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Abstract:
Repetition of building blocks, from single amino acids to entire domains, is a central mechanism in the emergence of new proteins and is likely to have played a critical role in the emergence of the first folded proteins during the transition from the RNA-peptide to the DNA-protein world. In order to explore the ability of polypeptides that are structured on an RNA scaffold to achieve autonomous folding, independently of the scaffold, we studied the potential of ribosomal protein fragments to form coiled-coils. For this, we used the ribosomal proteins of Thermus thermophilus as a source of such fragments and chose six that showed an elevated coiled-coil propensity in computational predictions. We took two approaches in parallel: we inserted the fragments between GCN4 N16V adaptors in order to establish their basic compatibility with coiled-coil structure, and we amplified them by repetition in order to explore whether they could yield autonomously folded forms. GCN4 N16V is a leucine zipper that can equally form dimers and trimers, thus not obscuring the oligomeric preference of the insert. Fragments inserted between such adaptors yielded highly stable oligomeric species with a high content of α-helical structure, mostly trimeric, but, in the case of a fragment from the L29 protein, dimeric. These results showed that the fragments we chose were fundamentally compatible with the coiled-coil fold. Concurrently, we investigated their ability to fold autonomously upon repetition. Of the six fragments, two (from L10 and L29) were able to fold autonomously upon duplication. In agreement with our previous results, the duplicated L10 fragment formed trimers in solution and the duplicated L29 fragment dimers. Since the native L29 protein forms a monomeric, aniparallel helical hairpin with local knobs-into-holes packing in the context of the ribosome, the ability of an L29 fragment to form parallel dimers is maybe not as surprising as the ability of the L10 fragment to form a parallel trimer, substantially different from its structure in the ribosome. These results show that there is indeed cryptic potential to form new structures in ribosomal proteins.
