SCREENING SCORPION, SPIDER, SNAKE, SNAIL TOXINS FOR BINDING TO K+ CHANNELS

筛选蝎子、蜘蛛、蛇、蜗牛毒素与 K 通道的结合

• 批准号: 8361482
• 负责人: Brian T Chait
• 金额: $ 5.22万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361482
• 关键词: Affinity Chromatography; Animals; Binding; Cysteine; Funding; Grant; Hydroxyproline; Libraries; Mass Spectrum Analysis; Messenger RNA; Methodology; Methods; N-terminal; National Center for Research Resources; Paper; Peptides; Potassium Channel; Principal Investigator; Proteins; Publishing; Research; Research Infrastructure; Resources; Scorpion Venoms; Scorpions; Screening procedure; Sequence Analysis; Snail Venoms; Snails; Snake Venoms; Snakes; Source; Spider Venoms; Spiders; Streptomyces lividans; Toxin; Tryptophan; United States National Institutes of Health; Venoms; Work; amidation; cost; extracellular; inhibitor/antagonist; macromolecule; molecular mass; programs; rapid technique; tool; voltage gated channel

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The extracellular entryway of the bacterial potassium channel of Streptomyces lividans (KcsA) is homologous to eukaryotic voltage gated channels. For this reason, KcsA is used as a template for the binding of extracellular pore blockers. Animal venoms such as snake, spider, scorpion and snail venoms are de facto libraries of naturally occurring toxins. Varrious venoms were screened against immobilized K+ channels using affinity chromatography. Following extensive washes, the channels were eluted along with specifically bound toxins. Mass spectrometry was used as a tool to quickly identify small protein toxins from their molecular mass and fragmentation spectra. This approach provides a rapid method for identifying potential inhibitors of eukaryotic potassium channels. We are developing mass spectrometric methods for rapidly determining the primary sequences of newly discovered channel-binding toxins. In particular, we have stablished methodology and workflow for toxin sequencing, including determination of number of cysteines and have developied a derivatization strategy to facilitate sequence analysis by ETD. Several toxins known and previously unknown have been identified. Several of the previously unknown toxins have sequenced mRNAs. PTMs like hydroxyproline, N-terminal amidation, and bromination of tryptophan were identified. We have also wriiten a program called TOXFINDER, which facilitates this analysis. We have published a paper describing this work (B.M. Ueberheide, D. Feny¿, P.F. Alewood, B.T. Chait "Rapid, sensitive analysis of peptide venom components" Proc Natl Acad Sci, 106 (2009) 6910-5).

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 浅青紫链霉菌 (KcsA) 的细菌钾通道 (KcsA) 的细胞外入口与真核电压门控通道同源。因此，KcsA 被用作细胞外孔阻断剂结合的模板。蛇、蜘蛛、蝎子和蜗牛等动物毒液实际上是天然毒素的库。使用亲和层析针对固定 K+ 通道筛选各种毒液。经过大量洗涤后，通道与特定结合的毒素一起被洗脱。质谱法被用作从分子质量和碎片谱中快速识别小蛋白质毒素的工具。该方法提供了一种快速鉴定真核钾通道潜在抑制剂的方法。我们正在开发质谱方法，用于快速确定新发现的通道结合毒素的一级序列。特别是，我们建立了毒素测序的方法和工作流程，包括半胱氨酸数量的测定，并开发了衍生化策略以促进 ETD 进行序列分析。 已经鉴定出几种已知和以前未知的毒素。 一些以前未知的毒素已经对 mRNA 进行了测序。 鉴定出羟脯氨酸、N 末端酰胺化和色氨酸溴化等 PTM。 我们还编写了一个名为 TOXFINDER 的程序，它有助于进行这种分析。 我们发表了一篇描述这项工作的论文（B.M. Ueberheide、D. Feny¿、P.F. Alewood、B.T. Chait“肽毒液成分的快速、灵敏分析”Proc Natl Acad Sci, 106 (2009) 6910-5）。