Extending Chemical Synthesis to Ion Channel Proteins

描述（由申请人提供）：拟议研究的长期目标是开发离子通道蛋白的化学合成策略，并利用化学合成来研究离子通道功能的机制。要理解蛋白质的原子结构与生物功能之间的关系，就需要能够以精确的方式扰乱蛋白质结构。化学合成促进了各种侧链和肽主链修饰的结合，从而能够精确地修饰蛋白质的结构和电子性质。传统的诱变方法无法进行类似的修饰，这使得化学合成成为研究蛋白质结构和功能的重要工具。蛋白质的大小一直是限制使用化学合成来研究蛋白质的主要因素。在膜蛋白领域，化学合成迄今为止只完成了相对较小的（< 150个氨基酸）蛋白质。感兴趣的蛋白质，如电压门控离子通道要大得多，目前还不能进行化学合成。为了克服这一限制，我们提出了开发可用于化学合成大（bbb150个氨基酸）膜蛋白的方法，从而使我们能够使用化学合成来研究这些蛋白质。我们将努力实现三大目标。目的1)化学合成电压门控K+通道KvAP和非选择性通道NaK。我们在这个目标中开发的化学合成方案不仅对研究这些特定的蛋白质有用，而且也将在其他重要类别的膜蛋白的化学合成中找到适用性。目的2)我们将研究KvAP通道的缓慢失活过程。了解缓慢失活的过程很重要，因为通道进入（和退出）失活状态的速率可以显著改变可用通道的数量，从而改变细胞的电学性质。目的3)我们将利用化学合成研究二价离子与NaK通道外前庭的结合。NaK通道在序列和功能上与环核苷酸门控离子通道相似。因此，我们在NaK通道的研究中发现的机制将有助于理解二价离子与CNG通道之间重要的生理相互作用。公共卫生相关性：离子通道功能是许多重要生物过程的基础，如神经和肌肉细胞的兴奋、激素的分泌和感觉转导。这项研究具有重要意义，因为它将为二价离子缓慢失活和阻断的生理重要过程提供更深入的理解。此外，该研究将建立化学合成作为理解离子通道结构和功能的重要工具。

DESCRIPTION (provided by applicant): The long range goal of the proposed research is to develop strategies for the chemical synthesis of ion channel proteins and to use chemical synthesis to investigate the mechanisms of ion channel function. Understanding the relationship between the atomic structure of a protein and the biological function requires the ability to perturb the protein structure in a precise manner. Chemical synthesis facilitates the incorporation of a wide variety of side chain and peptide backbone modifications that enables precise modifications of the structural and electronic properties of the protein. Similar modifications are not possible using conventional mutagenesis making chemical synthesis an important asset in investigations of protein structure and function. The size of the protein has been a major factor limiting the use of chemical synthesis to investigate proteins. In the field of membrane proteins, chemical synthesis has so far been accomplished only for relatively small (< 150 amino acids) proteins. Proteins of interest such as voltage gated ion channels are much bigger and are presently not amenable to chemical synthesis. To overcome this limitation, we propose developing methods that can be used for the chemical synthesis of large (> 150 amino acid) membrane proteins thereby enabling us to use chemical synthesis to investigate these proteins. We will pursue three major specific aims. Aim 1) To develop a chemical synthesis of the voltage gated K+ channel KvAP, and the non-selective channel, NaK. The chemical synthesis protocols that we develop in this aim will be useful not only in investigating these specific proteins but will also find applicability in the chemical synthesis of other important classes of membrane proteins. Aim 2) We will investigate the slow inactivation process in the KvAP channel. Understanding the process of slow inactivation is important because the rate of entry (and exit) of channels into the inactivated state can significantly alter the number of channels available and therefore the electrical properties of the cell. Aim 3) We will investigate the binding of divalent ions to the outer vestibule of the NaK channel using chemical synthesis. The NaK channel shows sequence and functional similarity to cyclic nucleotide gated ion channels. Therefore, the mechanisms that we uncover in our investigations of the NaK channel will be relevant in understanding the physiologically important interactions of divalent ions with CNG channels. PUBLIC HEALTH RELEVANCE: Ion channel function underlies many important biological processes such as the excitation of nerve and muscle cells, the secretion of hormones, and sensory transduction. The research proposed is significant because it will provide a deeper understanding of the physiologically important processes of slow inactivation and block by divalent ions. Further, the research is important as it will establish chemical synthesis as an important tool for understanding ion channel structure and function.