MRI: Acquisition of a Microscale Thermophoresis Instrument

MRI：购买微型热泳仪器

• 批准号: 1427465
• 负责人: Daryl Eggers
• 金额: $ 15.05万
• 依托单位: San Jose State University Foundation
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1427465&HistoricalAwards=false
• 关键词: MRI; Acquisition; Microscale; Thermophoresis; Instrument

Non Technical Description: This award to San Jose State University will provide funding for purchase of a specialized instrument that detects the movement of fluorescent molecules when introduced to a small localized temperature gradient, a process known as thermophoresis. Because the physical properties of a molecule change when bound to another molecule, thermophoresis experiments can be used to measure the binding affinity of two interacting partners, as long as one of the molecules is fluorescent or labeled with a fluorescent probe. Initial experiments will focus on three projects by three different investigators. The Skovran laboratory will use thermophoresis to characterize the expression and regulation of the key enzymes that allow microorganisms to utilize methane or methanol as a carbon source; this project could enhance metabolic engineering efforts that aim to design microorganisms for the production of biofuels and other desired chemicals. The Rascon laboratory will use thermophoresis to study certain digestive enzymes from mosquitos that bind and cleave blood-meal proteins; this project could lead to new strategies of controlling mosquito populations that carry serious diseases like the Dengue fever pathogen. The Eggers laboratory plans to use thermophoresis to test a new theory regarding the role of water in binding reactions; results from this project may be transformative for the field of biothermodynamics. Numerous undergraduate and MS/MA students will be trained in operation of the thermophoresis instrument, including minority students supported by other programs at SJSU.Technical Description: The microscale thermophoresis (MST) instrument to be purchased with this award employs a fluorescence detector with a built-in infrared laser that heats a nanoliter-sized volume of sample at the exact same location where fluorescence emission is monitored. The phenomenon of thermophoresis refers to the migration of species into/out of the heated zone due to differences in molecular size, surface chemistry, and hydration properties. If one of two binding species is fluorescently labeled, MST may be used to separate and measure the bound complex relative to the free reactants, yielding an equilibrium binding constant. Initial MST experiments will focus on three projects: (1) characterization of the key binding events that lead to expression and regulation of methanol dehydrogenases in a model organism, Methylobacterium extorquens; (2) the investigation of proteases from the mosquito Aedes aegypti to test the hypothesis that enzyme expression and activity are organized in a proteolytic cascade to optimize blood-meal protein digestion; and (3) the study of model enzyme-inhibitor interactions to test a new thermodynamic framework that incorporates the role of desolvation in the governing equations for binding equilibria. The use of microscale thermophoresis in these projects has several advantages over other binding techniques, including low reagent consumption, an unrestricted range of operating concentrations, quick analysis time, and the potential to analyze macromolecules in cell lysates without prior purification.

非技术描述：授予圣何塞州立大学的这一奖项将为购买一种专门的仪器提供资金，该仪器在引入小的局部温度梯度时检测荧光分子的运动，这一过程被称为热泳。因为当一个分子与另一个分子结合时，其物理性质会发生变化，所以热泳实验可以用来测量两个相互作用的伴侣的结合亲和力，只要其中一个分子是荧光的或用荧光探针标记。最初的实验将集中在三个不同的研究人员的三个项目。Skovran实验室将使用热泳来表征允许微生物利用甲烷或甲醇作为碳源的关键酶的表达和调节;该项目可以加强旨在设计用于生产生物燃料和其他所需化学品的微生物的代谢工程工作。 Rascon实验室将使用热泳来研究蚊子的某些消化酶，这些消化酶结合和切割血粉蛋白;该项目可能导致控制携带登革热病原体等严重疾病的蚊子种群的新策略。埃格斯实验室计划使用热泳来测试一种关于水在结合反应中的作用的新理论;该项目的结果可能会改变生物热力学领域。许多本科生和MS/MA学生将接受热泳仪器操作的培训，包括SJSU其他项目支持的少数民族学生。技术说明：该奖项将购买的微尺度热泳（MST）仪器采用内置红外激光器的荧光检测器，在荧光发射监测的同一位置加热纳升大小的样品体积。热泳现象是指由于分子大小、表面化学和水合性质的差异，物质迁移进入/离开加热区。如果两种结合物质中的一种被荧光标记，则MST可用于分离和测量结合的复合物相对于游离反应物，产生平衡结合常数。最初的MST实验将集中在三个项目上：（1）表征导致模式生物扭脱甲基杆菌中甲醇脱氢酶表达和调节的关键结合事件;（2）研究来自埃及伊蚊的蛋白酶，以检验酶表达和活性以蛋白水解级联方式组织以优化血粉蛋白消化的假设;和（3）模型酶抑制剂相互作用的研究，以测试一个新的热力学框架，结合平衡的控制方程中的去溶剂化的作用。在这些项目中使用微尺度热泳具有优于其他结合技术的几个优点，包括低试剂消耗、不受限制的操作浓度范围、快速分析时间以及在没有预先纯化的情况下分析细胞裂解物中的大分子的潜力。