Extending Chemical Synthesis to Ion Channel Proteins

将化学合成扩展到离子通道蛋白

• 批准号: 8055543
• 负责人: Francis Valiyaveetil
• 金额: $ 27.17万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8055543
• 关键词: Amino Acids; Binding; Binding Sites; Biological Process; Cell secretion; Cells; Electronics; Gated Ion Channel; Goals; Health; Hormones; Investigation; Ion Channel; Ion Channel Protein; Ions; Measurement; Membrane Proteins; Methodology; Methods; Modification; Muscle Cells; Mutagenesis; Neurons; Peptides; Potassium Channel; Process; Property; Proteins; Protocols documentation; Research; Sensory; Side; Site-Directed Mutagenesis; Structure; Vertebral column; Vestibule; Voltage-Gated Potassium Channel; chemical synthesis; cyclic-nucleotide gated ion channels; electrical property; interest; peptide chemical synthesis; protein structure; protein structure function; tool; voltage; Amino Acids; Binding; Binding Sites; Biological Process; Cell secretion; Cells; Electronics; Gated Ion Channel; Goals; Health; Hormones; Investigation; Ion Channel; Ion Channel Protein; Ions; Measurement; Membrane Proteins; Methodology; Methods; Modification; Muscle Cells; Mutagenesis; Neurons; Peptides; Potassium Channel; Process; Property; Proteins; Protocols documentation; Research; Sensory; Side; Site-Directed Mutagenesis; Structure; Vertebral column; Vestibule; Voltage-Gated Potassium Channel; chemical synthesis; cyclic-nucleotide gated ion channels; electrical property; interest; peptide chemical synthesis; protein structure; protein structure function; tool; voltage

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.

描述（由申请人提供）：拟议研究的远距离目标是制定离子通道蛋白化学合成的策略，并使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与生物学功能之间的关系需要精确的方式扰动蛋白质结构。化学合成促进了多种侧链和肽主链修饰的融合，从而可以对蛋白质的结构和电子特性进行精确修饰。使用常规诱变使化学合成成为研究蛋白质结构和功能研究中的重要资产，因此无法进行类似的修饰。蛋白质的大小一直是限制化学合成研究蛋白质的主要因素。在膜蛋白领域，到目前为止，仅针对相对较小的（<150个氨基酸）蛋白才能完成化学合成。感兴趣的蛋白质（例如电压门控离子通道）大得多，目前不适合化学合成。为了克服这一限制，我们提出了开发方法，可用于大型（> 150个氨基酸）膜蛋白的化学合成，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的特定目标。目的1）开发电压门控k+通道KVAP的化学合成，以及非选择性通道Nak。我们在此目标中开发的化学合成方案不仅在研究这些特定蛋白质中很有用，而且还将发现适用于其他重要类别的膜蛋白的化学合成。目标2）我们将研究KVAP通道中缓慢的灭活过程。了解缓慢失活的过程很重要，因为进入灭活状态的通道的进入率（和退出）可以显着改变可用的通道数量，因此可以改变细胞的电气性能。目的3）我们将使用化学合成研究二价离子与NAK通道外侧前庭的结合。 NAK通道显示与环状核苷酸门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与理解二价离子与CNG通道的生理重要相互作用有关。公共卫生相关性：离子通道功能是许多重要的生物学过程的基础，例如神经和肌肉细胞的激发，激素的分泌以及感觉转导。提出的研究很重要，因为它将更深入地了解慢速失活的生理重要过程，并通过二价离子阻塞。此外，这项研究很重要，因为它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。