![]() ![]() Water is the most abundant molecule inside living cells. These two binding sites function independently ( 4). In particular, the 5’-UGC bases wrap around a single phenylalanine side-chain. Specifically, the UG-3’ is bound canonically by the β-sheet while the 5’-UGCA interacts with residues of the β1/α1 protein loop ( 4). In the case of the Fox-1 RRM, the RNA binding mode is mixed. However, the RRM is extremely versatile and RNA binding patterns utilizing other parts of the domain (such as the α-helices, the protein loops or the unstructured chain termini) have been observed ( 7– 10). The exposed surface of the β-sheet is the most common (‘canonical’) RRM binding site for RNA. It typically assumes a β 1α 1β 2β 3α 2β 4 topology with the two α-helices packed against the four-stranded antiparallel β-sheet surface ( 6). The RRM is the most common RNA binding motif observed in proteins. At the molecular level, the Fox-1 protein functions by recognizing a 5΄-UGCAUG-3΄ sequence of the target mRNA through a single RNA recognition motif (RRM) domain which is present and highly conserved in all the tissue-specific variants of the Fox-1 protein ( 4, 5). The Fox-1 gene was first identified in the Caenorhabditis elegans as a sex-determining element ( 3) and numerous homologs have subsequently been observed in many different organisms ( 2), including humans ( 4). ![]() Their exact effects are tissue-specific and their proper function is essential for a developing cell ( 2). The Fox-1 family is a group of proteins that regulate alternative splicing ( 1). Hydration is not easily detectable in NMR experiments but can affect stability of protein/RNA complexes. In conclusion, MD is an effective tool for predicting and interpreting the hydration patterns of protein/RNA complexes. The S155 hydration site is evolutionarily conserved within the RRM domains. A quantitative agreement between theory and experiment is achieved for the S155A substitution but not for the S122A mutant. Both hydration sites are experimentally confirmed and their abolishment reduces the binding free-energy. We characterize two of them using NMR spectroscopy, RNA binding with switchSENSE and free-energy calculations of mutant proteins. MD predicts intricate hydration sites with water-binding times extending up to hundreds of nanoseconds. Simulations of the protein/RNA complex show hydration consistent with the isolated protein complemented by hydration sites specific to the protein/RNA interface. The individual monomers of the X-ray structure show diverse hydration patterns, however, MD excellently reproduces the most occupied hydration sites. We use this and the nuclear magnetic resonance (NMR) structure of the Fox-1 protein/RNA complex for molecular dynamics (MD) analyses of the structured hydration. We report a 1.8 Å X-ray structure of the free Fox-1 containing six distinct monomers. The Fox-1 RNA recognition motif (RRM) domain is an important member of the RRM protein family. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |