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。了解蛋白质的原子结构和生物功能之间的关系需要能够以精确的方式扰动蛋白质结构。化学合成有利于多种侧链和肽主链修饰的结合，从而能够精确修饰蛋白质的结构和电子特性。使用常规诱变不可能进行类似的修饰，这使得化学合成成为蛋白质结构和功能研究的重要资产。蛋白质的大小一直是限制使用化学合成研究蛋白质的主要因素。在膜蛋白领域，迄今为止仅完成了相对较小（< 150 个氨基酸）蛋白质的化学合成。感兴趣的蛋白质（例如电压门控离子通道）要大得多，目前不适合化学合成。为了克服这一限制，我们建议开发可用于化学合成大型（> 150 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。我们将追求三个主要具体目标。目标 1) 开发电压门控 K 通道 KvAP 和非选择性通道 NaK 的化学合成方法。我们为此目的开发的化学合成方案不仅可用于研究这些特定蛋白质，而且还可用于其他重要类别膜蛋白的化学合成。目标 2) 我们将研究 KvAP 通道中的缓慢失活过程。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着改变可用通道的数量，从而改变细胞的电特性。目标 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 通道显示出与环核苷酸门控离子通道的序列和功能相似性。因此，我们在 NaK 通道研究中发现的机制将有助于理解二价离子与 CNG 通道的生理上重要的相互作用。公共健康相关性：离子通道功能是许多重要生物过程的基础，例如神经和肌肉细胞的兴奋、激素的分泌和感觉转导。这项研究意义重大，因为它将提供对二价离子缓慢失活和阻断的重要生理过程的更深入理解。此外，这项研究很重要，因为它将化学合成确立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.