The broad objective of this study is to combine methods of structural biology with genetic methods of directed evolution to further the understanding of protein mediated molecular recognition. The elements of a protein structure necessary to discriminate between two related ligands will be determined by evolving a protein specific for one ligand into a variant with specific affinity for the second ligand. The protein mediated discrimination between the nucleotide guanosine (G) and its alkylated derivative N 7 -methylguanosine (m 7 G) shall be examined through this strategy. These nucleotides are nearly identical in their shape and hydrogen bonding patterns; however, they differ dramatically in their electronic structure. Methylation of the N 7 nitrogen introduces a positive charge that is delocalized throughout the heterocyclic p-ring system of the guanosine base. Eukaryotic cells utilize this alkylated derivative of guanosine as a tag for the 5' end of messenger RNAs suggesting that this moiety provides a unique recognition element. Four model protein systems are under consideration; two cap-binding proteins and two G-binding proteins. These protein systems have been chosen in order to facilitate the subsequent determination of the three-dimensional structures of any variants generated. Two methods of directed protein evolution, phage display coupled with affinity chromatography and bacterial display coupled with fluorescence-activated cell sorting, will be assessed for their ability to generate proteins with contrary ligand specificities within the same protein context. Both the primary and three-dimensional structures of the variant protein are then determined and compared to those of the wild-type protein. Comparing the structures of two ligand-binding sites with different specificities in the same protein context will clearly delineate the essential features of the binding site which are necessary for discrimination between the two related ligands. The general aim of this work is to provide a foundation for the design of macromolecules with high specificity. This approach will reveal whether different, but related, ligand specificities can be evolved into a single protein scaffold. By evolving multiple protein models to recognize the same ligands, the compatibility of different protein scaffolds in accommodating designed ligand specificities will be determined. Two different methods of directed protein evolution, phage display and bacterial display, will be compared for their utility in the design of specific ligand binding sites. Also, a general method for defining the structural elements essential to ligand discrimination will be tested. Finally, insight into the mechanism by which a protein may discriminate between an uncharged heterocylic base (guanosine) and a charged, alkylated base (7-methylguanosine) will be gained. The study of guanosine and 7-methylguanosine discrimination may elucidate the unique properties of m 7 G recognition that led to its use as a 5' mRNA tag by eukaryotic cells.
2. Non-technical
With the compartmentalization of genetic material within a nuclear envelope, eukaryotic cells have by necessity evolved unique mechanisms for chaperoning the transfer of genetic information from the nucleus to the cytoplasm. The genetic instructions encoded in messenger RNA must be carried by protein 'couriers' from the information center of the nucleus to the workshop of the cytoplasm. To facilitate this transfer of genetic information, the messenger RNA of eukaryotes is tagged at both ends to provide 'handles' for the protein couriers to recognize. At the mRNA 5' end, 7-methylguanosine (m 7 G) is attached to the transcript forming a cap structure. A number of proteins can specifically discriminate between the relatively rare m 7 G cap structure that identifies the 5'-end of the RNA and the common unmethylated guanosine (G) nucleotide that is used throughout the RNA message. In this study, the structural basis for the recognition of the 5' m 7 G cap will be elucidated by utilizing 'directed evolution' methods. Through recombinant methods, this study is designed to direct the evolution of a mRNA cap binding protein into a protein that recognizes unmethylated guanosine. In turn, a protein that specifically recognizes unmethylated guanosine will be evolved into a cap-binding protein. These new 'evolved' proteins will be compared to their parent versions to determine what elements of the parent structure were important in the discrimination between the mRNA cap and unmodified guanosine.
