Molecular Architecture of the Na+/Glucose Cotransporter

描述（由申请方提供）：Na+/葡萄糖协同转运蛋白（SGLT 1）是典型的协同转运蛋白，其利用跨膜Na+电化学电位梯度中储存的能量将营养物质驱动到细胞中。SGLT 1主要在小肠刷状缘膜中表达，负责每天吸收180 g膳食葡萄糖和半乳糖。SGLT 1的糖、盐和水转运偶联为口服补液疗法用于治疗感染性腹泻提供了理论依据。编码SGLT 1的基因突变导致葡萄糖-半乳糖-吸收不良。该项目的目标是确定SGLT 1如何耦合钠，糖和水的运输。本发明的目的是1）使用光谱方法如ATR-FTIR、CD和荧光来监测和确定配体诱导的蛋白质中的构象变化的程度，2）使用光亲和标记和质谱来鉴定底物结合位点，以及3）使用冷冻断裂电子显微镜来筛选2D和3D晶体来启动长期研究以解析蛋白质的结构。这些实验将在大肠杆菌中过表达的人SGLT 1和相关转运蛋白上进行。coli中，纯化并重构为脂质体。结果将提供钠/糖共转运交替访问模型的直接测试。

DESCRIPTION (provided by applicant): The Na+/glucose cotransporter (SGLT1) is the archetypical cotransporter that uses the energy stored in Na+ electrochemical potential gradients across membranes to drive nutrients into cells. SGLT1 is expressed mainly in the brush border membrane of the small intestine where it is responsible for the absorption of 180g of dietary glucose and galactose per day. The coupling of sugar, salt and water transport by SGLT1 provides the rational for the Oral Rehydration Therapy used to treat infectious diarrhea. Mutations in the gene coding for SGLT1 cause Glucose-Galactose-Malabsorption. The goal of this project is to determine how SGLT1 couples sodium, sugar and water transport. The present aims are to 1) use spectroscopic methods such as ATR-FTIR, CD and fluorescence to monitor and determine the extent of ligand induced conformational changes in the protein, 2) identify the substrate- binding sites using photo-affinity labeling and mass spectrometry, and 3) initiate long term studies to resolve the structure of the protein using freeze-fracture electron microscopy to screen for 2D and 3D crystals. These experiments will be carried out on human SGLT1 and related transporters over expressed in E. coli, purified and reconstituted into liposomes. The results will provide a direct test of the alternating access model for sodium/sugar cotransport.