The chaperone function of Nucleosome Assembly Protein 1

描述（由申请人提供）：核小体组装蛋白1（Nap 1）是一种组蛋白伴侣蛋白，通过与核心组蛋白和接头组蛋白H1相互作用参与组蛋白调控，以及参与核小体形成和基因调控。Nap 1被认为通过在体外和体内阻止非核小体组蛋白-DNA相互作用来组装核小体[4]。Nap 1是由两个48 kDa多肽组成的同源二聚体，其具有出现在圆顶形结构下侧的无序N-和C-末端尾[10]。初步数据显示，酸性C-末端尾是解决非核小体组蛋白-DNA相互作用所必需的。通过在体外的亲和力测量，与电泳迁移率变化测定（EMSA）配对，我们已经确定，C-末端尾是野生型Nap 1功能所需的。我们还确定它有助于屏蔽抑制性N-末端尾。目前还不清楚这种机制是否适用于其他Nap 1家族成员，如人类Nap 1（hNap 1）和SET，这将在这里进行研究。已经表明Nap 1主要是H2 A/H2 B组蛋白伴侣[4]。由于Nap 1主要与H2 A/H2 B一起发挥作用，因此我们的研究重点是确定其机制。使用氢-氘交换耦合质谱（H/DX-MS），我们的实验室最近表明，H2 A/H2 B快速采样部分未折叠状态（S。D'Arcy，submitted）.当与酵母Nap 1（yNap 1）结合时，H2 A/H2 B然后稳定在折叠构象（S. D'Arcy，submitted）.目前还不清楚DNA是否也会像yNap 1一样稳定H2 A/H2 B的折叠构象。同样地，yNap 1是否也稳定连接组蛋白H1的折叠状态？通过使用H/DX，我们将能够回答这些问题。由于H/DX只是一种中等分辨率的技术，我们只能收集三级结构的信息。为了获得更高的分辨率，将使用X射线晶体学。它将提供大量的分子相互作用的细节，可以提高我们的知识，组蛋白伴侣在核小体动力学的作用。

DESCRIPTION (provided by applicant): Nucleosome assembly protein 1 (Nap1) is a histone chaperone implicated in histone regulation through interactions with core histones and linker histone H1, as well as in nucleosome formation and gene regulation. Nap1 is thought to assemble nucleosomes through the prevention of non-nucleosomal histone-DNA interactions in vitro and in vivo [4]. Nap1 is a homo-dimer composed of two 48 kDa polypeptides with disordered N- and C-terminal tails that emerge at the underside of the dome-shaped structure [10]. Preliminary data shows the acidic C-terminal tail is essential for resolving non-nucleosomal histone-DNA interactions. Through affinity measurements in vitro, paired with Electrophoretic Mobility Shift Assays (EMSA), we have determined that the C-terminal tail is required for wild type Nap1 function. We also determined it aids in shielding the inhibitory N-terminal tail. It is unknown whether this mechanism applies to other Nap1-family members such as human Nap1 (hNap1) and SET, and this will be investigated here. It has been shown that Nap1 is primarily a H2A/H2B histone chaperone [4]. Since Nap1 primarily functions with H2A/H2B, our research has focused on determining the mechanism. Using hydrogen-deuterium exchange coupled to Mass Spectrometry (H/DX-MS), our laboratory has recently shown that H2A/H2B rapidly samples a partially unfolded state (S. D'Arcy, submitted). When bound to yeast Nap1 (yNap1), H2A/H2B is then stabilized in a folded conformation (S. D'Arcy, submitted). It is unknown whether DNA will also stabilize H2A/H2B in a folded conformation like yNap1 does. Likewise, does yNap1 also stabilize the folded state of linker histone H1? Through the use of H/DX we will be able to answer these questions. Since H/DX is only a medium resolution technique, we can only gather information on tertiary structure. To obtain greater resolution, x-ray crystallography will used. It will provide great detail of the molecular interactions that can enhance our knowledge of the role that histone chaperones have in nucleosome dynamics.