Dynamic Aspects of Nucleic Acid-Protein Recognition

描述(由申请人提供)：我们建议通过利用溶液和固态核磁共振波谱在广泛的时间范围内探测动态过程来研究蛋白质和核酸的动力学如何对分子识别做出贡献。我们的目标是将这两种互补的实验方法相互结合，并开发新的计算方法，通过描述发生运动的势能面，定量了解蛋白质和核酸动力学在分子识别中所起的作用。通过我们的联合方法，将有可能涵盖生物分子相互结合时出现的广泛的时间尺度和运动特征，从而全面描述结合所涉及的动力学。 我们将研究天然DNA和两个具有突出生物学意义的蛋白质-RNA复合体，在这些复合体中，运动已被证明发挥着重要的功能作用。我们已经定义了关于运动的作用的问题和假设，以认识到我们准备通过我们提出的实验计划进行测试。 我们的目标是通过使用我们以前研究中开发的共同实验方法来专注于两个特定的目标来实现这一建议。具体地说，我们的目标是使用溶液核磁共振和固体核磁共振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固体核磁共振研究的继续，现在我们在其中增加了分辨率核磁共振弛豫技术。作为特定的目标2，我们将比较两个不同的RRM蛋白质识别RNA的例子。在一种情况下，运动在结合时被抑制，以允许非常高的特异性由人U1A；在另一种情况下，相反，在CstF-结合时产生运动，以允许建立弥漫性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.