MRI: Acquisition of a Microscale Thermophoresis Instrument

非技术描述：授予圣何塞州立大学的这一奖项将提供资金，用于购买一种专门的仪器，当引入到小的局部温度梯度时，该仪器可以检测荧光分子的运动，这一过程被称为热电泳法。因为当一个分子与另一个分子结合时，分子的物理性质会发生变化，热电泳法实验可以用来测量两个相互作用的伙伴的结合亲和力，只要其中一个分子是荧光的或用荧光探针标记的。最初的实验将集中在三个不同研究人员的三个项目上。斯科夫兰实验室将使用热泳法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控；该项目可能会加强代谢工程努力，旨在设计微生物生产生物燃料和其他所需化学品。Rascon实验室将使用热渗透技术来研究蚊子的某些消化酶，这些酶可以结合和分解血粉蛋白；这个项目可能会带来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电泳法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对生物热力学领域产生革命性的影响。许多本科生和MS/MA学生将接受热导入仪器的操作培训，包括由上海理工大学其他项目支持的少数学生。技术说明：与该奖项一起购买的微型热电导入(MST)仪器使用带有内置红外激光器的荧光探测器，在监测荧光发射的完全相同的位置加热纳升大小的样品。热浸现象指的是由于分子大小、表面化学和水化性质的不同，物种迁入/迁出加热区的现象。如果两个结合物种中的一个被荧光标记，则可以使用MST来分离和测量相对于自由反应物的结合络合物，从而产生平衡结合常数。最初的MST实验将集中在三个项目上：(1)表征导致甲醇脱氢酶在模式生物甲氧基乳酸杆菌中表达和调控的关键结合事件；(2)研究来自埃及伊蚊的蛋白酶，以验证酶的表达和活性是在蛋白水解级联中组织的假设，以优化血粉蛋白的消化；以及(3)模型酶-抑制物相互作用的研究，以测试一个新的热力学框架，该框架在结合平衡的控制方程中引入了脱溶的作用。与其他结合技术相比，在这些项目中使用微型热电泳法具有几个优点，包括试剂消耗低、操作浓度范围不受限制、分析时间短，以及无需事先纯化即可分析细胞裂解物中的大分子。

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.