Conformational Changes in Secondary Active Transporters

次级活性转运蛋白的构象变化

• 批准号: 8746800
• 负责人: Joseph A Mindell
• 金额: $ 72.26万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8746800
• 关键词: Address; Affect; Affinity; Age; Area; Aspartate; Automobile Driving; Bacteria; Binding; Biochemical; Biological Models; Characteristics; Charge; Chemicals; Collaborations; Coupled; Coupling; Cut protein; Cysteine; Development; Drosophila genus; Electrons; Environment; Family; Glutamate Transporter; Goals; Homologous Gene; Homologous Protein; Insulin Resistance; Ions; Knowledge; Lead; Link; Longevity; Mammals; Methods; Modeling; Monitor; Movement; Mus; Mutation; Nature; Neuraxis; Neurons; Neurotransmitters; New York; Obesity; Paper; Play; Positioning Attribute; Process; Protein Dynamics; Proteins; Publications; Reporting; Role; Scanning; Shapes; Site; Spectrum Analysis; Stimulus; Succinates; Synapses; Therapeutic Agents; Time; Transmembrane Domain; Transport Process; Universities; Work; Writing; base; citrate carrier; experience; extracellular; fly; protein structure; reconstitution

This year we expanded this project to include both GltPh, a model for the EAAT family of glutamate transporters and a new protein, vcINDY, a succinate transproter which has been implicated in longevity and obesity. It is critical to understand the fundamental mechanisms by which there transporters function because such knowledge could lead to the development of therapeutic agents active against these proteins. We seek to analyze the dynamic movements of the functioning transporter on the way to a detailed understanding of its mechanism. Our approach is to analyze the details of transport in model transporters obtained from bacteria. These can be expressed and purified in large quantities and are amenable to biophysical methods not available for their mammalian cousins. We have continued our work using EPR spectroscopy to monitor conformational changes in GltPh. This work has identified local changes in the protein that may be important for coupling between the driving ion, Na+, and the substrate, aspartate. We are continuing work to identify the nature of this change. We recently reported that a extracellular loop of gltPH must be intact for effective transport. This year we probed the mechanism of this effect in detail and found that when the 34 loop is cut the proteins maintains substrate affinities but maximal transport is significantly reduced. We demonstrated that this effect relates to the activation energy of the substrate translocation step, implicating the loop in the piston like movement of the translocation domain. This year we also found that only the translocation of the substrate-bound form of the protein is affected--the apo, substrate-free transporter is unaffected by 34 loop cleavage. We have performed important controls eliminating alternative explanations for these effects and a paper describing this work is under review. This year we also began work on a new transporter, vcINDY, which is important for longevity in flies and is involved in obesity and insulin resistance in mammals. We performed the first successful functional reconstitution of vcINDY and directly demonstrated that it is a Na+ coupled succinate transporter. We have performed an extensive screen for substrates and identified that the protein carries three Na ions per succinate transpoted, that it is electrogenic (but does not have an uncoupled Cl- conductance) and that it primarily transports the doubly charged form of succinate. We are currently writing this work up for publication.

今年，我们扩大了这个项目，将GltPh（谷氨酸转运蛋白EAAT家族的模型）和vcINDY（琥珀酸转运蛋白，与长寿和肥胖有关）纳入其中。了解这些转运蛋白发挥作用的基本机制是至关重要的，因为这些知识可能导致开发针对这些蛋白质的治疗药物。我们试图在详细了解其机制的过程中分析功能转运体的动态运动。我们的方法是分析从细菌中获得的模型转运体中的转运细节。它们可以大量表达和纯化，并且适用于它们的哺乳动物表亲无法使用的生物物理方法。