VCU Bioinformatics and Bioengineering Summer Institute
Virginia Commonwealth University
Research Proposal

    Crystallographic Analysis of U1A RNA Binding Protein Interactions with U1 snRNA
    Introduction

    As our knowledge of protein-protein and protein-DNA interactions has been greatly increased with the availability of a large number of cocrystal structures, protein-RNA interactions remain hard to predict even for interactions in which only a single amino acid mutation has been introduced. With protein-protein and protein-DNA interactions, the large amount of incoming structural data is beneficial to measuring the accuracy of the current structural prediction tools, yet tools that use protein-RNA interactions to predict structure still require more development for active structural prediction. Understanding protein-RNA interactions is essential to predicting structures for large complex molecules that contain both protein-protein interactions and protein-RNA interactions. This study focuses on a mutation (Phe56Ala) of a particular protein-RNA interaction between the N-terminal RNP domain of the U1A RNA binding protein and stem loop 2 of U1 snRNA, which is just one of the many interactions occurring within a larger molecule the spliceosome. This mutation results in a five fold decrease in binding affinity for the interaction, which is a significant change due to a single amino acid mutation. By comparing the mutant structural and thermodynamic data with that of the wild type, we can develop predictions that will help to predict structural changes in protein-RNA complexes. Of course, the result of this study alone will not solve all protein-RNA structures, but the valuable in depth study of this particular interaction (rather than attempting to extract patterns from a database of information) will help to answer one of the most important structural biology questions to date: namely how can the tertiary and quaternary structure of a protein be predicted from primary structure? Answering this question could provide invaluable information in nearly every scientific field, especially comparative genomics and rational drug design.

    Methods

    Our goal is to take thermodynamic data for both the wild type and mutant protein-RNA complexes and account for the changes between the two by comparing their structures. Most of the thermodynamic data collection has already been completed and the wild type cocrystal has already been characterized (for structural analysis). In order to complete the picture, we must develop a free protein crystal as well as a complexed cocrystal for structural analysis via X-ray crystallography. Once the structure has been completed, the HINT (Hydropathic INTeraction) program, developed by Dr. Kellogg of Virginia Commonwealth University, will be applied to the structural data in an attempt to correlate structural changes with changes in the interactions between molecules, which can be extracted through thermodynamic analysis. The structural and thermodynamic data from the wild type and Phe56Ala mutant will be compared, and the thermodynamic interactions caused by the "induced fit" of the two molecules will be analyzed between the free and complexed forms of the mutant. By attributing certain changes in structure with certain changes observed in the interaction between the protein and RNA, we will be able to develop methods within the HINT program that can predict structural changes for any protein-RNA interaction.

    Predicted Results

    This project attempts to take pieces of known relationships (structural and thermodynamic) and relate the two to form a coherent method of predicting three dimensional structures. Since this method of analysis is still being developed, the accuracy of the predictions will be unknown until applied to multiple structures. Since the method of crystal development will be the same as was used for the wild type protein-RNA complex, the attainment of a high resolution crystal is not expected to be a problem. The main focus of the project will most likely fall upon the role of structural water molecules, which are important in a number of intermolecular forces that have not been fully characterized thermodynamically. Water that is buried within the protein-RNA complex obviously affects the hydropathic interactions near that region. Sometimes changes in conformation can trap water molecules within a hydrophobic region, significantly destabilizing the region. Water can also participate by forming a "bridge" of stability between two residues with hydrogen bonds. The accurate description of structural changes based upon the thermodynamic interactions of water molecules is the key area of development that must be described in order to accurately predict three dimensional structures. Since the modeling of protein-RNA interactions is an ongoing process, the time to complete the project is highly variable, although we think significant headway will be made describing the interactions between the N-terminal RNP domain of the U1A RNA binding protein and stem loop 2 of U1 snRNA in the 15 month period spanning the BBSI (Bioinformatics and Bioengineering Summer Institute) time period. Regardless of the success of the proposed model, the results obtained from this study will help forward the progress of obtaining tertiary and quaternary structure from the amino acid sequence. Only by solving and characterizing more protein-RNA interactions will scientists be able to make comparative predictions for similar interactions in the future. The basis of rational drug design relies most upon the ability to make these predictions.

    References:

    Kellogg, Glen and Donald Abraham. "Hydrophobicity: is LogPo/w more than the sum of its parts?" Eur. J. Med. Chem. 35 (2000) 651-661.
    Blackaj, Dukagjin et. al. "Molecular Dynamics and Thermodynamics of Protein-RNA Interactions: Mutation of a Conserved Aromatic Residue Modifies Stacking Interactions and Structural Adaptation in the U1A-Stem Loop 2 RNA Complex." J. Am. Chem. Soc. 2001, 123, 2548-2551.
    Oubridge, Chris et. al. "Crystallisation of RNA-Protein Complexes II. The Application of Protein Engineering for Crystallisation of the U1A Protein-RNA Complex." J. Mol. Biol. (1995) 249, 409-423.
    Tang, Yun and Lennart Nilsson. "Molecular Dynamics Simulations of the Complex between Human U1A Protein and Hairpin II of U1 Small Nuclear RNA and of Free RNA in Solution." Biophysical Journal, 77 (1999), 1284-1305.
    This project is funded by grants received from both the National Science Foundation (NSF) and the National Institutes of Health (NIH) awarded to the BBSI program at Virginia Commonwealth University.