Biochemistry. 2016 Aug 9;55(31):4333-43. doi: 10.1021/acs.biochem.6b00691.

Direct Monitoring of β-Sheet Formation in the Outer Membrane Protein TtoA Assisted by TtOmp85.

Henke K, Welte W, Hauser K.
  • Department of Chemistry, ‡Department of Biology, and §Konstanz Research School Chemical Biology, University of Konstanz , 78457 Konstanz, Germany.

Abstract:

Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was applied to investigate the folding of an outer membrane protein, TtoA, assisted by TtOmp85, both from the thermophilic eubacterium Thermus thermophilus. To directly monitor the formation of β-sheet structure in TtoA and to analyze the function of TtOmp85, we immobilized unfolded TtoA on an ATR crystal. Interaction with TtOmp85 initiated TtoA folding as shown by time-dependent spectra recorded during the folding process. Our ATR-FTIR experiments prove that TtOmp85 possesses specific functionality to assist β-sheet formation of TtoA. We demonstrate the potential of this spectroscopic approach to study the interaction of outer membrane proteins in vitro and in a time-resolved manner.

PMID: 27400268

 

Supplement:

Outer membrane proteins (OMPs) play a fundamental role in various cellular functions, e.g. protection of a cell against environmental conditions, signal transduction and control of metabolite in- and efflux. The survival of gram-negative bacteria depends on the correct folding and insertion of OMPs into the outer membrane, and OMP dysfunction in mitochondria is linked to severe diseases. Thus understanding the process of OMP folding and membrane insertion is of fundamental interest in life sciences. In vitro studies and appropriate experimental approaches can reduce the complexity of a cell and facilitate the analysis of individual proteins. We designed a novel folding experiment using infrared (IR) spectroscopy and could demonstrate its unique sensitivity to monitor structure formation and interaction of OMPs.

It has been shown previously that OMPs which are kept unfolded in denaturants are able to spontaneously adopt β-barrel structure upon dilution of the denaturant and addition of liposomes or detergent. However, other studies also showed that the presence of another outer membrane protein is important in order to insert into a membrane. These proteins are called insertases and belong to the Omp85 family of outer membrane proteins in bacteria and eukaryotes. Omp85 insertases work with or without accessory proteins but share the common features of a 16-stranded transmembrane β-barrel and a central pore. Different hypotheses on the role of these insertases in OMP folding exist. These focus on the importance of the barrel, the pore and/or the interface between barrel and outer membrane. An example for an interacting pair of an Omp85 and a substrate are SAM50 and TOM40 in mitochondria (1). The TOM complex is a translocation machinery for mitochondrial proteins from the cytosol to the interior of the mitochondria and closely linked to neurodegenerative diseases such as Alzheimer’s and Parkinson’s Disease (2).

 

 

Fig. 1:Schematic representation of theinfrared (ATR-FTIR) spectroscopic approach to monitor folding mechanisms of the outer membrane proteins TtoA and TtOmp85. Unfolded TtoA (green) is immobilized on the surface of the ATR crystal via its His-tag and a covalently attached linker (left). Time-limited incubation with TtOmp85 (blue) results in folded TtoA (right). The folding reaction can be detected by time-dependent IR-spectra. Our experiments support the assumption that interaction of TtOmp85 and TtoA occurs via β-augmentation.

 

In order to shed light on the interaction mechanism of Omp85 insertases and their substrates we studied an Omp85/substrate pair that functions without any accessory proteins and is thus a suitable model system. This pair is TtOmp85 and its substrate TtoA from Thermus thermophilus. We hypothesized that TtoA folding and membrane insertion happen simultaneously and via β-augmentation with the TtOmp85-barrel (3). For our folding experiments we had to keep TtoA in high concentrations of SDS to prevent spontaneous folding. These experimental conditions inspired us to design an ATR-FTIR experiment in which we could monitor the interaction of two outer membrane proteins in a membrane-free environment (Figure 1). We attached unfolded, His-tagged TtoA to the ATR crystal via a silane-NTA linker that we synthesized directly on the crystal. The immobilized TtoA could now be exposed to different solutions. These solutions could be added and removed from the crystal without removing TtoA. This technique allowed us to add TtOmp85 for a restricted period of time and to monitor structural changes in TtoA via IR-spectroscopy. The folding experiments showed that, even under denaturing conditions and in absence of a membrane or stabilizing detergent, TtOmp85 was able to promote β-structure formation in TtoA (Figure 2). The outer membrane does not seem to play a vital role for the initiation of TtoA folding by TtOmp85. The interaction with TtOmp85 without a membrane environment is probably insufficient to fold TtoA completely into its native structure, but we could demonstrate that TtoA adopts a native-like b-structure. Our experiments are in accordance with the β-augmentation model.

 

Importance of the Study:

Outer membrane proteins (OMPs) are crucial for the survival of gram-negative bacteria. They are also essential for the function of eukaryotic organelles. Severe diseases are linked to OMP dysfunction. Acquiring a deeper understanding on the biosynthesis of OMPs and the control and regulation of their insertion into the outer membrane is therefore essential for our understanding of the survival and vitality of organisms. The infrared spectroscopic approach that we developed to conduct folding experiments on OMPs can potentially be applied in presence and absence of membranes and is furthermore suitable to analyze protein-protein interaction and protein function.

 

Fig. 2:TtOmp85-assisted folding of TtoA. The interaction with TtOmp85 induces conformational changes of TtoA from an unfolded structure (left, before incubation with TtOmp85) to a native-like b-sheet structure (right, after incubation with TtOmp85). Our ATR-FTIR experiments indicate that the assisted folding of TtoA is TtOmp85-specific.

 

References

  1. Kozjak V, Wiedemann N, Milenkovic D, Lohaus C, Meyer HE, Guiard B, Meisinger C, Pfanner N 2003 An essential role of Sam50 in the protein sorting and assembly machinery of the mitochondrial outer membrane. J Biol Chem. 278, 48520-48523
  2. Gottschalk WK, Lutz MW, He YT, Saunders AM, Burns DK, Roses AD, Chiba-Falek O 2014 The broad impact of TOM40 on neurodegenerative diseases in aging. Journal Parkinsons Dis Alzheimers Dis. 1:12
  3. Estrada Mallarino, L, Fan, E, Odermatt, M, Müller, M, Lin, M, Liang, J, Heinzelmann, M, Fritsche, F, Apell, HJ, Welte, W 2015 TtOmp85, a β-barrel assembly protein, functions by barrel augmentation. Biochemistry 54, 844-852

 

 

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