Bioengineering, University of Illinois at Chicago, Chicago, IL 60607, USA.

Abstract

Biogenesis of β-barrel membrane proteins is a complex, multistep, and as yet incompletely characterized process. The bacterial porin family is perhaps the best-studied protein family among β-barrel membrane proteins that allows diffusion of small solutes across the bacterial outer membrane. In this study, we have identified residues that contribute significantly to the protein-protein interaction (PPI) interface between the chains of outer membrane protein F (OmpF), a trimeric porin, using an empirical energy function in conjunction with an evolutionary analysis. By replacing these residues through site-directed mutagenesis either with energetically favorable residues or substitutions that do not occur in natural bacterial outer membrane proteins, we succeeded in engineering OmpF mutants with dimeric and monomeric oligomerization states instead of a trimeric oligomerization state. Moreover, our results suggest that the oligomerization of OmpF proceeds through a series of interactions involving two distinct regions of the extensive PPI interface: two monomers interact to form a dimer through the PPI interface near G19. This dimer then interacts with another monomer through the PPI interface near G135 to form a trimer. We have found that perturbing the PPI interface near G19 results in the formation of the monomeric OmpF only. Thermal denaturation of the designed dimeric OmpF mutant suggests that oligomer dissociation can be separated from the process of protein unfolding. Furthermore, the conserved site near G57 and G59 is important for the PPI interface and might provide the essential scaffold for PPIs.

PMID:
22391420
PMCID:
PMC3772668
DOI:
10.1016/j.jmb.2012.02.043
[Indexed for MEDLINE]
Free PMC Article

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