Infrared spectroscopic folding studies of outer membrane proteins

Abstract

The Omp85/BamA family is a class of β-barrel proteins from the bacterial outer membrane that are thought to act as insertases for other outer membrane proteins (Omps). In this study, we expressed and purified two Omps with typical β-barrel structure: TtOmp85 and its outstandingly thermostable and denaturant-resistant substrate TtoA from the thermophilic bacterium Thermus thermophilus. Conductance studies in black lipid membranes indicate that TtoA is inserted via β-strand augmentation into the membrane. This process includes the formation of a large hybrid barrel. In order to shed further light on this mechanism, we have conducted FT-IR studies. After covalently attaching a silane linker to a silicon ATR (Attenuated Total Reflection) crystal we modified its head group by addition of Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate. Via the resulting Ni2+-binding molecule we were able to successfully immobilize unfolded, His-tagged TtoA. The unfolded protein was then exposed to TtOmp85 for a limited time. After removal of TtOmp85 an increase in β-sheet structure could be observed for TtoA. The results support that interaction with TtOmp85 induces folding of TtoA.