The Facility for Atomic Mutagenesis

原子诱变设施

基本信息

批准号：
10063065
负责人：
Christopher A Ahern
金额：
$ 24.79万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-12-01 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10063065
关键词：
Amino Acids Biophysics Caliber Cells Chemicals Chemistry Chicago Collaborations Communities Core Facility Custom DNA Data Ensure Environment Generations Goals Human Industrialization Infrastructure Integral Membrane Protein Ion Channel Iowa Journals Laboratories Letters Medicine Membrane Membrane Proteins Methods Mission Molecular Molecular Conformation Mutagenesis National Institute of Neurological Disorders and Stroke Neurosciences Online Systems Optics Peer Review Piper Potassium Channel Production Property Proteins Protocols documentation Publishing Reagent Records Reporting Research Research Personnel Resolution Resources Science Services Shipping Side Site Sodium Channel Standardization Structure Technical Expertise Techniques Technology Training Transfer RNA United States National Institutes of Health Universities Virginia Vision Work base college cost design experience experimental study improved interest member nervous system disorder next generation novel novel therapeutics outreach services professor protein structure response success tool unnatural amino acids voltage wiki

项目摘要

Ongoing advances in the elucidation of protein structures are leading to the production of large volumes of high-resolution data, in increasingly native environments. However, especially in the case of transmembrane proteins, experimental options for high-resolution functional analyses remain limited, and thus the usefulness of the structures in understanding human neurological diseases likewise remains limited. Moreover, the structures that are obtained represent single, static snapshots of proteins, although these likely have multiple conformations of functional significance. Thus, there is a growing need among the ion channel and membrane biophysics community, which is supported by the NINDS, for reagents that directly report on the functionality and dynamics of membrane proteins in live cells. An elegant solution to these problems is nonsense suppression, a method that makes it possible to encode any type of synthetic amino acid at a site of interest within a protein. These so-called unnatural amino acids can take the form of residues with single atom substitutions, or side-chains with novel fluorescent properties. Although multiple experimental avenues for the encoding of unnatural amino acids exist, each is associated with significant technical challenges. As such, this powerful approach remains inaccessible to most investigators studying molecular neuroscience. In 2014, in response to numerous inquiries by other investigators, the Ahern lab at the University of Iowa (UI) set out to simplify the dissemination of acylated orthogonal tRNAs, key components in nonsense suppression, for experiments involving eukaryotic membrane proteins. To this end, we made improvements to the underlying chemistry, making it more robust and allowing for the encoding of a more chemically diverse set of amino acids. In addition, these new reagents display vastly improved stability profiles thus allowing for easy shipping to laboratories throughout the U.S. With our reagents and guidance, a number of new user groups have successfully applied this previously difficult approach to a variety of membrane proteins relevant to the NINDS mission. Overall, these efforts have produced a high-functioning collaborative outreach service, “The Facility for Atomic Mutagenesis.” This resource will provide broad access to custom reagents for nonsense suppression, and this facility is able to quickly adapt or design synthetic approaches to meet the interests of an application by new users. As the technologies become more standardized and our user group expands, we will scale accordingly, taking advantage of infrastructure present with the UI Carver College of Medicine and local industrial partners such as Integrated DNA Technologies. These collaborations will ultimately support more efficient dissemination of these research tools, to answer diverse questions in molecular neuroscience. Their use will be buoyed by a growing user base, annual training seminars, web-based forums and published protocols in open-access peer-reviewed journals. Our Advisory Board – Kossiakoff, Perozo, Koide, Nakamoto – will ensure efficient stewardship of key resources and the alignment of our strategic vision to NINDS.

阐明蛋白质结构的持续进展导致产生大量高分辨率数据，在越来越多的天然环境中。但是，特别是在跨膜的情况下蛋白质，高分辨率功能分析的实验选择仍然有限，因此有用性理解人类神经疾病的结构同样仍然有限。而且，获得的结构代表蛋白质的单个，静态快照，尽管这些结构可能具有多个功能意义的构象。那就是离子通道和膜之间的需求越来越大由NINDS支持的生物物理学社区，用于直接报告功能的试剂和活细胞中膜蛋白的动力学。解决这些问题的优雅解决方案是胡说八道抑制，一种可以在感兴趣的位点编码任何类型的合成氨基酸的方法在蛋白质内。这些所谓的非天然氨基酸可以采用单个原子的残留形式取代或具有新型荧光性能的侧链。虽然多种实验途径存在不自然的氨基酸的编码，每种氨基酸都与重大的技术挑战有关。因此，这个对于大多数研究分子神经科学的研究者来说，强大的方法仍然无法访问。 2014年，在对其他调查员的众多询问的回应，爱荷华大学（UI）的Ahern实验室开始简化酰化正交trNA的传播，无义抑制中的关键组成部分涉及真核膜蛋白的实验。为此，我们对基础进行了改进化学，使其更强大，并允许编码更化学多样的氨基酸。此外，这些新试剂显示出巨大改进的稳定性轮廓，从而可以轻松运输向美国各地的实验室提供了我们的试剂和指导，许多新用户群体成功地将这种先前困难的方法应用于与Ninds相关的各种膜蛋白使命。总体而言，这些努力产生了高功能的协作外展服务，“该设施对于原子诱变。抑制作用，该设施能够快速适应或设计合成方法，以满足新用户的申请。随着技术变得更加标准化，我们的用户组扩展，我们将因此，比例比例，利用UI Carver医学院和本地的基础设施工业合作伙伴，例如集成的DNA技术。这些合作最终将支持更多这些研究工具的有效传播，以回答分子神经科学中的潜水员问题。他们的使用的用户群，年度培训中心，基于Web的论坛并发布开放访问同行评审期刊中的协议。我们的顾问委员会 - Kossiakoff，Perozo，Koide，Nakamoto - 将确保有效地管理关键资源，并使我们的战略愿景与Ninds保持一致。