Membrane protein targeting is achieved by the signal recognition particle (SRP) and its receptor (SR). Currently, we do not know how these factors work at the molecular level to discriminate signal sequences from other hydrophobic sequences (for instance from mitochondrial targeting sequences) and how they are regulated to ensure timely delivery of the translating ribosome at the translocation machinery SecYEG in the membrane.
In order to better understand the fidelity and timing of protein targeting by the ribosome, SRP, SR and SecYEG, we will combine biophysical and biochemical methods with high-resolution structure solution by cryo-electron microscopy (cryo-EM). We aim to solve the atomic structures of important complexes involved in targeting and thus shed light on discrete conformational states along the targeting pathway.
This project combines state-of-the-art biochemistry/biophysics, protein engineering and cutting edge cryo-EM and single particle analysis for the study of co-translational targeting of membrane proteins. We have engineered tailor-made nascent chain constructs and established an efficient in vitro transcription-translation system for the large scale production of translating ribosomes . We will produce stable, homogenous ribosomal complexes for cryo-EM and single particle analysis [2,3,4]. Computational analysis of conformational flexibility in the sample will be instrumental to obtain structures of high resolution which we can interpret at atomic level . We will apply fluorescence-resonance energy transfer experiments and mutational analyses to corroborate structure-based findings.
By taking the interdisciplinary approach proposed here, we expect to obtain a detailed mechanistic understanding of co-translational targeting, its fidelity and timing. This process is conserved in all kingdoms of life. It is crucial to analyse membrane protein targeting as membrane proteins are at the core of many diseases and their treatment.
1. Schaffitzel, C., Oswald, M., Berger, I., Ishikawa, T., Abrahams, J.P., Koerten, H.K., Koning, R.I. & Ban, N. (2006) Structure of the E. coli signal recognition particle bound to a translating ribosome. Nature 444, 503-506.
2. Estrozi, L.F., Boehringer, D., Shan, S.O., Ban, N. & Schaffitzel, C. (2011) Structure of the E. coli Co-translational Targeting Complex in the Stable Early Conformation. Nat. Struct. Mol. Biol. 18, 88-90.
3. von Loeffelholz, O., Knoops, K., Ariosa, A., Zhang, X., Karuppasamy, M., Huard, K., Schoehn, G., Berger, I., Shan, S.O. & Schaffitzel, C. (2013) Structural Basis of Signal Sequence Surveillance and Selection by the SRP-SR Complex. Nat. Struct. Mol. Biol. 20, 604-610.
4. Von Loeffelholz, O., Jiang, Q., Airosa, A., Karuppasamy, M., Huard, K., Berger, I., Shan, SO & Schaffitzel, C. (2015) The ribosome-SRP-FtsY co-translational targeting complex in the closed state. Proc. Natl. Acad. Sci. U.S.A. 112, 3943-3948.