Theoretical and MD Simulation Studies of U1A-RNA Binding and Specificity

描述（由申请人提供）：该项目的目标是通过分子动力学（MD）模拟和相应的连续静电和自由能计算获得的动力学结构计算模型，利用几种方法作为交叉检查结果，阐明U1A蛋白茎环RNA结合的机制。U1A是一类大型RNA结合蛋白的原型，它利用由三明治折叠组成的特征结构基序，形成由两个螺旋支撑的四股反平行片，称为RNA识别基序（RRM）或RNA结合域（RBD）。U1A RNA络合过程似乎是一个多步骤的过程，其中突出的问题是结构适应机制（诱导契合或构象捕获），局部U1A - RNA接触与结合事件中的协同效应，溶剂释放和所涉及的熵的大小，以及各种化学力对结合亲和力的贡献。将计算得到的U1A和RNA在溶液中的动态结构与络合状态下的动态结构进行比较，研究结构适应效应。参与RNA结合的蛋白质残基的合作网络将基于计算的原子波动的相互关系来研究，这些相互关系通过空间域相关性揭示。动力学结构和功能能量之间的联系将通过加法自由能分量计算来建立，自由能、焓和熵分解为各种氨基酸、核糖核苷酸碱基的贡献，以及由于溶质和溶剂化而分解为静电、范德华斥力和分散项。拟议的研究将首先关注U1A RNA和一组蛋白质突变体以及对观察到的亲和性和特异性至关重要的区域的RNA碱基替换，并在后期扩展到一系列相关复合物，其中初始结构确定和观察到的结合亲和性和特异性表明，这对于充分了解RRM/RBD RNA复合物的形成至关重要。本研究与卫斯理大学化学系和分子生物物理训练项目正在进行的基于生物有机和生物物理化学的并行实验、时间分辨荧光各向异性和核磁共振U1A RNA及相关系统的结构测定密切相关。

DESCRIPTION (provided by applicant): The objective of the proposed project is to elucidate the mechanism of U1A protein stem loop RNA binding based on computational models of dynamical structures obtained from molecular dynamics (MD) simulation and corresponding continuum electrostatics and free energy calculations using several methods as a cross check on results. U1A is a prototype of a large class of RNA binding proteins which utilize a characteristic structural motif comprised of an sandwich fold that forms a four-stranded antiparallel -sheet supported by two -helices known as the RNA recognition motif (RRM) or RNA binding domain (RBD). The U1A RNA complexation process appears to be multistep affair in which the outstanding problems are the mechanism of structural adaptation (induced fit or conformational capture), local U1A - RNA contacts vs. cooperative effects in the binding event, solvent release and magnitude of the entropy involved, and the contribution of various chemical forces to binding affinity. Structural adaptation effects will be studied by comparison of the calculated dynamical structures for U1A and RNA free in solution with those of the complexed state. The cooperative networks of protein residues involved in RNA binding will be investigated based on calculated cross correlations of atomic fluctuations from the MD, which reveal through space domain correlations. The link between dynamical structure and functional energetics will be established using an additive free energy component calculation of free energy, enthalpy and entropy resolved into contributions from various amino acids, ribonucleotide bases and into electrostatic, van der Waals repulsions and dispersion terms due to solute and solvation. The proposed research will focus initially on U1A RNA and a set of protein mutants and RNA base replacements in regions critical to observed affinity and specificity and in a later phase be extended to a series of related complexes for which initial structural determination and observed binding affinities and specificities indicate to be critical for a full understanding of RRM/RBD RNA complex formation. The proposed research is closely correlated with concurrent experiment based bio-organic and biophysical chemistry, time resolved fluorescence anisotropy and NMR structure determination on U1A RNA and related systems currently in progress in the Chemistry Department and Molecular Biophysics Training Program at Wesleyan University.