Identification of functional similarities between proteins using directed evolution.
Protein sequences are often highly redundant and evolution can change them beyond recognition. It can therefore be difficult to identify proteins with functional or structural similarities by inspection of their sequences. Here we have used an experimental evolutionary approach to detect hidden similarities between the antisense RNA-binding protein Rop and other proteins. We created an envelope of functional Rop mutants by combinatorial mutagenesis, used the compilation of mutant sequences to search a database of protein structures, and thereby identified a segment of the enzyme valyl-tRNA-synthetase (ValRS). Further inspection revealed that the structures of the RNA-binding sites of both proteins are highly related, as indeed are the RNA ligands. From the known 3D structure of the ValRS in complex with tRNA, we were able to build a model of an RNA hairpin pair in complex with Rop that has proved to be consistent with the biochemical and NMR data for the interaction between Rop and RNA hairpins. We suggest that this approach (mutational envelope scanning), by generating sequence information de novo, can help uncover hidden similarities between proteins.
Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom. duc@mrc-lmb.cam.ac.uk
Daniel
D
Christ
Greg
G
Winter
D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D019992:Databases as Topic; D004926:Escherichia coli / Q000201:enzymology; D015723:Gene Library; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D008958:Models, Molecular; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D016372:RNA, Antisense / Q000737:chemistry; D012343:RNA, Transfer / Q000378:metabolism; D013997:Time Factors; D014637:Valine-tRNA Ligase / Q000737:chemistry
D000596:Amino Acids; D011506:Proteins; D016372:RNA, Antisense; D012343:RNA, Transfer; D014637:Valine-tRNA Ligase