Dynamic Aspects of Nucleic Acid-Protein Recognition

描述（由申请人提供）：我们建议研究蛋白质和核酸的动力学如何通过溶液和固态NMR光谱在宽范围的时间尺度上探测动态过程来促进分子识别。我们的目标是将这两种互补的实验方法相互整合，并利用新的计算方法，通过提供运动发生的势能表面的描述，来定量了解蛋白质和核酸动力学在分子识别中的作用。通过我们的联合方法，将有可能涵盖生物分子相互结合时发生的广泛的时间尺度和运动特征，从而提供对结合中所涉及的动力学的全面描述。 我们将研究天然DNA和两个蛋白质-RNA复合物具有突出的生物学重要性，其中运动已被证明发挥重要的功能作用。我们已经定义了关于运动作用的问题和假设，并准备通过我们提出的实验方案进行测试。 我们的目标是通过使用我们以前研究中开发的常见实验方法专注于2个特定目标来实现这一建议。具体来说，我们的目标是使用溶液NMR和固态NMR来研究运动如何影响DNA甲基化。这个特定的目标基本上是早期的固态NMR研究的延续，我们现在添加分辨率NMR弛豫技术。作为具体目标2，我们将比较RRM蛋白识别RNA的两个不同实例。在一种情况下，运动在结合时被淬灭，以允许通过人U1 A的非常高的特异性;在另一种情况下，运动反而在通过CstF-64结合时产生，以允许建立扩散特异性和瞬时相互作用。

DESCRIPTION (provided by applicant): We propose to study how dynamics of proteins and nucleic acids contribute to molecular recognition by probing dynamic processes over a wide range of time scales by means of solution and solid state NMR spectroscopy. We aim to integrate these two complementary experimental approaches with each other and exploit new computational methods to gain a quantitative understanding of the role that protein and nucleic acid dynamics play in molecular recognition by providing a description of the potential energy surfaces upon which motion occurs. Through our joint approach, it will be possible to cover the wide range of time scale and motional characteristics that occurs in biological molecules as they bind each other, and therefore provide a comprehensive description of the dynamics involved in binding. We will study native DNA and two protein-RNA complexes with outstanding biological importance where motion has been shown to play an essential functional role. We have defined questions and hypotheses concerning the role of motion in recognition that we are poised to test through the experimental program we propose. We aim to fulfill this proposal by focusing on 2 specific aims using a common experimental approach developed in our previous studies. Specifically, we aim to use solution NMR and solid state NMR to study how motion affects DNA methylation. This specific aim is essentially a continuation of earlier solid state NMR studies to which we now add resolution NMR relaxation techniques. As specific aim 2 we will compare two different examples of RNA recognition by RRM proteins. In one case, motion is quenched upon binding to allow very high specificity by human U1A; in the other case, motion is instead generated upon binding by CstF-64 to allow diffuse specificity and transient interactions to be established.