Book of Abstracts: Albany 2005
NMR Structural Study of Wild Type and Mutant U1A Proteins
The RNA recognition motif (RRM) is one of the most common motifs found in RNA binding proteins. RRM-containing proteins are involved in transport and storage of RNA and play crucial roles in regulating gene expression. The RRM is 80 amino acids long with a βαββαβ topology. Two RNA binding regions on the surface of the β-sheet, RNP1 and RNP2, contain highly conserved amino acids that contact RNA.
The U1A protein is a good model system for investigating RRM-RNA recognition due its high affinity and specificity to stem loop 2 of U1 snRNA. The U1A protein binds RNA primarily through the N-terminal domain that contains one RRM. The U1A protein is part of the U1 small nuclear ribonucleoprotein particle, which is involved in RNA splicing. Structures of the free and bound wild type U1A, determined by NMR and X-ray crystallography have been reported.
Previously we showed that mutation of a highly conserved phenylalanine in RNP1 to alanine (F56A) results in a loss in stability of the complex of about 5 kcal/mol. We are using NMR methods to investigate the structure of U1A proteins (wild type and F56A mutant) in the free and bound state in order to identify the possible causes of the binding energy loss upon mutation. We have compared the overall backbone folding of the free and bound U1A wild type and F56A mutant. Ongoing NMR studies that focus on solving the NMR structure of the F56A mutant in the free and bound state will be presented.
Alina G. Britchi*
Department of Chemistry