Structural investigation of an immunoglobulin domain: biosynthesis on the ribosome and co-translational folding

Protein folding is the process by which a polypeptide acquires its correct threedimensional, biologically active function. During biosynthesis on the ribosome the elongating nascent chain emerges in a vectorial manner from the restricted
environment of the ribosomal exit tunnel and begins to explore conformational space. The structural understanding of the emerging nascent chain is sparse because its inherent dynamics is making it invisible to conventional structural and biophysical methods, including conventional NMR. Application of paramagnetic relaxation enhancement (PRE) NMR cannot only aid in structural characterization of the major populated state but can also aid in detecting and characterizing fast relaxing minor states which are inherent to co-translational folding.

The PRE method probes ensemble-averaged, transient contacts over distances as great as 15-24 Å. As previously described for isolated proteins, a nitroxide spin-label is attached to a region of a protein, and in its oxidized (paramagnetic) state, enhances relaxation of heteronuclear coherences. Relaxation enhancement scales as r-6 with label proximity, allowing the computation of distance restraints used to calculate ensembles of structures by MD simulations.

Using this approach, I will structurally investigate the inherently dynamic process of cotranslational folding using an immunoglobulin domain as my model protein and get a deeper understanding of this so far structurally invisible process.